NZ623181B2 - Compositions and methods for silencing aldehyde dehydrogenase - Google Patents
Compositions and methods for silencing aldehyde dehydrogenase Download PDFInfo
- Publication number
- NZ623181B2 NZ623181B2 NZ623181A NZ62318112A NZ623181B2 NZ 623181 B2 NZ623181 B2 NZ 623181B2 NZ 623181 A NZ623181 A NZ 623181A NZ 62318112 A NZ62318112 A NZ 62318112A NZ 623181 B2 NZ623181 B2 NZ 623181B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- lipid
- mol
- particle
- nucleic acid
- sirna
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 202
- 102000005369 Aldehyde Dehydrogenase Human genes 0.000 title claims abstract description 110
- 108020002663 Aldehyde Dehydrogenase Proteins 0.000 title claims abstract description 110
- 230000001743 silencing Effects 0.000 title claims description 21
- 108020004459 Small Interfering RNA Proteins 0.000 claims abstract description 330
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims abstract description 305
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 208
- 239000002773 nucleotide Substances 0.000 claims abstract description 178
- 230000002452 interceptive Effects 0.000 claims abstract description 162
- 230000000692 anti-sense Effects 0.000 claims abstract description 107
- 230000014509 gene expression Effects 0.000 claims abstract description 85
- 229920000972 Sense strand Polymers 0.000 claims abstract description 45
- 230000000295 complement Effects 0.000 claims abstract description 38
- 239000002924 silencing RNA Substances 0.000 claims abstract 8
- 239000002245 particle Substances 0.000 claims description 394
- 150000002632 lipids Chemical class 0.000 claims description 375
- -1 cationic lipid Chemical class 0.000 claims description 153
- 150000007523 nucleic acids Chemical class 0.000 claims description 133
- 108020004707 nucleic acids Proteins 0.000 claims description 129
- HVYWMOMLDIMFJA-DPAQBDIFSA-N (3β)-Cholest-5-en-3-ol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 120
- 229940107161 Cholesterol Drugs 0.000 claims description 60
- 235000012000 cholesterol Nutrition 0.000 claims description 60
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 55
- 239000011780 sodium chloride Substances 0.000 claims description 54
- 229920001223 polyethylene glycol Polymers 0.000 claims description 50
- 150000003839 salts Chemical class 0.000 claims description 45
- 150000003904 phospholipids Chemical class 0.000 claims description 38
- 241000282414 Homo sapiens Species 0.000 claims description 36
- 241000124008 Mammalia Species 0.000 claims description 32
- KILNVBDSWZSGLL-KXQOOQHDSA-N Dipalmitoylphosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 claims description 25
- 238000004220 aggregation Methods 0.000 claims description 23
- 230000002776 aggregation Effects 0.000 claims description 23
- 208000007848 Alcoholism Diseases 0.000 claims description 22
- 201000007930 alcohol dependence Diseases 0.000 claims description 20
- 239000003814 drug Substances 0.000 claims description 14
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical class O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims description 9
- 239000003937 drug carrier Substances 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- XVUQPECVOGMPRU-ZPPAUJSGSA-N N,N-dimethyl-1,2-bis[(9Z,12Z)-octadeca-9,12-dienoxy]propan-1-amine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCOC(C)C(N(C)C)OCCCCCCCC\C=C/C\C=C/CCCCC XVUQPECVOGMPRU-ZPPAUJSGSA-N 0.000 claims description 2
- OXOWTLDONRGYOT-UHFFFAOYSA-M 4-(dimethylamino)butanoate Chemical compound CN(C)CCCC([O-])=O OXOWTLDONRGYOT-UHFFFAOYSA-M 0.000 claims 1
- 102000009645 Mitochondrial Aldehyde Dehydrogenase Human genes 0.000 claims 1
- 108010009513 Mitochondrial Aldehyde Dehydrogenase Proteins 0.000 claims 1
- OZBZDYGIYDRTBV-RSLAUBRISA-N N,N-dimethyl-1,2-bis[(9Z,12Z,15Z)-octadeca-9,12,15-trienoxy]propan-1-amine Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCCOC(C)C(N(C)C)OCCCCCCCC\C=C/C\C=C/C\C=C/CC OZBZDYGIYDRTBV-RSLAUBRISA-N 0.000 claims 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 239000004055 small Interfering RNA Substances 0.000 description 330
- 229920001985 Small interfering RNA Polymers 0.000 description 312
- 210000004027 cells Anatomy 0.000 description 94
- 125000002091 cationic group Chemical group 0.000 description 78
- 108020004461 Double-Stranded RNA Proteins 0.000 description 71
- 238000009472 formulation Methods 0.000 description 53
- 235000002639 sodium chloride Nutrition 0.000 description 51
- 239000002679 microRNA Substances 0.000 description 42
- 108020004999 Messenger RNA Proteins 0.000 description 41
- 229920002106 messenger RNA Polymers 0.000 description 41
- 238000000034 method Methods 0.000 description 36
- 230000001225 therapeutic Effects 0.000 description 35
- 238000004166 bioassay Methods 0.000 description 34
- 239000000969 carrier Substances 0.000 description 34
- 229920001239 microRNA Polymers 0.000 description 34
- 125000005647 linker group Chemical group 0.000 description 32
- 125000000217 alkyl group Chemical group 0.000 description 31
- 239000002585 base Substances 0.000 description 29
- 239000000758 substrate Substances 0.000 description 29
- 229920000272 Oligonucleotide Polymers 0.000 description 28
- 229920001891 Small hairpin RNA Polymers 0.000 description 25
- 230000015572 biosynthetic process Effects 0.000 description 25
- 150000002500 ions Chemical class 0.000 description 25
- IKHGUXGNUITLKF-UHFFFAOYSA-N acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 24
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 24
- 230000002194 synthesizing Effects 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 23
- 230000000694 effects Effects 0.000 description 23
- 229920000642 polymer Polymers 0.000 description 23
- 150000002148 esters Chemical class 0.000 description 22
- 238000003786 synthesis reaction Methods 0.000 description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 230000027455 binding Effects 0.000 description 21
- 238000000338 in vitro Methods 0.000 description 21
- 230000004048 modification Effects 0.000 description 21
- 238000006011 modification reaction Methods 0.000 description 21
- 125000002652 ribonucleotide group Chemical group 0.000 description 21
- 239000002253 acid Substances 0.000 description 20
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 20
- 239000000243 solution Substances 0.000 description 20
- 102100015294 DICER1 Human genes 0.000 description 19
- 101710039595 DICER1 Proteins 0.000 description 19
- 239000003795 chemical substances by application Substances 0.000 description 19
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 18
- 229920001914 Ribonucleotide Polymers 0.000 description 18
- 235000019441 ethanol Nutrition 0.000 description 18
- 238000009396 hybridization Methods 0.000 description 18
- 238000002156 mixing Methods 0.000 description 18
- 239000002336 ribonucleotide Substances 0.000 description 18
- 230000003308 immunostimulating Effects 0.000 description 17
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 125000002252 acyl group Chemical group 0.000 description 16
- 230000002401 inhibitory effect Effects 0.000 description 16
- 239000002502 liposome Substances 0.000 description 16
- 230000025458 RNA interference Effects 0.000 description 15
- 230000028993 immune response Effects 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 15
- 210000001519 tissues Anatomy 0.000 description 15
- 230000001809 detectable Effects 0.000 description 14
- 238000010790 dilution Methods 0.000 description 14
- 125000000623 heterocyclic group Chemical group 0.000 description 14
- 230000000670 limiting Effects 0.000 description 14
- 239000010452 phosphate Substances 0.000 description 14
- 229920000023 polynucleotide Polymers 0.000 description 14
- 239000002157 polynucleotide Substances 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 210000004185 Liver Anatomy 0.000 description 13
- 229920001850 Nucleic acid sequence Polymers 0.000 description 13
- 230000000875 corresponding Effects 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical class 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 12
- 238000002347 injection Methods 0.000 description 12
- 239000007924 injection Substances 0.000 description 12
- 101700043375 sing Proteins 0.000 description 12
- 239000000725 suspension Substances 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 11
- 230000015556 catabolic process Effects 0.000 description 11
- 230000004059 degradation Effects 0.000 description 11
- 238000006731 degradation reaction Methods 0.000 description 11
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 11
- 239000001257 hydrogen Substances 0.000 description 11
- 229910052739 hydrogen Inorganic materials 0.000 description 11
- 239000012528 membrane Substances 0.000 description 11
- 235000021317 phosphate Nutrition 0.000 description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 11
- 241000894007 species Species 0.000 description 11
- LVNGJLRDBYCPGB-LDLOPFEMSA-N (R)-1,2-distearoylphosphatidylethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-LDLOPFEMSA-N 0.000 description 10
- 229940067631 Phospholipids Drugs 0.000 description 10
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 10
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-K [O-]P([O-])([O-])=O Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 230000001264 neutralization Effects 0.000 description 10
- AUZONCFQVSMFAP-UHFFFAOYSA-N Disulfiram Chemical compound CCN(CC)C(=S)SSC(=S)N(CC)CC AUZONCFQVSMFAP-UHFFFAOYSA-N 0.000 description 9
- 125000003342 alkenyl group Chemical group 0.000 description 9
- 230000003321 amplification Effects 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 238000001990 intravenous administration Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 9
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 9
- 239000000523 sample Substances 0.000 description 9
- 229960002563 Disulfiram Drugs 0.000 description 8
- 229940088598 Enzyme Drugs 0.000 description 8
- 241000282412 Homo Species 0.000 description 8
- 101700080605 NUC1 Proteins 0.000 description 8
- 150000001982 diacylglycerols Chemical class 0.000 description 8
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 8
- 229940079593 drugs Drugs 0.000 description 8
- 238000005755 formation reaction Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 101700006494 nucA Proteins 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 210000000056 organs Anatomy 0.000 description 8
- 235000000346 sugar Nutrition 0.000 description 8
- OPTASPLRGRRNAP-UHFFFAOYSA-N Cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 7
- 229940067605 Phosphatidylethanolamines Drugs 0.000 description 7
- 239000004698 Polyethylene (PE) Substances 0.000 description 7
- DRTQHJPVMGBUCF-UCVXFZOQSA-N Uridine Natural products O[C@H]1[C@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UCVXFZOQSA-N 0.000 description 7
- 229940045145 Uridine Drugs 0.000 description 7
- 125000000304 alkynyl group Chemical group 0.000 description 7
- 125000004432 carbon atoms Chemical group C* 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 7
- 150000002431 hydrogen Chemical class 0.000 description 7
- 239000003446 ligand Substances 0.000 description 7
- 239000003607 modifier Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 206010049460 Abasia Diseases 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 239000004952 Polyamide Substances 0.000 description 6
- 210000002966 Serum Anatomy 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- 238000007792 addition Methods 0.000 description 6
- 125000003277 amino group Chemical group 0.000 description 6
- 125000000129 anionic group Chemical group 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 125000003118 aryl group Chemical group 0.000 description 6
- 150000001841 cholesterols Chemical class 0.000 description 6
- 239000005547 deoxyribonucleotide Substances 0.000 description 6
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 6
- 235000014113 dietary fatty acids Nutrition 0.000 description 6
- 230000003828 downregulation Effects 0.000 description 6
- 239000000839 emulsion Substances 0.000 description 6
- 239000000194 fatty acid Substances 0.000 description 6
- 150000004665 fatty acids Chemical class 0.000 description 6
- ZHNUHDYFZUAESO-UHFFFAOYSA-N formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 6
- 230000030279 gene silencing Effects 0.000 description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 6
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 6
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 6
- 229920002647 polyamide Polymers 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 210000004369 Blood Anatomy 0.000 description 5
- 229920000858 Cyclodextrin Polymers 0.000 description 5
- 210000002540 Macrophages Anatomy 0.000 description 5
- LKQLRGMMMAHREN-YJFXYUILSA-N N-stearoylsphingosine-1-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)N[C@@H](COP([O-])(=O)OCC[N+](C)(C)C)[C@H](O)\C=C\CCCCCCCCCCCCC LKQLRGMMMAHREN-YJFXYUILSA-N 0.000 description 5
- SNKAWJBJQDLSFF-YEUCEMRASA-N [2-({2,3-bis[(9Z)-octadec-9-enoyloxy]propyl phosphonato}oxy)ethyl]trimethylazanium Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-YEUCEMRASA-N 0.000 description 5
- 150000001412 amines Chemical class 0.000 description 5
- 150000001450 anions Chemical class 0.000 description 5
- 102000004965 antibodies Human genes 0.000 description 5
- 108090001123 antibodies Proteins 0.000 description 5
- 239000012472 biological sample Substances 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000005538 encapsulation Methods 0.000 description 5
- 230000002708 enhancing Effects 0.000 description 5
- 230000000799 fusogenic Effects 0.000 description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 5
- 238000007901 in situ hybridization Methods 0.000 description 5
- 210000004962 mammalian cells Anatomy 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 230000003000 nontoxic Effects 0.000 description 5
- 231100000252 nontoxic Toxicity 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 5
- 229920000765 poly(2-oxazolines) Polymers 0.000 description 5
- 230000002633 protecting Effects 0.000 description 5
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 5
- OPCHFPHZPIURNA-MFERNQICSA-N (2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide Chemical compound CCCCCCCCCCCCCCCCCCN(CC(=O)NC(=O)[C@H](CCCNCCCN)NCCCN)CCCCCCCCCCCCCCCCCC OPCHFPHZPIURNA-MFERNQICSA-N 0.000 description 4
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 4
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 4
- SLKDGVPOSSLUAI-UHFFFAOYSA-N 2-azaniumylethyl 2,3-di(hexadecanoyloxy)propyl phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-UHFFFAOYSA-N 0.000 description 4
- OIRDTQYFTABQOQ-SXVXDFOESA-N Adenosine Natural products Nc1ncnc2c1ncn2[C@@H]3O[C@@H](CO)[C@H](O)[C@@H]3O OIRDTQYFTABQOQ-SXVXDFOESA-N 0.000 description 4
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 4
- 210000000170 Cell Membrane Anatomy 0.000 description 4
- 229920002676 Complementary DNA Polymers 0.000 description 4
- 210000000805 Cytoplasm Anatomy 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 4
- 108020004388 MicroRNAs Proteins 0.000 description 4
- 210000003819 Peripheral blood mononuclear cell Anatomy 0.000 description 4
- 210000002381 Plasma Anatomy 0.000 description 4
- 229920002873 Polyethylenimine Polymers 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N Thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 229960005305 adenosine Drugs 0.000 description 4
- 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 4
- 230000001476 alcoholic Effects 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- KXDHJXZQYSOELW-UHFFFAOYSA-M carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 4
- 238000003113 dilution method Methods 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 230000002068 genetic Effects 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 125000005842 heteroatoms Chemical group 0.000 description 4
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 description 4
- 230000002209 hydrophobic Effects 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-L oxalate Chemical compound [O-]C(=O)C([O-])=O MUBZPKHOEPUJKR-UHFFFAOYSA-L 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- RZVAJINKPMORJF-UHFFFAOYSA-N p-acetaminophenol Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 4
- 230000036961 partial Effects 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000011528 polyamide (building material) Substances 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000002829 reduced Effects 0.000 description 4
- 230000002987 rna-interference Effects 0.000 description 4
- 238000004513 sizing Methods 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- KWVJHCQQUFDPLU-YEUCEMRASA-N 2,3-bis[[(Z)-octadec-9-enoyl]oxy]propyl-trimethylazanium Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KWVJHCQQUFDPLU-YEUCEMRASA-N 0.000 description 3
- 125000003601 C2-C6 alkynyl group Chemical group 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate dianion Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 210000003169 Central Nervous System Anatomy 0.000 description 3
- 229920001405 Coding region Polymers 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 229940104302 Cytosine Drugs 0.000 description 3
- UYTPUPDQBNUYGX-UHFFFAOYSA-N Guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 3
- 206010019233 Headache Diseases 0.000 description 3
- 210000003494 Hepatocytes Anatomy 0.000 description 3
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 3
- 241000229754 Iva xanthiifolia Species 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
- 239000004472 Lysine Substances 0.000 description 3
- 210000001616 Monocytes Anatomy 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 229920002224 Peptide nucleic acid Polymers 0.000 description 3
- 229920000954 Polyglycolide Polymers 0.000 description 3
- 241000288906 Primates Species 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- 101710040537 TNF Proteins 0.000 description 3
- 229940035295 Ting Drugs 0.000 description 3
- 230000002730 additional Effects 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 108010081577 aldehyde dehydrogenase (NAD(P)+) Proteins 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000000903 blocking Effects 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 150000001720 carbohydrates Chemical group 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 230000001808 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 238000010511 deprotection reaction Methods 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000002255 enzymatic Effects 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 150000002367 halogens Chemical class 0.000 description 3
- 231100000869 headache Toxicity 0.000 description 3
- 229920001477 hydrophilic polymer Polymers 0.000 description 3
- 238000001114 immunoprecipitation Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 238000007834 ligase chain reaction Methods 0.000 description 3
- 230000001404 mediated Effects 0.000 description 3
- 102000005614 monoclonal antibodies Human genes 0.000 description 3
- 108010045030 monoclonal antibodies Proteins 0.000 description 3
- 125000001419 myristoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 229920000747 poly(lactic acid) polymer Polymers 0.000 description 3
- 229920000768 polyamine Polymers 0.000 description 3
- 239000004633 polyglycolic acid Substances 0.000 description 3
- 239000004626 polylactic acid Substances 0.000 description 3
- 150000003230 pyrimidines Chemical class 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 125000001424 substituent group Chemical group 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 230000003612 virological Effects 0.000 description 3
- NJGIRBISCGPRPF-KXQOOQHDSA-N (2-aminoethoxy)[(2R)-2-(icosanoyloxy)-3-(pentadecanoyloxy)propoxy]phosphinic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(=O)O[C@@H](COP(O)(=O)OCCN)COC(=O)CCCCCCCCCCCCCC NJGIRBISCGPRPF-KXQOOQHDSA-N 0.000 description 2
- 125000006527 (C1-C5) alkyl group Chemical group 0.000 description 2
- 125000006656 (C2-C4) alkenyl group Chemical group 0.000 description 2
- 125000006650 (C2-C4) alkynyl group Chemical group 0.000 description 2
- 125000006729 (C2-C5) alkenyl group Chemical group 0.000 description 2
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-dilinoleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 description 2
- KZKAYEGOIJEWQB-UHFFFAOYSA-N 1,3-dibromopropane;N,N,N',N'-tetramethylhexane-1,6-diamine Chemical compound BrCCCBr.CN(C)CCCCCCN(C)C KZKAYEGOIJEWQB-UHFFFAOYSA-N 0.000 description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(Z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- CGDNFXSLPGLMHK-UHFFFAOYSA-N 2-[2-(2-methylprop-2-enoyloxy)ethyldisulfanyl]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCSSCCOC(=O)C(C)=C CGDNFXSLPGLMHK-UHFFFAOYSA-N 0.000 description 2
- 101710027066 ALB Proteins 0.000 description 2
- 102100001249 ALB Human genes 0.000 description 2
- 230000036912 Bioavailability Effects 0.000 description 2
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 2
- 241000282465 Canis Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N Deoxyribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000005977 Ethylene Substances 0.000 description 2
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 210000002216 Heart Anatomy 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006822 Human Serum Albumin Proteins 0.000 description 2
- 241000282619 Hylobates lar Species 0.000 description 2
- JGJLWPGRMCADHB-UHFFFAOYSA-N Hypobromite Chemical compound Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- 210000001865 Kupffer Cells Anatomy 0.000 description 2
- 241000283953 Lagomorpha Species 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 210000004072 Lung Anatomy 0.000 description 2
- 210000001165 Lymph Nodes Anatomy 0.000 description 2
- 210000003563 Lymphoid Tissue Anatomy 0.000 description 2
- ZUHZZVMEUAUWHY-UHFFFAOYSA-N N,N-dimethylpropan-1-amine Chemical compound CCCN(C)C ZUHZZVMEUAUWHY-UHFFFAOYSA-N 0.000 description 2
- 206010028813 Nausea Diseases 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N Nicotinamide adenine dinucleotide Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 2
- 241000282579 Pan Species 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-M Perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 2
- UNJJBGNPUUVVFQ-ZJUUUORDSA-N Phosphatidylserine Chemical compound CCCC(=O)O[C@H](COC(=O)CC)COP(O)(=O)OC[C@H](N)C(O)=O UNJJBGNPUUVVFQ-ZJUUUORDSA-N 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 229940005581 Sodium Lactate Drugs 0.000 description 2
- NGSFWBMYFKHRBD-UHFFFAOYSA-M Sodium lactate Chemical compound [Na+].CC(O)C([O-])=O NGSFWBMYFKHRBD-UHFFFAOYSA-M 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N Spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N Spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 210000000952 Spleen Anatomy 0.000 description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N Stearic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 102100009534 TNF Human genes 0.000 description 2
- 229940113082 Thymine Drugs 0.000 description 2
- 229920001949 Transfer RNA Polymers 0.000 description 2
- 229940035893 Uracil Drugs 0.000 description 2
- 210000003462 Veins Anatomy 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- RBAWEGYJSDPEHX-SNVBAGLBSA-N [(2R)-2,3-diformyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound C[N+](C)(C)CCOP([O-])(=O)OC[C@H](OC=O)COC=O RBAWEGYJSDPEHX-SNVBAGLBSA-N 0.000 description 2
- 230000001154 acute Effects 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- 238000004115 adherent culture Methods 0.000 description 2
- 229940050528 albumin Drugs 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229960000070 antineoplastic Monoclonal antibodies Drugs 0.000 description 2
- 125000002619 bicyclic group Chemical group 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000035514 bioavailability Effects 0.000 description 2
- 238000002306 biochemical method Methods 0.000 description 2
- 238000006065 biodegradation reaction Methods 0.000 description 2
- 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 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- SXDBWCPKPHAZSM-UHFFFAOYSA-M bromate Chemical compound [O-]Br(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-M 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 239000006172 buffering agent Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L cacl2 Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000001413 cellular Effects 0.000 description 2
- 150000001784 cerebrosides Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- XTEGARKTQYYJKE-UHFFFAOYSA-M chlorate Chemical compound [O-]Cl(=O)=O XTEGARKTQYYJKE-UHFFFAOYSA-M 0.000 description 2
- 150000003841 chloride salts Chemical class 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 125000004093 cyano group Chemical group *C#N 0.000 description 2
- 230000016396 cytokine production Effects 0.000 description 2
- 239000000412 dendrimer Substances 0.000 description 2
- 229920000736 dendritic polymer Polymers 0.000 description 2
- 230000001419 dependent Effects 0.000 description 2
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 2
- SNQXJPARXFUULZ-UHFFFAOYSA-N dioxolane Chemical compound C1COOC1 SNQXJPARXFUULZ-UHFFFAOYSA-N 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000007429 general method Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 150000004676 glycans Polymers 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth media Substances 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-M hydrogensulfate Chemical compound OS([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 230000000415 inactivating Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 230000002045 lasting Effects 0.000 description 2
- 125000005645 linoleyl group Chemical group 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000011068 load Methods 0.000 description 2
- 239000007937 lozenge Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 229920001427 mPEG Polymers 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- YACKEPLHDIMKIO-UHFFFAOYSA-L methylphosphonate(2-) Chemical compound CP([O-])([O-])=O YACKEPLHDIMKIO-UHFFFAOYSA-L 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229960000060 monoclonal antibodies Drugs 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 238000007899 nucleic acid hybridization Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 150000008103 phosphatidic acids Chemical class 0.000 description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 229920000962 poly(amidoamine) Polymers 0.000 description 2
- 229920000058 polyacrylate Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 150000004804 polysaccharides Polymers 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 230000002335 preservative Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 108091007167 primary miRNA Proteins 0.000 description 2
- 239000001294 propane Substances 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002213 purine nucleotide Substances 0.000 description 2
- 150000003212 purines Chemical class 0.000 description 2
- 239000002719 pyrimidine nucleotide Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000003307 reticuloendothelial Effects 0.000 description 2
- 235000003441 saturated fatty acids Nutrition 0.000 description 2
- 150000004671 saturated fatty acids Chemical class 0.000 description 2
- 231100000486 side effect Toxicity 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000001540 sodium lactate Substances 0.000 description 2
- 235000011088 sodium lactate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000003595 spectral Effects 0.000 description 2
- 150000003408 sphingolipids Chemical class 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 230000001954 sterilising Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 150000003432 sterols Chemical class 0.000 description 2
- 235000003702 sterols Nutrition 0.000 description 2
- 230000004936 stimulating Effects 0.000 description 2
- 125000000547 substituted alkyl group Chemical group 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 125000003507 tetrahydrothiofenyl group Chemical group 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000002103 transcriptional Effects 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 239000000277 virosome Substances 0.000 description 2
- 238000011179 visual inspection Methods 0.000 description 2
- QYIXCDOBOSTCEI-QCYZZNICSA-N (5α)-cholestan-3β-ol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCCC(C)C)[C@@]2(C)CC1 QYIXCDOBOSTCEI-QCYZZNICSA-N 0.000 description 1
- VDYVTMXBGOIUMS-KWXKLSQISA-N (6Z,9Z,29Z,32Z)-19-[(dimethylamino)methyl]octatriaconta-6,9,29,32-tetraene-18,21-dione Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)CC(CN(C)C)C(=O)CCCCCCC\C=C/C\C=C/CCCCC VDYVTMXBGOIUMS-KWXKLSQISA-N 0.000 description 1
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 description 1
- SLKDGVPOSSLUAI-PGUFJCEWSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-PGUFJCEWSA-N 0.000 description 1
- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 description 1
- BIABMEZBCHDPBV-MPQUPPDSSA-N 1,2-palmitoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCCCC BIABMEZBCHDPBV-MPQUPPDSSA-N 0.000 description 1
- YAMUFBLWGFFICM-PTGWMXDISA-N 1-O-oleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C YAMUFBLWGFFICM-PTGWMXDISA-N 0.000 description 1
- PLKOSISDOAHHCI-QYCRHRGJSA-N 1-[2,3-bis[(9Z,12Z)-octadeca-9,12-dienoxy]propyl]-4-methylpiperazine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCOCC(OCCCCCCCC\C=C/C\C=C/CCCCC)CN1CCN(C)CC1 PLKOSISDOAHHCI-QYCRHRGJSA-N 0.000 description 1
- NKHPSESDXTWSQB-WRBBJXAJSA-N 1-[3,4-bis[(Z)-octadec-9-enoxy]phenyl]-N,N-dimethylmethanamine Chemical compound CCCCCCCC\C=C/CCCCCCCCOC1=CC=C(CN(C)C)C=C1OCCCCCCCC\C=C/CCCCCCCC NKHPSESDXTWSQB-WRBBJXAJSA-N 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- FHQVHHIBKUMWTI-ZCXUNETKSA-N 1-palmitoyl-2-oleoyl phosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC FHQVHHIBKUMWTI-ZCXUNETKSA-N 0.000 description 1
- RLOQBKJCOAXOLR-UHFFFAOYSA-N 1H-pyrrole-2-carboxamide Chemical class NC(=O)C1=CC=CN1 RLOQBKJCOAXOLR-UHFFFAOYSA-N 0.000 description 1
- VGONTNSXDCQUGY-RRKCRQDMSA-N 2'-deoxyinosine Chemical group C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 VGONTNSXDCQUGY-RRKCRQDMSA-N 0.000 description 1
- WALUVDCNGPQPOD-UHFFFAOYSA-M 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCCCCCCCC WALUVDCNGPQPOD-UHFFFAOYSA-M 0.000 description 1
- MPCAJMNYNOGXPB-UHFFFAOYSA-N 2-(hydroxymethyl)oxane-3,4,5-triol Chemical class OCC1OCC(O)C(O)C1O MPCAJMNYNOGXPB-UHFFFAOYSA-N 0.000 description 1
- NEZDNQCXEZDCBI-UHFFFAOYSA-N 2-azaniumylethyl 2,3-di(tetradecanoyloxy)propyl phosphate Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCC NEZDNQCXEZDCBI-UHFFFAOYSA-N 0.000 description 1
- 125000000069 2-butynyl group Chemical group [H]C([H])([H])C#CC([H])([H])* 0.000 description 1
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- 125000006029 2-methyl-2-butenyl group Chemical group 0.000 description 1
- FTBBGQKRYUTLMP-UHFFFAOYSA-N 2-nitro-1H-pyrrole Chemical compound [O-][N+](=O)C1=CC=CN1 FTBBGQKRYUTLMP-UHFFFAOYSA-N 0.000 description 1
- 125000006024 2-pentenyl group Chemical group 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K 2qpq Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- BVZVICBYYOYVEP-MAZCIEHSSA-N 3-[bis[(9Z,12Z)-octadeca-9,12-dienyl]amino]propane-1,2-diol Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCN(CC(O)CO)CCCCCCCC\C=C/C\C=C/CCCCC BVZVICBYYOYVEP-MAZCIEHSSA-N 0.000 description 1
- KUQZVISZELWDNZ-UHFFFAOYSA-N 3-aminopropyl dihydrogen phosphate Chemical compound NCCCOP(O)(O)=O KUQZVISZELWDNZ-UHFFFAOYSA-N 0.000 description 1
- 125000004080 3-carboxypropanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C(O[H])=O 0.000 description 1
- HYCSHFLKPSMPGO-UHFFFAOYSA-L 3-hydroxypropyl phosphate Chemical compound OCCCOP([O-])([O-])=O HYCSHFLKPSMPGO-UHFFFAOYSA-L 0.000 description 1
- LOJNBPNACKZWAI-UHFFFAOYSA-N 3-nitro-1H-pyrrole Chemical compound [O-][N+](=O)C=1C=CNC=1 LOJNBPNACKZWAI-UHFFFAOYSA-N 0.000 description 1
- GICMHQJTKXNMGB-UHFFFAOYSA-N 4-(5-methoxy-1-benzofuran-2-yl)aniline Chemical compound C=1C2=CC(OC)=CC=C2OC=1C1=CC=C(N)C=C1 GICMHQJTKXNMGB-UHFFFAOYSA-N 0.000 description 1
- JVVRCYWZTJLJSG-UHFFFAOYSA-N 4-Dimethylaminophenol Substances CN(C)C1=CC=C(O)C=C1 JVVRCYWZTJLJSG-UHFFFAOYSA-N 0.000 description 1
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-dimethylaminopyridine Substances CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 1
- LZINOQJQXIEBNN-UHFFFAOYSA-N 4-hydroxybutyl dihydrogen phosphate Chemical compound OCCCCOP(O)(O)=O LZINOQJQXIEBNN-UHFFFAOYSA-N 0.000 description 1
- LAVZKLJDKGRZJG-UHFFFAOYSA-N 4-nitro-1H-indole Chemical compound [O-][N+](=O)C1=CC=CC2=C1C=CN2 LAVZKLJDKGRZJG-UHFFFAOYSA-N 0.000 description 1
- CWSZBVAUYPTXTG-UHFFFAOYSA-N 5-[6-[[3,4-dihydroxy-6-(hydroxymethyl)-5-methoxyoxan-2-yl]oxymethyl]-3,4-dihydroxy-5-[4-hydroxy-3-(2-hydroxyethoxy)-6-(hydroxymethyl)-5-methoxyoxan-2-yl]oxyoxan-2-yl]oxy-6-(hydroxymethyl)-2-methyloxane-3,4-diol Chemical compound O1C(CO)C(OC)C(O)C(O)C1OCC1C(OC2C(C(O)C(OC)C(CO)O2)OCCO)C(O)C(O)C(OC2C(OC(C)C(O)C2O)CO)O1 CWSZBVAUYPTXTG-UHFFFAOYSA-N 0.000 description 1
- XYVLZAYJHCECPN-UHFFFAOYSA-L 6-aminohexyl phosphate Chemical compound NCCCCCCOP([O-])([O-])=O XYVLZAYJHCECPN-UHFFFAOYSA-L 0.000 description 1
- PSWCIARYGITEOY-UHFFFAOYSA-N 6-nitro-1H-indole Chemical compound [O-][N+](=O)C1=CC=C2C=CNC2=C1 PSWCIARYGITEOY-UHFFFAOYSA-N 0.000 description 1
- GYDJEQRTZSCIOI-UHFFFAOYSA-N AMCHA Chemical compound NCC1CCC(C(O)=O)CC1 GYDJEQRTZSCIOI-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Natural products NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229960000643 Adenine Drugs 0.000 description 1
- 206010001584 Alcohol abuse Diseases 0.000 description 1
- 206010053164 Alcohol withdrawal syndrome Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 229940059260 Amidate Drugs 0.000 description 1
- SWLVFNYSXGMGBS-UHFFFAOYSA-N Ammonium bromide Chemical compound [NH4+].[Br-] SWLVFNYSXGMGBS-UHFFFAOYSA-N 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 102000008682 Argonaute Proteins Human genes 0.000 description 1
- 108010088141 Argonaute Proteins Proteins 0.000 description 1
- DJHGAFSJWGLOIV-UHFFFAOYSA-K Arsenate Chemical compound [O-][As]([O-])([O-])=O DJHGAFSJWGLOIV-UHFFFAOYSA-K 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 229940072107 Ascorbate Drugs 0.000 description 1
- 229960001230 Asparagine Drugs 0.000 description 1
- 241000282706 Ateles Species 0.000 description 1
- 241000726103 Atta Species 0.000 description 1
- 206010063659 Aversion Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 230000037177 Biodistribution Effects 0.000 description 1
- 210000001772 Blood Platelets Anatomy 0.000 description 1
- 210000004204 Blood Vessels Anatomy 0.000 description 1
- 210000000988 Bone and Bones Anatomy 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 210000004556 Brain Anatomy 0.000 description 1
- 210000000621 Bronchi Anatomy 0.000 description 1
- 241000195940 Bryophyta Species 0.000 description 1
- 210000001217 Buttocks Anatomy 0.000 description 1
- 125000006374 C2-C10 alkenyl group Chemical group 0.000 description 1
- TTWXVHUYMARJHI-KWXKLSQISA-N CCCCC\C=C/C\C=C/CCCCCCCCC(CN(C)C)C(OC(N)=O)CCCCCCCC\C=C/C\C=C/CCCCC Chemical compound CCCCC\C=C/C\C=C/CCCCCCCCC(CN(C)C)C(OC(N)=O)CCCCCCCC\C=C/C\C=C/CCCCC TTWXVHUYMARJHI-KWXKLSQISA-N 0.000 description 1
- QCMYYKRYFNMIEC-UHFFFAOYSA-M COP([O-])=O Chemical class COP([O-])=O QCMYYKRYFNMIEC-UHFFFAOYSA-M 0.000 description 1
- 241000204432 Candidatus Sodalis pierantonius str. SOPE Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 210000001736 Capillaries Anatomy 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 210000003855 Cell Nucleus Anatomy 0.000 description 1
- 229940106189 Ceramides Drugs 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 210000004507 Chromosomes, Artificial Anatomy 0.000 description 1
- 206010009192 Circulatory collapse Diseases 0.000 description 1
- 206010010305 Confusional state Diseases 0.000 description 1
- 210000002808 Connective Tissue Anatomy 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N DEOXYTHYMIDINE Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- XULFJDKZVHTRLG-JDVCJPALSA-N DOSPA trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCNC(=O)C(CCCNCCCN)NCCCN)OCCCCCCCC\C=C/CCCCCCCC XULFJDKZVHTRLG-JDVCJPALSA-N 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N Dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 229920002491 Diethylaminoethyl-dextran Polymers 0.000 description 1
- REZZEXDLIUJMMS-UHFFFAOYSA-M Dimethyldioctadecylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC REZZEXDLIUJMMS-UHFFFAOYSA-M 0.000 description 1
- 241000668709 Dipterocarpus costatus Species 0.000 description 1
- 206010013754 Drug withdrawal syndrome Diseases 0.000 description 1
- 102000007698 EC 1.1.1.1 Human genes 0.000 description 1
- 108010021809 EC 1.1.1.1 Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 210000001163 Endosomes Anatomy 0.000 description 1
- 210000002889 Endothelial Cells Anatomy 0.000 description 1
- XEHVFKKSDRMODV-UHFFFAOYSA-N Ethynyl radical Chemical group C#[C] XEHVFKKSDRMODV-UHFFFAOYSA-N 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 229960000304 Folic Acid Drugs 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 102100006425 GAPDH Human genes 0.000 description 1
- 101710008404 GAPDH Proteins 0.000 description 1
- MSWZFWKMSRAUBD-GASJEMHNSA-N Galactosamine Chemical compound N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- MNQZXJOMYWMBOU-UHFFFAOYSA-N Glyceraldehyde Chemical compound OCC(O)C=O MNQZXJOMYWMBOU-UHFFFAOYSA-N 0.000 description 1
- NYHBQMYGNKIUIF-PXMDKTAGSA-N Guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1[C@H]1O[C@@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-PXMDKTAGSA-N 0.000 description 1
- 229940029575 Guanosine Drugs 0.000 description 1
- 210000004837 Gut-associated lymphoid tissue Anatomy 0.000 description 1
- 229940031574 HYDROXYMETHYL CELLULOSE Drugs 0.000 description 1
- 230000036499 Half live Effects 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 206010073071 Hepatocellular carcinoma Diseases 0.000 description 1
- 229950007870 Hexadimethrine Bromide Drugs 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 210000004408 Hybridomas Anatomy 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 229940072221 IMMUNOGLOBULINS Drugs 0.000 description 1
- 210000000987 Immune System Anatomy 0.000 description 1
- 102000018358 Immunoglobulins Human genes 0.000 description 1
- 108060003951 Immunoglobulins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010022114 Injury Diseases 0.000 description 1
- UGQMRVRMYYASKQ-KMPDEGCQSA-N Inosine Natural products O[C@H]1[C@H](O)[C@@H](CO)O[C@@H]1N1C(N=CNC2=O)=C2N=C1 UGQMRVRMYYASKQ-KMPDEGCQSA-N 0.000 description 1
- 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 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 210000002977 Intracellular Fluid Anatomy 0.000 description 1
- 241001527806 Iti Species 0.000 description 1
- 210000003734 Kidney Anatomy 0.000 description 1
- TYQCGQRIZGCHNB-JLAZNSOCSA-N L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(O)=C(O)C1=O TYQCGQRIZGCHNB-JLAZNSOCSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 229940001447 Lactate Drugs 0.000 description 1
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 description 1
- 229920000126 Latex Polymers 0.000 description 1
- 229940067606 Lecithin Drugs 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 210000000265 Leukocytes Anatomy 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 108060001084 Luciferase family Proteins 0.000 description 1
- BVJSKAUUFXBDOB-RTWAWAEBSA-M Lysophosphatidylglycerol Chemical class CCCCCCCCCCCCCCCC(=O)OC[C@H](O)COP([O-])(=O)OC[C@H](O)CO BVJSKAUUFXBDOB-RTWAWAEBSA-M 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FEWJPZIEWOKRBE-XIXRPRMCSA-N Mesotartaric acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-XIXRPRMCSA-N 0.000 description 1
- 230000036740 Metabolism Effects 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N Methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 210000000274 Microglia Anatomy 0.000 description 1
- 210000000865 Mononuclear Phagocyte System Anatomy 0.000 description 1
- 229940051866 Mouthwash Drugs 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- MAFHEURJBRFHIT-YEUCEMRASA-N N,N-dimethyl-1,2-bis[(Z)-octadec-9-enoxy]propan-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCOC(C)C(N(C)C)OCCCCCCCC\C=C/CCCCCCCC MAFHEURJBRFHIT-YEUCEMRASA-N 0.000 description 1
- HNPWJTONLANQJY-UHFFFAOYSA-N N,N-dimethyl-1,2-dioctadecoxypropan-1-amine Chemical compound CCCCCCCCCCCCCCCCCCOC(C)C(N(C)C)OCCCCCCCCCCCCCCCCCC HNPWJTONLANQJY-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-ACETYL-D-GALACTOSAMINE Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- VODZWWMEJITOND-OWWNRXNESA-N N-Stearoylsphingosine Chemical compound CCCCCCCCCCCCCCCCCC(=O)NC(CO)C(O)\C=C\CCCCCCCCCCCCC VODZWWMEJITOND-OWWNRXNESA-N 0.000 description 1
- 229940052665 NADH Drugs 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 210000004940 Nucleus Anatomy 0.000 description 1
- 229940049964 Oleate Drugs 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 210000002741 Palatine Tonsil Anatomy 0.000 description 1
- 210000000496 Pancreas Anatomy 0.000 description 1
- LLKYUHGUYSLMPA-UHFFFAOYSA-N Phosphoramidite Chemical compound NP([O-])[O-] LLKYUHGUYSLMPA-UHFFFAOYSA-N 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- RKCAIXNGYQCCAL-UHFFFAOYSA-N Porphin Chemical compound N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 RKCAIXNGYQCCAL-UHFFFAOYSA-N 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 108020004412 RNA 3' Polyadenylation Signals Proteins 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N Rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 102000003661 Ribonuclease III Human genes 0.000 description 1
- 108010057163 Ribonuclease III Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000581682 Sanguisorba Species 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 229920000978 Start codon Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N Sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-L Sulphite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 1
- 206010042602 Supraventricular extrasystoles Diseases 0.000 description 1
- 206010042772 Syncope Diseases 0.000 description 1
- MPLHNVLQVRSVEE-UHFFFAOYSA-N Texas Red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 description 1
- 229920000401 Three prime untranslated region Polymers 0.000 description 1
- 210000001541 Thymus Gland Anatomy 0.000 description 1
- UIERETOOQGIECD-ONEGZZNKSA-N Tiglic acid Chemical compound C\C=C(/C)C(O)=O UIERETOOQGIECD-ONEGZZNKSA-N 0.000 description 1
- AOBORMOPSGHCAX-DGHZZKTQSA-N Tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 231100000765 Toxin Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H Tricalcium phosphate Chemical class [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 108020004417 Untranslated RNA Proteins 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- VCGDSYLTBQKCQN-CHWSQXEVSA-N [(2R)-3-[[3-[[(2R)-2,3-diformyloxypropoxy]-hydroxyphosphoryl]oxy-2-hydroxypropoxy]-hydroxyphosphoryl]oxy-2-formyloxypropyl] formate Chemical compound O=COC[C@@H](OC=O)COP(O)(=O)OCC(O)COP(O)(=O)OC[C@@H](COC=O)OC=O VCGDSYLTBQKCQN-CHWSQXEVSA-N 0.000 description 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N [(2R)-3-acetyloxy-2-hydroxypropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- LJGMGXXCKVFFIS-IATSNXCDSA-N [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl] decanoate Chemical compound C([C@@H]12)C[C@]3(C)[C@@H]([C@H](C)CCCC(C)C)CC[C@H]3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)CCCCCCCCC)C1 LJGMGXXCKVFFIS-IATSNXCDSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(E)-octadec-9-enoyl]oxypropyl] (E)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- RQKBYNZCLOWPDE-UHFFFAOYSA-N [5-(2-amino-6-oxo-3H-purin-9-yl)-4-hydroxy-2-(phosphonooxymethyl)oxolan-3-yl] [3-[[3-[[5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)-4-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(2,4-d Chemical compound O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OC2C(C(OC2COP(O)(=O)OC2C(C(OC2COP(O)(=O)OC2C(C(OC2COP(O)(O)=O)N2C3=C(C(N=C(N)N3)=O)N=C2)O)N2C(NC(=O)C=C2)=O)O)N2C3=NC=NC(N)=C3N=C2)O)O1.O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(=O)OC2C(C(OC2COP(O)(=O)OC2C(C(OC2COP(O)(=O)OC2C(C(OC2COP(O)(O)=O)N2C3=C(C(N=C(N)N3)=O)N=C2)O)N2C(NC(=O)C=C2)=O)O)N2C3=NC=NC(N)=C3N=C2)O)O1 RQKBYNZCLOWPDE-UHFFFAOYSA-N 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- VUBTYKDZOQNADH-UHFFFAOYSA-N acetyl hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OC(C)=O VUBTYKDZOQNADH-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-M acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 1
- 231100000494 adverse effect Toxicity 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 201000003082 alcohol use disease Diseases 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 125000003282 alkyl amino group Chemical group 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 150000001350 alkyl halides Chemical class 0.000 description 1
- 125000005103 alkyl silyl group Chemical group 0.000 description 1
- 150000008052 alkyl sulfonates Chemical class 0.000 description 1
- 125000005466 alkylenyl group Chemical group 0.000 description 1
- 230000000172 allergic Effects 0.000 description 1
- 229940087168 alpha Tocopherol Drugs 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N alpha-Tocopherol Natural products OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
- 125000002714 alpha-linolenoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 125000000909 amidinium group Chemical group 0.000 description 1
- 125000000747 amidyl group Chemical group [H][N-]* 0.000 description 1
- 229940077484 ammonium bromide Drugs 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000000844 anti-bacterial Effects 0.000 description 1
- 230000000111 anti-oxidant Effects 0.000 description 1
- 230000000890 antigenic Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000008365 aqueous carrier Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 125000004097 arachidonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940000489 arsenate Drugs 0.000 description 1
- OWTFKEBRIAXSMO-UHFFFAOYSA-N arsenite(3-) Chemical compound [O-][As]([O-])[O-] OWTFKEBRIAXSMO-UHFFFAOYSA-N 0.000 description 1
- 125000005228 aryl sulfonate group Chemical group 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 201000001320 atherosclerosis Diseases 0.000 description 1
- 125000004429 atoms Chemical group 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-M benzoate Chemical compound [O-]C(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-M 0.000 description 1
- 125000003310 benzodiazepinyl group Chemical class N1N=C(C=CC2=C1C=CC=C2)* 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 210000003008 brain-resident macrophage Anatomy 0.000 description 1
- 125000001246 bromo group Chemical group Br* 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 125000002837 carbocyclic group Chemical group 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 230000005591 charge neutralization Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- QBWCMBCROVPCKQ-UHFFFAOYSA-M chlorite Chemical compound [O-]Cl=O QBWCMBCROVPCKQ-UHFFFAOYSA-M 0.000 description 1
- 229910001919 chlorite Inorganic materials 0.000 description 1
- 229910052619 chlorite group Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- GGCLNOIGPMGLDB-GYKMGIIDSA-N cholest-5-en-3-one Chemical compound C1C=C2CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 GGCLNOIGPMGLDB-GYKMGIIDSA-N 0.000 description 1
- ZCDOYSPFYFSLEW-UHFFFAOYSA-N chromate(2-) Chemical compound [O-][Cr]([O-])(=O)=O ZCDOYSPFYFSLEW-UHFFFAOYSA-N 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- 230000002759 chromosomal Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000536 complexating Effects 0.000 description 1
- 201000006233 congestive heart failure Diseases 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 210000004748 cultured cells Anatomy 0.000 description 1
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 description 1
- XFXPMWWXUTWYJX-UHFFFAOYSA-N cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000000596 cyclohexenyl group Chemical group C1(=CCCCC1)* 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 125000002433 cyclopentenyl group Chemical group C1(=CCCC1)* 0.000 description 1
- 125000001511 cyclopentyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- 125000001559 cyclopropyl group Chemical group [H]C1([H])C([H])([H])C1([H])* 0.000 description 1
- 230000002354 daily Effects 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 230000000368 destabilizing Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229920005994 diacetyl cellulose Polymers 0.000 description 1
- 125000004663 dialkyl amino group Chemical group 0.000 description 1
- 150000001985 dialkylglycerols Chemical class 0.000 description 1
- RNPXCFINMKSQPQ-UHFFFAOYSA-N dicetyl hydrogen phosphate Chemical compound CCCCCCCCCCCCCCCCOP(O)(=O)OCCCCCCCCCCCCCCCC RNPXCFINMKSQPQ-UHFFFAOYSA-N 0.000 description 1
- 229940093541 dicetylphosphate Drugs 0.000 description 1
- 229940042935 dichlorodifluoromethane Drugs 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 1
- RGLFGXMUJWSIQF-WRBBJXAJSA-N dimethyl-bis[(Z)-octadec-9-enyl]azanium Chemical compound CCCCCCCC\C=C/CCCCCCCC[N+](C)(C)CCCCCCCC\C=C/CCCCCCCC RGLFGXMUJWSIQF-WRBBJXAJSA-N 0.000 description 1
- DSNRWDQKZIEDDB-CLFAGFIQSA-N dioleoyl phosphatidylglycerol Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-CLFAGFIQSA-N 0.000 description 1
- 239000002612 dispersion media Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- JZKFHQMONDVVNF-UHFFFAOYSA-N dodecyl sulfate;tris(2-hydroxyethyl)azanium Chemical compound OCCN(CCO)CCO.CCCCCCCCCCCCOS(O)(=O)=O JZKFHQMONDVVNF-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 235000021271 drinking Nutrition 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- XNWFRZJHXBZDAG-UHFFFAOYSA-N ethylene glycol monomethyl ether Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- 125000005469 ethylenyl group Chemical group 0.000 description 1
- NPUKDXXFDDZOKR-LLVKDONJSA-N etomidate Chemical compound CCOC(=O)C1=CN=CN1[C@H](C)C1=CC=CC=C1 NPUKDXXFDDZOKR-LLVKDONJSA-N 0.000 description 1
- 229960001690 etomidate Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 150000002190 fatty acyls Chemical group 0.000 description 1
- 230000004634 feeding behavior Effects 0.000 description 1
- SRMBQCVUAVULDJ-UHFFFAOYSA-N ferrioxamine B Chemical compound [Fe+3].CC(=O)N([O-])CCCCCNC(=O)CCC(=O)N([O-])CCCCCNC(=O)CCC(=O)N([O-])CCCCCN SRMBQCVUAVULDJ-UHFFFAOYSA-N 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 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 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 150000002224 folic acids Chemical class 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N fumaric acid Chemical compound OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000003976 glyceryl group Chemical group [H]C([*])([H])C(O[H])([H])C(O[H])([H])[H] 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-M glycolate Chemical compound OCC([O-])=O AEMRFAOFKBGASW-UHFFFAOYSA-M 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical group NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- FJLUATLTXUNBOT-UHFFFAOYSA-N hexadecan-1-amine Chemical compound CCCCCCCCCCCCCCCCN FJLUATLTXUNBOT-UHFFFAOYSA-N 0.000 description 1
- NFPRBYPETYFMKO-UHFFFAOYSA-N hexan-1-amine Chemical group [CH2]CCCCCN NFPRBYPETYFMKO-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 150000007857 hydrazones Chemical class 0.000 description 1
- KLGZELKXQMTEMM-UHFFFAOYSA-N hydride Chemical compound [H-] KLGZELKXQMTEMM-UHFFFAOYSA-N 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxyl anion Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 150000002466 imines Chemical class 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003834 intracellular Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-M iodide Chemical compound [I-] XMBWDFGMSWQBCA-UHFFFAOYSA-M 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000005468 isobutylenyl group Chemical group 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M isothiocyanate Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- LVHBHZANLOWSRM-UHFFFAOYSA-L itaconate(2-) Chemical compound [O-]C(=O)CC(=C)C([O-])=O LVHBHZANLOWSRM-UHFFFAOYSA-L 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 125000005644 linolenyl group Chemical group 0.000 description 1
- 125000002669 linoleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000002479 lipoplex Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 235000019359 magnesium stearate 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
- VZCYOOQTPOCHFL-UPHRSURJSA-L maleate(2-) Chemical compound [O-]C(=O)\C=C/C([O-])=O VZCYOOQTPOCHFL-UPHRSURJSA-L 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-M mandelate Chemical compound [O-]C(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-M 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 media Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000035786 metabolism Effects 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000009126 molecular therapy Methods 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 108091021642 mouse interleukin-6 Proteins 0.000 description 1
- 235000011929 mousse Nutrition 0.000 description 1
- 239000002324 mouth wash Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 239000005445 natural product Substances 0.000 description 1
- 230000002887 neurotoxic Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- TWXTWZIUMCFMSG-UHFFFAOYSA-N nitride(3-) Chemical compound [N-3] TWXTWZIUMCFMSG-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229920001894 non-coding RNA Polymers 0.000 description 1
- 238000007826 nucleic acid assay Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-M oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC([O-])=O ZQPPMHVWECSIRJ-KTKRTIGZSA-M 0.000 description 1
- 125000002811 oleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 230000003287 optical Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 239000000668 oral spray Substances 0.000 description 1
- 150000002891 organic anions Chemical class 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 125000003566 oxetanyl group Chemical group 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 125000000466 oxiranyl group Chemical group 0.000 description 1
- 125000004043 oxo group Chemical group O=* 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 230000000149 penetrating Effects 0.000 description 1
- 230000002093 peripheral Effects 0.000 description 1
- NPDODHDPVPPRDJ-UHFFFAOYSA-N permanganate Chemical compound [O-][Mn](=O)(=O)=O NPDODHDPVPPRDJ-UHFFFAOYSA-N 0.000 description 1
- 230000003617 peroxidasic Effects 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000275 pharmacokinetic Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L phosphate Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- NJRWNWYFPOFDFN-UHFFFAOYSA-L phosphonate(2-) Chemical compound [O-][P]([O-])=O NJRWNWYFPOFDFN-UHFFFAOYSA-L 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000068 poly(2-ethyl-2-oxazoline) Polymers 0.000 description 1
- 229920000083 poly(allylamine) Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 108010055896 polyornithine Proteins 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920001289 polyvinyl ether Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000001144 postural Effects 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000000750 progressive Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylaxis Effects 0.000 description 1
- 125000005470 propylenyl group Chemical group 0.000 description 1
- 125000002568 propynyl group Chemical group [*]C#CC([H])([H])[H] 0.000 description 1
- 230000001681 protective Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000000717 retained Effects 0.000 description 1
- 229920002973 ribosomal RNA Polymers 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229920002033 ribozyme Polymers 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- 229960001860 salicylate Drugs 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000005549 size reduction Methods 0.000 description 1
- KISFEBPWFCGRGN-UHFFFAOYSA-M sodium;2-(2,4-dichlorophenoxy)ethyl sulfate Chemical compound [Na+].[O-]S(=O)(=O)OCCOC1=CC=C(Cl)C=C1Cl KISFEBPWFCGRGN-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 235000015096 spirit Nutrition 0.000 description 1
- 230000000087 stabilizing Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 125000003696 stearoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N stearylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 201000009032 substance abuse Diseases 0.000 description 1
- 125000005017 substituted alkenyl group Chemical group 0.000 description 1
- 125000004426 substituted alkynyl group Chemical group 0.000 description 1
- 125000003107 substituted aryl group Chemical group 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
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 229960001663 sulfanilamide Drugs 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000004853 tetrahydropyridinyl group Chemical group N1(CCCC=C1)* 0.000 description 1
- 125000004632 tetrahydrothiopyranyl group Chemical group S1C(CCCC1)* 0.000 description 1
- XVTUQDWPJJBEHJ-KZCWQMDCSA-N tetrastearoyl cardiolipin Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCCCC)CO[P@@](O)(=O)OCC(O)CO[P@](O)(=O)OC[C@H](OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC XVTUQDWPJJBEHJ-KZCWQMDCSA-N 0.000 description 1
- 235000019529 tetraterpenoid Nutrition 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 239000005451 thionucleotide Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical class [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 230000000699 topical Effects 0.000 description 1
- 239000006208 topical dosage form Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 108020003112 toxins Proteins 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000004450 types of analysis Methods 0.000 description 1
- 238000009424 underpinning Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 229920003169 water-soluble polymer Polymers 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-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
Classifications
-
- 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
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
- A61P25/32—Alcohol-abuse
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
- C12N2310/3521—Methyl
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
Abstract
Disclosed is a composition comprising an interfering RNA that silences aldehyde dehydrogenase (ALDH) gene expression, wherein the interfering RNA comprises a sense strand and a complementary antisense strand, and wherein the interfering RNA comprises a double-stranded region of about 15 to about 60 nucleotides in length, wherein the interfering RNA is selected from the group consisting of siRNA Identifiers of the sequences as defined in the specification. Also disclosed is their use in treatment. nucleotides in length, wherein the interfering RNA is selected from the group consisting of siRNA Identifiers of the sequences as defined in the specification. Also disclosed is their use in treatment.
Description
ITIONS AND METHODS FOR SILENCING ALDEHYDE
DEHYDROGENASE
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit under 35 U.S.C.§ll9(e) of US. provisional patent
ation no. 61/599,238 filed on February 15, 2012 and US. provisional patent application no.
61/543,700 filed on October 5, 201 l, the contents of which are incorporated herein by reference in
their entireties. NZ 712990 is a divisional out of the present application. The description of the
t invention and the invention ofNZ 712990 is retained herein for clarity and completeness.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in
lieu of a paper copy, and is hereby orated by reference into the specification. The name of the
text file containing the Sequence Listing is TEKM_074_02WO_ST25.txt. The text file is 12 KB, was
created on October 4, 2012, and is being submitted electronically via EFS-Web.
BACKGROUND OF THE INVENTION
Alcoholism is the addiction to or dependency upon drinking excessive amounts of
alcoholic beverages such as beer, wine and distilled spirits. Alcoholism is sometimes also referred to
as alcohol abuse or alcohol dependence.
The biological mechanisms underpinning alcoholism are ain, however risk
factors include stress, mental health problems, and genetic position. Long-term l abuse
produces physiological changes in the brain that result in alcohol withdrawal syndrome upon
discontinuation of alcohol consumption. Alcohol damages almost every organ in the body, and the
alcoholic risks ing l and atric disorders.
ent of alcoholism is problematic and lly includes alcohol detoxification
to withdraw the alcoholic person from drinking alcohol. Neurologically active drugs, such as
benzodiazepines, may be used to manage alcohol withdrawal symptoms. Post-medical care, such as
psychological therapy, is usually required to maintain alcoholic abstention.
Disulfiram is a drug that causes an acute sensitivity to ingested alcohol (ethanol), and
is sometimes used in the treatment of chronic alcoholism. Alcohol is broken down in the liver by the
enzyme alcohol dehydrogenase to acetaldehyde, which is then converted by the enzyme dehyde
dehydrogenase to acetic acid. Disulfiram blocks the enzyme acetaldehyde ogenase. Thus,
disulfiram can cause the concentration of acetaldehyde in the blood of a human being who has
consumed alcohol to be substantially higher (e. g., 5 to 10 times higher) than that found in the blood of
a person who consumed the same amount of alcohol in the absence of disulfiram. Acetaldehyde is one
of the major causes of the symptoms of a "hangover", and so consumption of disulfiram produces
severe negative reaction to alcohol. Symptoms include flushing of the skin, accelerated heart rate,
ess of breath, nausea, vomiting, ing headache, Visual disturbance, mental confusion,
postural fainting, and circulatory collapse.
Disulfiram has clinical limitations, however, due to poor compliance by patients, and
a range of side-effects, such as drowsiness, headache and, less often, neurotoxicity. Disulfiram is
usually administered daily in order to be effective, and so it is easy for a t to discontinue use of
the drug.
Thus, there is a continuing need for compositions and methods for suppressing,
reducing and/or ating the ty of the acetaldehyde ogenase that is involved in
metabolising alcohol in a mammal, particularly a human being. Such compositions and methods can
be used, for example, in the ent of alcoholism. In particular, there is a need for compositions
and methods that suppress, reduce and/or eliminate the activity of acetaldehyde dehydrogenase for a
period of time (e.g., weeks or months) that is significantly longer than the effective period of
disulfiram, thereby making it difficult for a human subject to discontinue alcohol aversion therapy
after consuming such a relatively long lasting inhibitor of ALDH. There is also a particular need for
compositions and methods that suppress, reduce and/or eliminate the activity of acetaldehyde
dehydrogenase and that have fewer, or less severe, side effects than disulfuram.
BRIEF SUMMARY OF THE INVENTION
In one aspect of the present invention, there is provided a composition comprising an
interfering RNA that silences aldehyde dehydrogenase (ALDH) gene expression, wherein the
interfering RNA comprises a sense strand and a mentary antisense strand, and wherein the
interfering RNA comprises a double-stranded region of about 15 to about 60 tides in ,
wherein the interfering RNA is selected from the group consisting of siRNA Identifier 1 (SEQ ID
NOs:11 & 12), siRNA Identifier 2 (SEQ ID NOs:l3 & 12), siRNA Identifier 3 (SEQ ID NOs:l4 &
), siRNA Identifier 4 (SEQ ID NOszl6 & 15), siRNA Identifier S (SEQ ID NOs:l7 & l8) and
siRNA fier 6 (SEQ ID NOszl9 & 18).
[followed by page 2a]
[0009a] As described more fully herein, acetaldehyde dehydrogenase is a member of
the broader class of de dehydrogenase (ALDH) enzymes. A member of the ALDH family
that is believed to be primarily sible for converting acetaldehyde to acetic acid in the
human liver is the aldehyde dehydrogenase 2 (ALDH2) enzyme, although other isoforms of
ALDH, such as ALDHl, are also implicated in the metabolism of l in humans. It is an object
of the present invention to provide compositions and methods for inhibiting the expression of one
or more genes encoding one or more ALDH enzymes, in ular the ALDHZ enzyme, or to
at least provide the public with a useful choice. Inhibition is through the mechanism of RNA
interference. The compositions and methods of the present invention are thus useful, for
example, for treating alcoholism in a human being by inhibiting, or blocking, the conversion of
acetaldehyde to acetic acid, thereby sing the amount of acetaldehyde in the body of a human
[followed by page 3]
W0 2013i052677 PCT/U82012/058770
who consumes alcohol, and consequently intensifying the adverse effects associated with the presence
of acetaldehyde in the body (e.g., headache and nausea).
Thus, the present invention provides compositions comprising therapeutic nucleic acids
such as interfering RNA (e.g., dsRNA such as siRNA) that target aldehyde dehydrogenase (ALDH)
gene expression, lipid les comprising one or more (e.g., a cocktail) of the therapeutic nucleic
acids, methods of making the lipid particles, and methods of ring and/or administering the lipid
particles (e.g., for treating alcoholism).
More particularly, the invention provides compositions comprising unmodified and
chemically modified interfering RNA (e. g., siRNA) molecules which inhibit or silence ALDH gene
expression. The present ion also provides serum-stable nucleic acid—lipid particles (e.g.,
SNALP) and formulations thereof comprising one or more (e.g., a cocktail) of the interfering RNA
(e.g., siRNA) described herein, a cationic lipid, and a non-cationic lipid, which can r comprise a
conjugated lipid that inhibits aggregation of particles. Examples of interfering RNA molecules
include, but are not limited to, double-stranded RNA (dsRNA) such as siRNA, Dicer—substrate
dsRNA, shRNA, aiRNA, RNA, and ations f.
In one aspect, the present invention provides an ering RNA that targets ALDH gene
expression, wherein the interfering RNA comprises a sense strand and a complementary antisense
strand, and wherein the interfering RNA comprises a double—stranded region of about 15 to about 60
nucleotides in length. In n ments, the present invention provides compositions
comprising a combination (e.g., a cocktail) of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
interfering RNA molecules that target the same and/or different regions of the ALDH genome. The
interfering RNA of the invention are capable of inhibiting or silencing ALDH gene expression in. vitro
and in viva.
Non—limiting examples of ALDH2 transcript sequences that can be used, for example, in
the design of siRNA molecules that inhibit ALDH2 gene expression are set forth in Genbank
(www.ncbi.nlm.nih.gov/genbank) as Accession Nos. NM_000690.3 (Gene ID 217, isoform 1) and
NM 0012048891 (Gene ID 217, isoform 2).
Non-limiting es ofALDHI transcript sequences that can be used, for example, in
the design of siRNA molecules that inhibit ALDH] gene expression are set forth in k
cbi.nlm.nih.gov/genbank) as Accession Nos. NM_000689.4 (ALDHlAl, Gene ID 216), and
NM_000692.4 (ALDHlBl, Gene ID 219).
In another aspect, the present invention provides an interfering RNA that targets
aldehyde ogenase (ALDH) gene expression, wherein the interfering RNA comprises a sense
PCT/U82012/058770
strand and a complementary antisense strand, and wherein the interfering RNA comprises a double-
stranded region of about 15 to about 60 nucleotides in . In certain embodiments, the present
invention provides compositions sing a combination (e.g., a cocktail) of at least about 2, 3, 4,
, 6, 7, 8, 9, 10, or more ering RNA molecules that target the same and/or ent s of
the ALDH gene. The interfering RNA of the invention are capable of inhibiting or completely
silencing ALDH gene expression in vitro and in viva.
Each of the interfering RNA sequences present in the compositions of the invention may
independently comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more
modified nucleotides such as 2’0Me nucleotides, e.g., in the sense and/or antisense strand of the
—stranded region. Preferably, e and/or guanosine nucleotides are modified with 2’OMe
nucleotides. In ular embodiments, each of the interfering RNA sequences present in the
compositions of the invention comprises at least one 2’OMe—uridine nucleotide and at least one
2’OMe—guanosine nucleotide in the sense and/or antisense strands.
Examples of double—stranded siRNA molecules that are useful in the practice of
the present invention for inhibiting ALDH2 gene expression include the double-stranded
siRNA molecules (numbered 1 through 6) having the ally modified sense and
antisense strand sequences set forth in Table A of Example 4 herein. The double-stranded
siRNA molecules set forth in Table A of Example 4 are chemically d by the presence
of a 2’—0—methyl moiety on the ribonucleotide units identified with the letter “m”. The
present ion also includes double-stranded siRNA molecules having the sense and
antisense sequences set forth in Table A of Example 4, wherein the sense and antisense
strands are not chemically modified. The present invention also includes isolated, single-
stranded, nucleic acid molecules having any one of the sense strand sequences (chemically
modified or not chemically modified) set forth in Table A of Example 4. The present
invention also includes ed, -stranded, nucleic acid molecules having any one of
the antisense strand sequences (chemically modified or not chemically modified) set forth in
Table A ofExample 4.
The present invention also provides a pharmaceutical composition comprising one or a
cocktail of interfering RNA (e.g., siRNA) molecules that target ALDH gene expression, and a
pharmaceutically acceptable r.
In another aspect, the present invention es a nucleic acid—lipid particle that targets
ALDH gene expression. The nucleic acid—lipid particle typically comprises one or more unmodified
and/or modified interfering RNA that silence ALDH gene sion, a cationic lipid, and a non—
ic lipid. In certain instances, the nucleic acid-lipid particle further comprises a conjugated lipid
that inhibits aggregation of particles. In preferred embodiments, the nucleic acid-lipid particle
ses one or more unmodified and/or modified interfering RNA that silence ALDH gene
expression, a cationic lipid, a tionic lipid, and a conjugated lipid that inhibits aggregation of
particles.
In other embodiments, the interfering RNA molecules of the invention are fully
encapsulated in the nucleic acid—lipid particle (e.g., SNALP). With respect to formulations
comprising a cocktail of interfering RNA, the different types of interfering RNA molecules may be
apsulated in the same nucleic acid-lipid particle, or each type of interfering RNA species
IO present in the cocktail may be encapsulated in its own particle.
The present invention also provides pharmaceutical compositions comprising a nucleic
acid—lipid particle and a pharrnaceutically acceptable carrier.
The nucleic acid~lipid particles of the invention are useful for the prophylactic or
therapeutic delivery of interfering RNA (e.g., dsRNA) molecules that silence the expression of one or
more ALDH genes (e.g., ALDH2 gene). In some embodiments, one or more of the interfering RNA
molecules described herein are formulated into nucleic acid~lipid les, and the particles are
administered to a mammal (e.g., a human) requiring such treatment. In certain instances, a
therapeutically ive amount of the nucleic acid—lipid particle can be administered to the ,
e. g., for preventing or treating lism in a human being). The nucleic acid—lipid particles of the
2O invention are particularly useful for targeting liver cells in humans which is the site of most ALDH2
gene expression. Administration of the nucleic acid-lipid particle can be by any route known in the
art, such as, e.g., oral, intranasal, intravenous, intraperitoneal, uscular, intra—articular,
intralesional, intratracheal, subcutaneous, or intradermal. In ular embodiments, the nucleic acid—
lipid particle is administered ically, e.g., via enteral or parenteral routes of administration.
[0023] In some embodiments, downregulation of ALDH gene expression is determined by
ing ALDH RNA or protein levels in a biological sample from a mammal after nucleic ipid
particle administration. In other embodiments, downregulation of ALDH gene expression is
determined by detecting ALDH mRNA or protein levels in a biological sample from a mammal after
nucleic acid-lipid particle administration. In n embodiments, downregulation of ALDH or
ALDH gene expression is ed by monitoring symptoms associated with l withdrawal in a
mammal after particle administration.
W0 2013f052677 PCT/U82012/058770
In r ment, the t invention provides methods for introducing an
interfering RNA that es ALDH gene expression into a cell, the method comprising the step of
contacting the cell with a nucleic ipid particle of the present invention.
In another embodiment, the present invention provides methods for silencing ALDH
gene expression in a mammal (e.g., a human) in need thereof, wherein the methods each include the
step of administering to the mammal a nucleic acid—lipid le of the present invention.
In another aspect, the present invention provides methods for treating and/or
ameliorating one or more symptoms associated with alcoholism in a human, wherein the methods
each include the step of administering to the human a therapeutically effective amount of a nucleic
acid—lipid particle of the present invention.
In another aspect, the present invention provides methods for inhibiting the expression of
ALDH in a mammal in need thereof (e.g., a human suffering from alcoholism), wherein the methods
each include the step of administering to the mammal a therapeutically effective amount of a nucleic
acid—lipid particle of the present invention.
[0028] In a r aspect, the present invention es methods for preventing and/or treating
alcoholism in a human, wherein the methods each include the step of administering to the human a
therapeutically effective amount of a nucleic acid-lipid particle ofthe present invention.
Other objects, features, and advantages of the present ion will be apparent to one
of skill in the art from the following detailed description and figures.
DETAILED DESCRIPTION OF THE INVENTION
1. Introduction
The interfering RNA (e.g., siRNA) drug therapy described herein advantageously
provides significant new itions and methods for treating alcoholism in human beings.
Embodiments of the present invention can be administered, for example, once per day, once per week,
or once every several weeks (e.g., once every two, three, four, five or six weeks). It is more difficult
for an alcoholic person to discontinue alcohol on therapy after consuming a composition of the
present invention that is ive to inhibit ALDH for a period of several days or weeks.
Furthermore, the lipid particles bed herein (e.g., SNALP) enable the effective
delivery of a nucleic acid drug such as an interfering RNA into target s and cells within the
body. The ce of the lipid particle confers protection from nuclease degradation in the
PCT/U82012/058770
bloodstream, allows preferential accumulation in target tissue and provides a means of drug entry into
the cellular cytoplasm where the siRNAs can perform their intended function of RNA interference.
II. Definitions
As used herein, the following terms have the meanings ascribed to them unless specified
otherwise.
The term “aldehyde dehydrogenase” (abbreviated as ALDH) means an enzyme that
catalyzes the oxidation (dehydrogenation) of an de (e.g., acetaldehyde) to a carboxylic acid
(e.g., acetic acid). A family of structurally and functionally related aldehyde dehydrogenases exists in
mammals, and includes aldehyde dehydrogenase 2 (ALDH2) that is a mitochondrially localized
enzyme that is ed to be mainly responsible for the conversion of acetaldehyde to acetic acid in
human beings.
The term “interfering RNA” or “RNAi” or “interfering RNA sequence” as used herein
includes single—stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotides, ssDNAi
ucleotides) or -stranded RNA (12a, duplex RNA such as siRNA, substrate dsRNA,
shRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target
gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which
are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as
the target gene or sequence. Interfering RNA thus refers to the single-stranded RNA that is
complementary to a target mRNA sequence or to the double—stranded RNA formed by two
complementary strands or by a single, self—complementary strand. ering RNA may have
substantial or complete ty to the target gene or sequence, or may comprise a region of mismatch
(i.e., a mismatch motif). The sequence of the interfering RNA can correspond to the full—length target
gene, or a subsequence f. Preferably, the ering RNA les are chemically
synthesized.
[0035] Interfering RNA includes “small—interfering RNA” or “siRNA,” e.g., interfering RNA of
about 15-60, 15-50, or 15—40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-
x) nucleotides in length, and is preferably about 20—24, 21-22, or 21-23 (duplex) nucleotides
in length (e.g., each complementary sequence of the double-stranded siRNA is 15—60, 15-50, 15-40,
—30, 15—25, or 19—25 nucleotides in length, preferably about 20-24, 21-22, or 21—23 tides in
, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base
pairs in length, preferably about 18-22, 19—20, or 19-21 base pairs in length). siRNA duplexes may
comprise 3’ overhangs of about 1 to about 4 nucleotides or abOut 2 to about 3 nucleotides and 5’
phosphate termini. Examples of siRNA include, without limitation, a double—stranded polynucleotide
molecule assembled from two te stranded molecules, wherein one strand is the sense strand and
the other is the complementary antisense strand; a double-stranded polynucleotide molecule
assembled from a single stranded molecule, where the sense and antisense regions are linked by a
nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with
a hairpin secondary structure having self—complementary sense and antisense regions; and a circular
single-stranded polynucleotide molecule with two or more loop structures and a stem having self—
complementary sense and antisense regions, where the circular polynucleotide can be processed in
vivo or in vitro to generate an active —stranded siRNA molecule.
Preferably, siRNA are ally synthesized. siRNA can also be generated by
cleavage of longer dsRNA (e.g., dsRNA greater than about 25 tides in length) with the E. coli
RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e. g.,
Yang et al, Proc. Natl. Acad. Sci. USA, 99:9942—9947 (2002); Calegari et al., Proc. Natl. Acad. Sci.
USA, 99:14236 (2002); Byrom el al., Ambion TechNoteS, 10(1):4~6 (2003); Kawasaki er al., Nucleic
Acids Res, 31:981—987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al, J.
Biol. Chem, 243282 (1968)). Preferably, dsRNA are at least 50 nucleotides to about 100, 200, 300,
400, or 500 tides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in
length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene ript. In
certain instances, siRNA may be encoded by a plasmid (e.g., transcribed as sequences that
automatically fold into duplexes with hairpin loops).
[0037] As used , the term “mismatch motif" or “mismatch region” refers to a portion of an
ering RNA (e.g., siRNA) sequence that does not have 100 % complementarity to its target
ce. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch
regions. The mismatch regions may be contiguous or may be ted by l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
ll, 12, or more nucleotides. The mismatch motifs or regions may se a single nucleotide or
may comprise two, three, four, five, or more nucleotides.
The phrase “inhibiting sion of a target gene” refers to the ability of an interfering
RNA (e. g., siRNA) of the invention to silence, reduce, or inhibit expression of a target gene (e.g.,
ALDH gene). To e the extent of gene silencing, a test sample (e.g., a biological sample from
an organism of interest expressing the target gene or a sample of cells in culture sing the target
gene) is contacted with an interfering RNA (e. g., siRNA) that silences, reduces, or inhibits expression
of the target gene. Expression of the target gene in the test sample is compared to expression of the
target gene in a control sample (e.g., a biological sample from an organism of interest expressing the
target gene or a sample of cells in culture expressing the target gene) that is not contacted with the
interfering RNA (e. g., siRNA). Control samples (e.g., s expressing the target gene) may be
assigned a value of 100%. In particular embodiments, silencing, tion, or reduction of expression
PCT/U52012/058770
of a target gene is achieved when the value of the test sample relative to the control sample is about
95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%,
%, or 0%. Suitable assays include, without limitation, examination of protein or mRNA levels using
ques known to those of skill in the art, such as, e.g., dot blots, rn blots, in situ
hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to
those of skill in the art.
An “effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid
such as an interfering RNA is an amount sufficient to produce the desired effect, e.g., an tion of
expression of a target sequence in comparison to the normal expression level detected in the absence
of an interfering RNA. In particular embodiments, inhibition of expression of a target gene or target
sequence is achieved when the value obtained with an interfering RNA relative to the control is about
95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,
%, 5%, or 0%. le assays for measuring the expression of a target gene or target sequence
include, but are not d to, examination of protein or mRNA levels using techniques known to
those of skill in the art, such as, e. g., dot blots, Northern blots, in situ hybridization, ELISA,
immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
By ase,” asing,9! 6‘reduce,”
or “reducing” of an immune response by an
interfering RNA is ed to mean a detectable decrease of an immune response to a given
interfering RNA (e.g., a modified interfering RNA). The amount of decrease of an immune response
by a modified interfering RNA may be determined relative to the level of an immune response in the
presence of an fied interfering RNA. A detectable se can be about 5%, 10%, 15%,
%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or
more lower than the immune response detected in the presence of the unmodified interfering RNA. A
decrease in the immune response to interfering RNA is typically measured by a decrease in cytokine
production (e.g., IFNy, IFNu, TNFa, IL—6, or IL—l2) by a responder cell in vitro or a decrease in
cytokine production in the sera of a mammalian subject after administration ofthe interfering RNA.
As used herein, the term “responder cell” refers to a cell, preferably a mammalian cell,
that produces a detectable immune response when contacted with an immunostimulatory interfering
RNA such as an unmodified siRNA. Exemplary responder cells include, e.g., tic cells,
macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Detectable
immune responses include, e.g., production of cytokines or growth factors such as TNF-a, IFN-a,
lFN—B, IFN-y, IL-1, IL-2, lL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof.
Detectable immune responses also include, e.g., induction of interferon-induced protein with
ricopeptide repeats 1 (IFITI) mRNA.
PCT/U82012/058770
“Substantial identity” refers to a sequence that hybridizes to a reference sequence under
stringent conditions, or to a ce that has a specified percent ty over a specified region of a
reference sequence.
The phrase “stringent hybridization conditions” refers to conditions under which a
nucleic acid will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence—dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular
y—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the
strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-
100C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which
50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
Stringent ions may also be achieved with the addition of destabilizing agents such as
formamide. For selective or specific ization, a ve signal is at least two times background,
preferably 10 times background hybridization.
Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5x
SSC, and 1% SDS, incubating at 420C, or, 5x SSC, 1% SDS, incubating at 650C, with wash in 0.2x
SSC, and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for low stringency
amplification, although annealing temperatures may vary between about 32°C and 480C depending
on primer length. For high stringency PCR amplification, 3 temperature of about 62°C is typical,
although high ency annealing temperatures can range from about 50°C to about 65°C,
depending on the primer length and specificity. l cycle conditions for both high and low
stringency amplifications include a denaturation phase of 50C for 30 sec. to 2 min, an
annealing phase lasting 30 sec. to 2 min, and an ion phase of about 72°C for l to 2 min.
Protocols and guidelines for low and high stringency amplification ons are provided, e. g., in
Innis et al, PCR Protocols, A Guide to s and Applications, ic Press, Inc. NY. (l990).
Nucleic acids that do not hybridize to each other under stringent conditions are still
substantially identical if the polypeptides which they encode are substantially identical. This occurs,
for example, when a copy of a nucleic acid is created using the m codon degeneracy permitted
by the genetic code. In such cases, the nucleic acids typically hybridize under tely stringent
hybridization conditions. Exemplary “moderately stringent hybridization ions” include a
W0 2013I052677
hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 370C, and a wash in 1X SSC at
45°C. A positive hybridization is at least twice ound. Those of ordinary skill will readily
recognize that alternative hybridization and wash conditions can be utilized to provide conditions of
similar stringency. Additional guidelines for determining hybridization parameters are provided in
numerous references, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.
The terms “substantially identical” or “substantial identity,” in the context of two or
more nucleic acids, refer to two or more sequences or subsequences that are the same or have a
specified tage of nucleotides that are the same (i.e., at least about 60%, preferably at least about
65%, 70%, 75%, 80%, 85%, 90%, or 95% ty over a specified region), when compared and
aligned for maximum correspondence over a ison window, or designated region as measured
using one of the following sequence comparison thms or by manual alignment and visual
inspection. This definition, when the context indicates, also refers analogously to the complement of
a sequence. ably, the substantial identity exists over a region that is at least about 5, 10, 15, 20,
, 30, 35, 40, 45, 50, 55, or 60 tides in length.
[0047] For sequence comparison, typically one sequence acts as a reference sequence, to which
test sequences are compared. When using a sequence comparison algorithm, test and reference
sequences are entered into a computer, subsequence nates are designated, if necessary, and
sequence algorithm program parameters are designated. Default m parameters can be used, or
ative ters can be designated. The sequence comparison algorithm then calculates the
percent sequence identities for the test sequences relative to the reference sequence, based on the
program parameters.
A “comparison window,” as used herein, includes reference to a segment of any one of a
number of contiguous positions selected from the group ting of from about 5 to about 60,
usually about 10 to about 45, more usually about 15 to about 30, in which a sequence may be
compared to a reference ce of the same number of contiguous positions after the two sequences
are optimally d. Methods of alignment of sequences for comparison are well known in the art.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology
algorithm of Smith and Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol, 48:443 (1970), by the search for rity
method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8522444 (1988), by computerized
entations of these algorithms (GAP, BESALDHIT, FASTA, and ALDHASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or
by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology,
Ausubel et al, eds. (1995 supplement)).
PCT/U82012/058770
miting examples of algorithms that are suitable for determining percent sequence
identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in
Altschul et (21., Nuc. Acids Res, 25 :3389-3402 (1977) and Altschul et (11., J. Mol. Biol., 215:403—410
(1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to
ine percent sequence identity for the nucleic acids of the invention. Software for performing
BLAST analyses is publicly available through the National Center for Biotechnology Information
(http://Www.ncbi.nlm.nih.gov/). Another example is a global alignment algorithin for determining
percent ce identiy such as the Needleman—Wunsch algorithm for aligning protein or nucleotide
(e.g., RNA) ces.
[0050] The BLAST algorithm also performs a statistical analysis of the rity between two
sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873—5787 (1993)). One
measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which
provides an indication of the probability by which a match n two nucleotide sequences would
occur by chance. For example, a nucleic acid is considered similar to a reference ce if the
st sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less
than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
The term ic acid” as used herein refers to a polymer ning at least two
deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA
and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed
DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes,
ic sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may
be in the form of small interfering RNA (siRNA), Dicer—substrate dsRNA, small hairpin RNA
(shRNA), asymmetrical interfering RNA ), microRNA (miRNA), mRNA, tRNA, rRNA,
tRNA, viral RNA (VRNA), and combinations thereof. Nucleic acids include nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages, which are tic, lly
occurring, and non—naturally occurring, and which have similar binding ties as the reference
nucleic acid. Examples of such analogs include, without limitation, phosphorothioates,
phosphoramidates, methyl phosphonates, chiral-methyl onates, 2’-O-methyl ribonucleotides,
and peptide-nucleic acids (PNAs). Unless cally limited, the term encompasses nucleic acids
containing known analogues of natural nucleotides that have similar binding ties as the
reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g., degenerate codon tutions), alleles,
orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
Specifically, rate codon substitutions may be achieved by generating sequences in which the
third position of one or more selected (or all) codons is substituted with mixed—base and/or
WO 2013052677
deoxyinosine residues (Batzer et al., Nucleic Acid Res, 1925081 (1991); Ohtsuka er al., J. Biol.
Chem, 260:2605-2608 (1985); Rossolini et (1]., M01. Cell. , 8:91-98 (1994)). “Nucleotides”
contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are
linked er h the ate groups. ” include purines and pyrimidines, which
further include natural compounds adenine, thymine, guanine, cytosine, uracil, e, and natural
analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to,
modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols,
carboxylates, and alkylhalides.
The term “gene” refers to a nucleic acid (e. g., DNA or RNA) sequence that comprises
partial length or entire length coding sequences ary for the production of a polypeptide or
precursor polypeptide.
“Gene product,” as used herein, refers to a product of a gene such as an RNA transcript
or a polypeptide.
The term “lipid” refers to a group of organic compounds that include, but are not limited
to, esters of fatty acids and are characterized by being ble in water, but soluble in many organic
solvents. They are usually divided into at least three classes: (1) “simple lipids,” which e fats
and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and
(3) “derived lipids” such as steroids.
The term “lipid particle” includes a lipid formulation that can be used to deliver a
therapeutic nucleic acid (e. g., interfering RNA) to a target site of interest (e. g., cell, tissue, organ, and
the like). In preferred embodiments, the lipid particle of the invention is a nucleic acid—lipid particle,
which is typically formed from a cationic lipid, a non-cationic lipid, and optionally a ated lipid
that prevents aggregation of the particle. In other preferred embodiments, the therapeutic nucleic acid
(e.g., interfering RNA) may be encapsulated in the lipid portion of the particle, thereby protecting it
from enzymatic degradation.
As used herein, the term “SNALP” refers to a stable nucleic acid—lipid le. A
SNALP represents a particle made from lipids (e.g., a cationic lipid, a tionic lipid, and
optionally a conjugated lipid that prevents aggregation of the particle), wherein the nucleic acid (e.g.,
interfering RNA) is fully encapsulated within the lipid. In certain instances, SNALP are extremely
useful for ic applications, as they can exhibit extended circulation lifetimes following
intravenous (iv) injection, they can accumulate at distal sites (e.g., sites physically separated from
the administration site), and they can mediate ing of target gene expression at these distal sites.
The nucleic acid may be complexed with a condensing agent and encapsulated within a SNALP as set
forth in PCT Publication No. WO 00/03683, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
The lipid les of the invention (e.g., SNALP) typically have a mean diameter of
from about 30 nm to about 150 nm, from about 40 run to about 150 nm, from about 50 nm to about
150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to
about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70
to about 90 nm, from about 80 run to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm,
nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nrn, 80 nm, 85 nm, 90 nm, 95 nm, 100
nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and
are substantially non—toxic. In addition, nucleic acids, when present in the lipid particles of the
present invention, are resistant in aqueous solution to degradation with a se. Nucleic acid-lipid
particles and their method of ation are disclosed in, e.g., U.S. Patent Publication Nos.
20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their
entirety for all purposes.
[0058] As used , “lipid encapsulated” can refer to a lipid particle that provides a
therapeutic nucleic acid such as an interfering RNA (e.g., siRNA), with full encapsulation, partial
encapsulation, or both. In a preferred embodiment, the nucleic acid (e.g., interfering RNA) is fully
encapsulated in the lipid particle (e.g., to form a SNALP or other nucleic acid-lipid particle).
The term “lipid conjugate” refers to a conjugated lipid that inhibits aggregation of lipid
particles. Such lipid conjugates include, but are not limited to, pid conjugates such as, e. g.,
PEG coupled to dialkyloxypropyls (e.g., A conjugates), PEG coupled to diacylglycerols
(e.g., PEG—DAG ates), PEG coupled to cholesterol, PEG coupled to
phosphatidylethanolamines, and PEG conjugated to ceramides (see, e.g., U.S. Patent No. 5,885,613),
cationic PEG lipids, polyoxazoline (POZ)—lipid conjugates (e.g., POZ-DAA conjugates; see, e.g., U.S.
Provisional Application No. 61/294,828, filed y 13, 2010, and U.S. Provisional Application No.
61/295, 140, filed January 14, 2010), polyamide oligomers (e.g., ATTA—lipid ates), and
mixtures thereof. Additional examples of POZ~lipid conjugates are described in PCT Publication No.
. PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid
via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be
used including, e. g., non-ester containing linker moieties and ester-containing linker moieties. In
certain preferred embodiments, non-ester ning linker es, such as amides or ates,
are used. The disclosures of each of the above patent documents are herein incorporated by reference
in their entirety for all purposes.
PCT/U52012/058770
The term pathic lipid” refers, in part, to any suitable material n the
hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of
polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl,
nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the ion of apolar
groups that include, but are not limited to, long-chain saturated and rated aliphatic hydrocarbon
groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and
sphingolipids.
[0061] entative examples of phospholipids include, but are not limited to,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, oyloleoyl phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
roylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in
orus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and B—acyloxyacids,
are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids
described above can be mixed with other lipids ing triglycerides and sterols.
The term “neutral lipid” refers to any of a number of lipid species that exist either in an
uncharged or neutral zwitterionic form at a ed pH. At physiological pH, such lipids include, for
example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin,
cephalin, cholesterol, cerebrosides, and diacylglycerols.
The term “non—cationic lipid” refers to any amphipathic lipid as well as any other neutral
lipid or anionic lipid.
The term “anionic lipid” refers to any lipid that is negatively charged at logical
pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins,
diacylphosphatidylserines, diacylphosphatidic acids, N—dodecanoyl phosphatidylethanolamines, N-
succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines,
lysylphosphatidylglycerols, oyloleyolphosphatidylglycerol (POPG), and other anionic
modifying groups joined to neutral lipids.
[0065] The term “hydrophobic lipid” refers to compounds having apolar groups that include, but
are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups
ally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable
PCT/U82012/058770
es include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-
diacyloxy—3-aminopropane, and l,2-dialkyl-3—aminopropane.
The terms “cationic lipid” and “amino lipid” are used interchangeably herein to include
those lipids and salts thereof having one, two, three, or more fatty acid or fatty alkyl chains and a pH-
titratable amino head group (e.g., an alkylamino or dialkylamino head group). The cationic lipid is
typically protonated (i.e., positively charged) at a pH below the pKa of the ic lipid and is
substantially neutral at a pH above the pKa. The cationic lipids of the invention may also be termed
titratable cationic lipids. In some ments, the cationic lipids comprise: a protonatable tertiary
amine (e.g., pH-titratable) head group; C18 alkyl chains, wherein each alkyl chain independently has 0
to 3 (e.g., 0, l, 2, or 3) double bonds; and ether, ester, or ketal linkages between the head group and
alkyl chains. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA,
DLenDMA, y—DLenDMA, DLin-K—DMA, DLin—K—CZ-DMA (also known as DLin—CZK-DMA,
XTC2, and C2K), DLin—K—C3—DMA, DLin-K-C4-DMA, DLen-CZK—DMA, y—DLen-C2K-DMA,
DLin—M—CZ—DMA (also known as MC2), and DLin-M-C3-DMA (also known as MC3).
[0067] The term “salts” includes any anionic and cationic complex, such as the complex formed
between a cationic lipid and one or more anions. Non-limiting examples of anions include inorganic
and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., alate),
phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate,
e, nitrite, nitride, te, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate,
e, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,
glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, thacrylate, perchlorate,
chlorate, chlorite, lorite, bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,
arsenate, arsenite, chromate, mate, cyanide, cyanate, thiocyanate, hydroxide, de,
permanganate, and es thereof. In ular embodiments, the salts of the cationic lipids
disclosed herein are crystalline salts.
The term ” includes a straight chain or branched, noncyclic or cyclic, saturated
tic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain
alkyls e, but are not limited to, methyl, ethyl, n-propyl, n-butyl, yl, n—hexyl, and the like,
while saturated branched alkyls include, without limitation, isopropyl, sec-butyl, isobutyl, tert-butyl,
isopentyl, and the like. Representative saturated cyclic alkyls e, but are not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, while unsaturated cyclic alkyls include,
without limitation, cyclopentenyl, cyclohexenyl, and the like.
The term “alkenyl” includes an alkyl, as defined above, containing at least one double
bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative
PCT/U82012/058770
ht chain and branched alkenyls include, but are not limited to, ethylenyl, propylenyl, l-butenyl,
nyl, isobutylenyl, l—pentenyl, 2—pentenyl, 3-methyl-l—butenyl, 2-methyl—2-butenyl, 2,3-
dimethyl-Z-butenyl, and the like.
The term “alkynyl” includes any alkyl or alkenyl, as defined above, which additionally
contains at least one triple bond between adjacent carbons. Representative straight chain and
branched alkynyls include, without limitation, acetylenyl, propynyl, l—butynyl, 2—butynyl, ynyl,
2—pentynyl, 3—methyl—1 l, and the like.
The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein the carbon at the point of
attachment is tuted with an 0x0 group, as defined below. The following are non-limiting
examples of acyl groups: —C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl.
The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7— to lO-membered
bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from
1 or 2 heteroatoms independently selected from en, oxygen and sulfur, and wherein the nitrogen
and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally
nized, including bicyclic rings in which any of the above heterocycles are fused to a benzene
ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include, but
are not limited to, heteroaryls as defined below, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,
piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, ydroprimidinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, ydrothiopyranyl, and the
like.
The terms “optionally substituted alkyl”, nally substituted alkenyl”, “optionally
substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” mean that,
when tuted, at least one hydrogen atom is replaced with a tuent. In the case of an 0x0
substituent (=0), two hydrogen atoms are replaced. In this regard, substituents include, but are not
limited to, oxo, halogen, heterocycle, -CN, —OR", »NRXRY, ~NRXC(=O)Ry, -NRX802Ry, -C(=O)R", —
C(=O)ORX, —C(=O)NRny, ~SOnR", and -SOnNRny, wherein n is 0, l, or 2, RK and Ry are the
same or ent and are independently hydrogen, alkyl, or heterocycle, and each of the alkyl and
heterocycle substituents may be fithher substituted with one or more of 0x0, halogen, -OH, —CN,
alkyl, -OR‘, heterocycle, -NR"Ry, -NRXC(=O)Ry, -NRXSOzRy, -C(=O)R", -C(=O)OR", —
C(=O)NR"Ry, -SOnR", and -SOnNR"Ry. The term “optionally tuted,” when used before a
list of substituents, means that each of the tuents in the list may be optionally substituted as
described herein.
W0 20131052677
The term “halogen” includes fluoro, chloro, bromo, and iodo.
The term enic” refers to the ability of a lipid particle, such as a SNALP, to fuse
with the membranes of a cell. The membranes can be either the plasma membrane or membranes
surrounding lles, e.g., endosome, nucleus, etc.
As used herein, the term “aqueous solution” refers to a composition comprising in
whole, or in part, water.
As used herein, the term “organic lipid solution” refers to a composition comprising in
whole, or in part, an organic solvent having a lipid.
“Distal site,” as used herein, refers to a physically separated site, which is not limited to
an adjacent capillary bed, but includes sites broadly distributed throughout an organism.
“Serum—stable” in relation to nucleic acid-lipid les such as SNALP means that the
particle is not significantly degraded after exposure to a serum or nuclease assay that would
cantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum
assay, a DNAse assay, or an RNAse assay.
[0080] “Systemic delivery,” as used herein, refers to delivery of lipid particles that leads to a
broad tribution of an active agent such as an interfering RNA (e.g., siRNA) within an organism.
Some techniques of administration can lead to the systemic delivery of certain agents, but not others.
Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most
parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the
agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid,
nonspecific cell g) before reaching a disease site distal to the site of administration. Systemic
delivery of lipid particles can be by any means known in the art including, for example, intravenous,
aneous, and intraperitoneal. In a preferred ment, systemic delivery of lipid particles is
by intravenous delivery.
[0081] “Local ry,” as used , refers to delivery of an active agent such as an
interfering RNA (e.g., siRNA) directly to a target site within an organism. For e, an agent can
be locally delivered by direct injection into a disease site, other target site, or a target organ such as
the liver, heart, pancreas, kidney, and the like.
The term “mammal” refers to any mammalian species such as a human, mouse, rat, dog,
cat, hamster, guinea pig, rabbit, livestock, and the like.
The term “reticuloendothelial system” or “RES” refers to the part of the immune system
that ns reticuloendothelial cells, including the phagocytic cells located in reticular connective
tissue such as monocytes and macrophages. These cells typically late in lymph nodes and the
spleen. The Kupffer cells of the liver and tissue cytes are also part of the RES. The RES is
divided into y and secondary lymphoid organs. Primary (“central”) lymphoid organs are the
sites where the cells of the RES are produced. The cells of the RES are produced in the bone .
The thymus is also included as it is the required site for T cell maturation. ary (“peripheral”)
lymphoid organs are the sites where the cells of the RES function. This includes the lymph nodes,
tonsils, spleen, and “MALT” (mucosa-associated lymphoid tissue). MALT is further divided into
“GALT” (gut—associated lymphoid tissue) and “BALT” (bronchus—associated lymphoid tissue). The
Kupffer cells of the liver act as part of this system, but are not organized into a tissue; rather, they are
sed throughout the liver sinusoids. The microglia of the central nervous system (CNS) can be
considered a part of the RES. They are scavenger cells that proliferate in response to CNS injury.
III. Description of the Embodiments
[0084] The present invention provides therapeutic nucleic acids such as interfering RNA (e.g.,
dsRNA such as siRNA) that target the expression of ALDH genes, in particular ALDH2 gene, lipid
particles comprising one or more (e.g., a cocktail) of the therapeutic nucleic acids, methods ofmaking
the lipid particles, and methods of delivering and/or administering the lipid particles (e.g., for the
treatment of alcoholism in humans).
[0085] In one aspect, the present ion provides interfering RNA molecules that target
ALDH gene expression. Non—limiting examples of interfering RNA molecules include double—
stranded RNA capable of mediating RNAi such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA,
pre—miRNA, and mixtures thereof. In certain instances, the present ion provides compositions
sing a combination (8g, a cocktail, pool, or mixture) of interfering RNAs that target ent
regions of the ALDH gene. In certain ces, the ering RNA (e.g., siRNA) molecules of the
invention are capable of silencing ALDH gene expression, inactivating ALDH, and/or inhibiting the
replication ofALDH in vitro or in viva.
In particular embodiments, the present invention provides an interfering RNA (e.g.,
siRNA) that silences ALDH gene expression, wherein the interfering RNA comprises a sense strand
and a complementary antisense strand, and wherein the interfering RNA comprises a double—stranded
region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-30, 15-25, 19-30, 19—25,
—60, 20—55, 20—50, 20—45, 20-40, 20—35, 20—30, 20-25, 21-30, 21-29, 22—30, 22—29, 22-28, 23—30, 23-
28, 24—30, 24—28, 25—60, 25-55, 25—50, 25—45, 25-40, 25-35, or 25-30 nucleotides in length, or about
PCT/U82012/058770
, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in
length).
In certain embodiments, the ering RNA (e.g., siRNA) of the present invention may
comprise at least one, two, three, four, five, six, seven, eight, nine, ten, or more modified tides
such as 2’OMe nucleotides, e.g., in the sense and/or antisense strand of the -stranded region of
the interfering RNA. Preferably, uridine and/or guanosine nucleotides in the interfering RNA are
modified with 2’OMe nucleotides. In certain instances, the interfering RNA contains 2’OMe
nucleotides in both the sense and antisense strands and comprises at least one 2’OMe—uridine
nucleotide and at least one 2’OMe-guanosine nucleotide in the double-stranded region. In some
ments, the sense and/or antisense strand of the interfering RNA may further comprise
modified (e.g., 2’OMe-modified) adenosine and/or modified (e.g., 2’OMe—modified) cytosine
nucleotides, e.g., in the double~stranded region of the interfering RNA.
In particular embodiments, the ering RNA (e.g., siRNA) les of the present
invention comprise a 3’ ng of 1, 2, 3, or 4 nucleotides in one or both strands. In certain
instances, the interfering RNA may contain at least one blunt end. In particular embodiments, the 3’
overhangs in one or both s of the interfering RNA may each independently comprise l, 2, 3, or
4 modified and/or unmodified deoxythymidine (“t” or “dT”) nucleotides, 1, 2, 3, or 4 d (e. g.,
2’OMe) and/or unmodified uridine (“U”) ribonucleotides, or 1, 2, 3, or 4 modified (e.g., 2’OMe)
and/or unmodified ribonucleotides or deoxyribonucleotides having complementarity to the target
sequence or the complementary strand thereof.
The present invention also provides a pharmaceutical composition sing one or
more (e.g., a cocktail) of the interfering RNAs described herein and a pharrnaceutically acceptable
carrier.
In another aspect, the present invention provides a nucleic acid—lipid particle (6g,
SNALP) that targets ALDH gene expression. The nucleic acid—lipid particles (e.g., SNALP) typically
comprise one or more (e. g., a cocktail) of the interfering RNAs described herein, a cationic lipid, and
a non-cationic lipid. In certain instances, the nucleic acid—lipid particles (e.g., SNALP) filrther
comprise a conjugated lipid that inhibits aggregation of particles. Preferably, the nucleic acid-lipid
particles (e.g., SNALP) comprise one or more (e.g., a il) of the ering RNAs described
herein, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of
les.
In some ments, the interfering RNAs (e.g., siRNAs) of the present invention are
fully encapsulated in the nucleic acid-lipid particle (e. g., SNALP). With respect to formulations
2012/058770
comprising an interfering RNA cocktail, the different types of interfering RNA species present in the
cocktail (e.g., interfering RNA compounds with ent sequences) may be co-encapsulated in the
same particle, or each type of ering RNA species present in the cocktail may be encapsulated in
a separate particle. The interfering RNA cocktail may be formulated in the particles described herein
using a mixture of two or more individual interfering RNAs (each having a unique sequence) at
identical, similar, or different concentrations or molar ratios. In one embodiment, a cocktail of
interfering RNAs (corresponding to a plurality of interfering RNAs with ent sequences) is
formulated using cal, similar, or ent concentrations or molar ratios of each interfering RNA
species, and the different types of interfering RNAs are co—encapsulated in the same particle. In
r embodiment, each type of interfering RNA species present in the il is encapsulated in
different particles at identical, similar, or different interfering RNA concentrations or molar ratios,
and the particles thus formed (each ning a ent interfering RNA d) are administered
separately (e.g., at different times in accordance with a therapeutic regimen), or are combined and
administered together as a single unit dose (e.g., with a phannaceutically acceptable carrier). The
les described herein are serum—stable, are resistant to nuclease degradation, and are substantially
non-toxic to mammals such as humans.
The cationic lipid in the nucleic acid—lipid particles of the invention (e.g., SNALP) may
se, e.g., one or more cationic lipids of Formula I-III bed herein or any other cationic lipid
species. In one particular embodiment, the cationic lipid is selected from the group consisting of 1,2-
dilinoleyloxy—N,N-dimethylaminopropane (DLinDMA), l ,2-dilinolenyloxy-N,N-
dimethylaminopropane (DLenDMA), l ,2~di-y-linolenyloxy-N,N-dimethylaminopropane (y—
DLenDMA), 2,2—dilinoley1-4—(2-dimethylaminoethyl)-[l,3]~dioxolane (DLin—K—CZ—DMA), 2,2—
dilinoleyl—4—dimethylaminomethyl-[ l ,3]—dicxolane (DLin—K~DMA), dilinoleylmethyl-3 -
dimethylaminopropionate (DLin—M-CZ—DMA), ,282,3 ptatriaconta-6,9,28,3 l—tetraen- l 9-
yl 4-(dimethylamino)butan0ate (DLin—M—C3—DMA), salts thereof, and mixtures thereof.
The non-cationic lipid in the nucleic acid—lipid particles of the present invention (e. g.,
SNALP) may comprise, e.g., one or more anionic lipids and/or neutral lipids. In some embodiments,
the non—cationic lipid comprises one of the following neutral lipid components: ( 1) a mixture of a
phospholipid and cholesterol or a derivative thereof; (2) cholesterol or a derivative thereof; or (3) a
phospholipid. In certain preferred embodiments, the olipid comprises
dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture f.
In a particularly preferred embodiment, the non-cationic lipid is a mixture of DPPC and cholesterol.
The lipid conjugate in the nucleic acid-lipid particles of the invention (e.g., SNALP)
inhibits aggregation of particles and may comprise, e.g., one or more of the lipid ates described
herein. In one particular embodiment, the lipid conjugate comprises a PEG—lipid conjugate.
WO 2013052677 PCT/U52012/058770
Examples of PEG-lipid conjugates include, but are not limited to, PEG—DAG conjugates, PEG—DAA
conjugates, and mixtures f. In certain embodiments, the A conjugate in the lipid
le may comprise a PBG-didecyloxypropyl (C10) ate, a PEG—dilauryloxypropyl (C12)
conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, a
PEG-distearyloxypropyl (C13) conjugate, or es thereof. In another embodiment, the lipid
conjugate ses a POZ—lipid conjugate such as a POZ—DAA conjugate.
In some embodiments, the present invention provides nucleic acid—lipid particles (e. g.,
SNALP) comprising: (a) one or more (e.g, a cocktail) ering RNA molecules that target ALDH
gene sion; (b) one or more cationic lipids or salts thereof comprising from about 50 mol % to
about 85 mol % of the total lipid t in the le; (0) one or more non—cationic lipids
comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and ((1)
one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol %
to about 2 mol % of the total lipid present in the particle.
In one aspect of this ment, the nucleic acid-lipid particle comprises: (a) one or
more (e.g., a cocktail) interfering RNA molecules that target ALDH gene expression; (b) a cationic
lipid or a salt thereof comprising from about 52 mol % to about 62 mol % of the total lipid present in
the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from
about 36 mol % to about 47 mol % of the total lipid present in the particle; and (d) a PEG-lipid
ate comprising from about 1 mol % to about 2 mol % of the total lipid present in the particle.
This embodiment of nucleic acid-lipid particle is generally referred to herein as the “1:57”
formulation. In one particular embodiment, the 1:57 formulation is a four—component system
comprising about 1.4 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA), about 57.1 mol %
cationic lipid (e.g., DLin—K—CZ—DMA) or a salt thereof, about 7.1 mol % DPPC (or DSPC), and about
34.3 mol % cholesterol (or derivative thereof).
[0097] In another aspect of this embodiment, the nucleic acid—lipid particle comprises: (a) one
or more (e.g., a cocktail) interfering RNA molecules that target ALDH gene sion; (b) a cationic
lipid or a salt thereof sing from about 56.5 mol % to about 66.5 mol % of the total lipid present
in the particle; (c) cholesterol or a derivative thereof comprising from about 31.5 mol % to about 42.5
mol % of the total lipid present in the particle; and (d) a PEG—lipid conjugate comprising from about 1
mol % to about 2 mol % of the total lipid present in the particle. This embodiment of nucleic acid-
lipid particle is generally referred to herein as the “1 :62” ation. In one particular ment,
the 1:62 formulation is a three-component system which is phospholipid-free and comprises about 1.5
mol % PEG-lipid conjugate (e.g., PEGZOOO-C—DMA), about 61.5 mol % cationic lipid (e.g., DLin-K-
C2—DMA) or a salt thereof, and about 36.9 mol % cholesterol (or derivative thereof).
PCT/U82012/058770
Additional embodiments related to the 1:57 and 1:62 formulations are bed in PCT
Publication No. W0 09/127060 and published US patent ation publication number US
2011/0071208 Al, the disclosures of which are herein incorporated by reference in their entirety for
all purposes.
In other embodiments, the present invention provides nucleic acid-lipid les (e.g.,
SNALP) comprising: (a) one or more (e. g., a cocktail) interfering RNA molecules that target ALDH
gene expression; (b) one or more cationic lipids or salts thereof comprising from about 2 mol % to
about 50 mol % of the total lipid t in the particle; (c) one or more non-cationic lipids
comprising from about 5 mol % to about 90 mol % of the total lipid t in the particle; and ((1)
one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol %
to about 20 mol % ofthe total lipid present in the le.
In one aspect of this ment, the nucleic acid-lipid particle comprises: (a) one or
more (tag, a cocktail) interfering RNA molecules that target ALDH gene expression; (b) a cationic
lipid or a salt thereof comprising from about 30 mol % to about 50 mol % of the total lipid present in
the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from
about 47 mol % to about 69 mol % of the total lipid present in the particle; and (d) a PEG—lipid
conjugate comprising from about 1 mol % to about 3 mol % of the total lipid present in the particle,
This embodiment of nucleic acid-lipid particle is generally referred to herein as the “2:40”
ation. In one particular embodiment, the 2:40 formulation is a four—component system which
comprises about 2 mol % PEG—lipid ate (e.g., PEGZOOO-C-DMA), about 40 mol % cationic
lipid (e.g., DLin—K—C2-DMA) or a salt thereof, about 10 mol % DPPC (or DSPC), and about 48 mol
% cholesterol (or derivative thereof).
In further embodiments, the present invention provides c acid-lipid particles (e. g.,
SNALP) comprising: (a) one or more (e.g., a cocktail) interfering RNA molecules that target ALDH
gene expression; (b) one or more cationic lipids or salts thereof comprising from about 50 mol % to
about 65 mol % of the total lipid present in the particle; (0) one or more non—cationic lipids
comprising from about 25 mol % to about 45 mol % of the total lipid t in the particle; and (d)
one or more conjugated lipids that inhibit aggregation of particles comprising from about 5 mol % to
about 10 mol % of the total lipid present in the le.
[00102] In one aspect of this embodiment, the nucleic acid-lipid particle comprises: (a) one or
more (e. g., a cocktail) interfering RNA molecules that target ALDH gene expression; (b) a ic
lipid or a salt f comprising from about 50 mol % to about 60 mol % of the total lipid present in
the particle; (c) a mixture of a phospholipid and cholesterol or a derivative thereof comprising from
about 35 mol % to about 45 mol % of the total lipid present in the particle; and (d) a PEG—lipid
WO 2013052677
conjugate comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle.
This embodiment of nucleic acid-lipid particle is generally referred to herein as the “7:54”
formulation. In certain instances, the non—cationic lipid mixture in the 7:54 formulation comprises:
(i) a phospholipid of from about 5 mol % to about 10 mol % of the total lipid t in the particle;
and (ii) cholesterol or a derivative f of fiom about 25 mol % to about 35 mol % of the total lipid
present in the le. In one particular embodiment, the 7:54 formulation is a four—component
system which comprises about 7 mol % PEG—lipid conjugate (e.g., —C-DMA), about 54 mol
% cationic lipid (e.g., DLin—K—CZ—DMA) or a salt f, about 7 mol % DPPC (or DSPC), and
about 32 mol % cholesterol (or derivative thereof).
[00103] In another aspect of this embodiment, the nucleic ipid particle comprises: (a) one
or more (e.g., a cocktail) interfering RNA molecules that target ALDI-I gene expression; (b) a cationic
lipid or a salt thereof comprising from about 55 mol % to about 65 mol % of the total lipid present in
the particle; (c) cholesterol or a derivative thereof comprising from about 30 mol % to ab0ut 40 mol
% ofthe total lipid present in the particle; and (d) a PEG—lipid conjugate comprising from about 5 mol
% to about 10 mol % of the total lipid present in the le. This embodiment of nucleic acid—lipid
particle is generally referred to herein as the “7:58” formulation. In one particular embodiment, the
7:58 formulation is a three—component system which is phospholipid-free and comprises about 7 mol
% PEG-lipid ate (e.g., PEG750-C-DMA), about 58 mol % cationic lipid (e.g., DLin-K-CZ-
DMA) or a salt f, and about 35 mol % cholesterol (or derivative thereof).
[00104] Additional embodiments related to the 7:54 and 7:58 formulations are described in
hed US patent application publication number US 2011/0076335 Al, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.
The present invention also provides pharmaceutical compositions comprising a nucleic
acid-lipid particle such as a SNALP and a pharmaceutically able carrier.
[00106] The nucleic acid—lipid particles of the present invention (e.g., SNALP) are useful for the
therapeutic delivery of interfering RNAs (e.g., siRNAs) that silence the expression of one or more
ALDH genes. In some embodiments, a cocktail of interfering RNAs that target different regions (e. g.,
overlapping and/or non-overlapping sequences) of an ALDI-I gene is formulated into the same or
ent nucleic acid-lipid particles, and the particles are administered to a mammal (e.g., a human)
ing such treatment. In certain instances, a eutically effective amount of the nucleic acid-
lipid les can be administered to the mammal, e. g., for treating alcoholism in a human.
In certain embodiments, the present invention provides a method for introducing one or
more interfering RNA (e. g., siRNA) molecules bed herein into a cell by contacting the cell with
W0 20131‘052677 PCT/U82012/058770
a nucleic acid—lipid particle described herein (e.g., a SNALP formulation). In one particular
embodiment, the cell is a loendothelial cell (e.g., monocyte or macrophage), fibroblast cell,
endothelial cell, or et cell.
In some embodiments, the nucleic acid-lipid particles described herein (e.g., SNALP) are
administered by one of the following routes of administration: oral, intranasal, intravenous,
eritoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and
intradermal. In particular embodiments, the c acid-lipid particles are administered systemically,
e.g., via enteral or parenteral routes of administration.
In particular ments, the nucleic acid—lipid les of the invention (e.g., SNALP)
can preferentially deliver a payload such as an interfering RNA (e.g., dsRNA) to the liver as
compared to other tissues, e.g., for the treatment of acute or chronic alcoholism.
In n aspects, the present invention provides methods for silencing ALDH gene
expression in a mammal (e.g., human) in need thereof, the method comprising administering to the
mammal a therapeutically effective amount of a nucleic acid—lipid particle (e.g., a SNALP
formulation) comprising one or more interfering RNAs (e.g., siRNAs) described herein (e.g., siRNAs
targeting one or more ALDH genes). In some embodiments, administration of nucleic acid-lipid
particles comprising one or more ALDH interfering RNAs reduces ALDH RNA levels by at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or
any range therein) relative to ALDH RNA levels detected in the absence of the interfering RNA (e.g.,
buffer l or vant non—ALDH targeting interfering RNA l). In other embodiments,
administration of nucleic acid-lipid particles comprising one or more ALDH-targeting interfering
RNAs reduces ALDH RNA levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or
more (or any range therein) relative to a ve control such as, e. g, a buffer control or an irrelevant
non—ALDH targeting interfering RNA control.
In other aspects, the present invention provides methods for silencing ALDH gene
expression in a mammal (e.g., human) in need thereof, the method comprising administering to the
mammal a therapeutically effective amount of a nucleic acid-lipid le (e.g., a SNALP
formulation) comprising one or more interfering RNAs (e.g., siRNAs) bed herein (e.g., siRNAs
targeting one or more regions of the ALDH gene). In some embodiments, stration of nucleic
acid—lipid les comprising one or more ALDH interfering RNAs reduces ALDH mRNA levels by
at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
W0 2013i052677
(or any range therein) relative to ALDH mRNA levels detected in the absence of the interfering RNA
(e.g., buffer control or irrelevant non-ALDH ing interfering RNA control). In other
embodiments, administration of nucleic ipid particles comprising one or more ALDH—targeting
interfering RNAs reduces ALDH mRNA levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
100 days or more (or any range therein) ve to a negative control such as, e. g., a buffer l or
an irrelevant non—ALDH targeting interfering RNA control.
In other aspects, the present ion provides methods for treating, preventing,
reducing the risk or likelihood of ping (e.g., reducing the susceptibility to), delaying the onset
of, and/or rating one or more symptoms associated with alcoholism in a mammal (e.g., human)
in need thereof, the method comprising administering to the mammal a therapeutically effective
amount of a nucleic acid-lipid particle (e.g., a SNALP formulation) comprising one or more
interfering RNA molecules (e.g., siRNAs) described herein that target ALDH gene expression.
In r s, the present invention provides a method for inactivating ALDH in a
mammal (e.g., human) in need thereof (e. g., a human ing from alcoholism), the method
comprising stering to the mammal a therapeutically effective amount of a nucleic acid—lipid
le (e.g., a SNALP formulation) comprising one or more interfering RNAs (e.g., siRNAs)
described herein that target ALDH gene expression. In some embodiments, administration of nucleic
acid-lipid les comprising one or more ALDH-targeting interfering RNAs , reduces, or
decreases ALDH enzyme levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% (or any range therein) relative to the ALDH enzyme levels
detected in the absence of the interfering RNA (e.g., buffer control or irrelevant non-ALDH targeting
interfering RNA control).
[00114] By way of example, ALDH2 mRNA can be measured using a branched DNA assay
(QuantiGene®; Affymetrix). The branched DNA assay is a sandwich nucleic acid hybridization
method that uses bDNA molecules to amplify signal from captured target RNA. Again by way of
example, ALDH enzymatic activity can be measured spectrophotometrically by adding NAD and a
substrate (e.g., acetaldehyde) to a protein sample and measuring the formation of NADH at 340 nm
(see, e.g., Sheppard, Albersheim & McCleam, J. Biol. Chem. 1970 245(11):2876; Arolfo et a!
Alcoholism: Clinical and Experimental Research 2009 33(11):l935; Garver et a1, Alcohol &
Alcoholism 2000 35(5):435)).
PCT/U82012/058770
IV. Therapeutic Nucleic Acids
The term “nucleic acid” includes any oligonucleotide or polynucleotide, with fragments
containing up to 60 nucleotides generally termed Oligonucleotides, and longer fragments termed
polynucleotides. In particular ments, oligonucletoides of the invention are from about 15 to
about 60 nucleotides in length. In some embodiments, nucleic acid is associated with a carrier system
such as the lipid particles described herein. In certain embodiments, the nucleic acid is fully
encapsulated in the lipid particle. Nucleic acid may be administered alone in the lipid les of the
invention, or in combination (e.g., co—administered) with lipid particles comprising peptides,
polypeptides, or small molecules such as tional drugs.
[00116] In the context of this invention, the terms “polynucleotide” and “oligonucleotide” refer to
a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally—occurring bases,
sugars and intersugar (backbone) linkages. The terms “polynucleotide” and “oligonucleotide” also
include polymers or oligomers comprising non—naturally ing monomers, or portions thereof,
which function similarly. Such modified or substituted Oligonucleotides are often preferred over
native forms because of properties such as, for example, enhanced cellular uptake, reduced
immunogenicity, and increased stability in the presence eases.
Oligonucleotides are generally classified as deoxyribooligonucleotides or
igonucleotides. A deoxyribooligonucleotide ts of a 5—carbon sugar called deoxyribose
joined covalently to phosphate at the 5’ and 3’ carbons of this sugar to form an alternating,
unbranched polymer. A ribooligonucleotide consists of a similar ing ure Where the 5-
carbon sugar is ribose.
The nucleic acid can be -stranded DNA or RNA, or double-stranded DNA or RNA,
or DNA-RNA hybrids. In preferred embodiments, the nucleic acid is double—stranded RNA.
Examples of double-stranded RNA are described herein and include, e. g., siRNA and other RNAi
agents such as substrate dsRNA, shRNA, aiRNA, and pre-miRNA. In other embodiments, the
nucleic acid is single-stranded. Single—stranded nucleic acids include, e.g., nse Oligonucleotides,
ribozymes, mature miRNA, and triplex—forming Oligonucleotides.
Nucleic acids of the invention may be of various s, generally dependent upon the
particular form of nucleic acid. For example, in particular embodiments, ds or genes may be
from about 1,000 to about 100,000 nucleotide es in length. In ular embodiments,
ucleotides may range from about 10 to about 100 nucleotides in length. In various related
embodiments, Oligonucleotides, both single—stranded, double—stranded, and triple—stranded, may range
in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20
PCT/U52012/058770
to about 50 tides, from about 15 to about 30 nucleotides, or from about 20 to about 30
nucleotides in length.
In particular embodiments, an oligonucleotide (or a strand thereof) of the invention
specifically hybridizes to or is complementary to a target polynucleotide sequence. The terms
fically izable” and “complementary” as used herein indicate a sufficient degree of
complementarity such that stable and specific binding occurs between the DNA or RNA target and the
oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its
target nucleic acid sequence to be specifically izable. In preferred embodiments, an
ucleotide is specifically hybridizable when binding of the oligonucleotide to the target sequence
interferes with the normal function of the target sequence to cause a loss of utility or sion
therefrom, and there is a sufficient degree of complementarity to avoid non—specific binding of the
oligonucleotide to non—target sequences under conditions in which specific binding is desired, i.e.,
under physiological conditions in the case of in viva assays or therapeutic treatment, or, in the case of
in vitro assays, under conditions in which the assays are conducted. Thus, the oligonucleotide may
include 1, 2, 3, or more base substitutions as compared to the region of a gene or mRNA sequence
that it is targeting or to which it specifically izes.
A. siRNA
] The fied and modified siRNA molecules of the invention are capable of silencing
ALDH gene expression. Each strand of the siRNA duplex is typically about 15 to about 60
nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments,
the siRNA comprises at least one modified tide. The modified siRNA is generally less
stimulatory than a corresponding unmodified siRNA sequence and retains RNAi activity
against the target gene of interest. In some embodiments, the d siRNA contains at least one
2’OMe purine or pyrimidine nucleotide such as a 2’0Me—guanosine, 2’OMe—uridine, 2’OMe-
adenosine, and/or 2’OMe—cytosine nucleotide. The modified nucleotides can be present in one strand
(i.e., sense or antisense) or both strands of the siRNA. In some red embodiments, one or more
of the e and/or guanosine nucleotides are modified (e. g., 2’OMe-modified) in one strand (i.e.,
sense or antisense) or both strands of the siRNA. In these embodiments, the modified siRNA can
further comprise one or more modified (e.g., 2’OMe—modified) adenosine and/or modified (e.g.,
2’OMe-modified) cytosine nucleotides. In other preferred embodiments, only uridine and/or
guanosine nucleotides are modified (e.g., Z’OMe-modified) in one strand (Le, sense or antisense) or
both strands of the siRNA. The siRNA ces may have overhangs (e. g., 3’ or 5’ overhangs as
described in Elbashir er al., Genes Dev., 151188 (2001) or Nykanen et al., Cell, 107:309 (2001)), or
may lack overhangs (tie, have blunt ends).
PCT/U52012/058770
In particular embodiments, the selective incorporation of modified nucleotides such as
2’OMe uridine and/or guanosine nucleotides into the double—stranded region of either or both strands
of the siRNA reduces or completely abrogates the immune response to that siRNA le. In
certain instances, the immunostimulatory properties of specific siRNA sequences and their ability to
silence gene expression can be balanced or optimized by the introduction of minimal and selective
2’OMe modifications within the double—stranded region of the siRNA duplex. This can be achieved
at therapeutically viable siRNA doses t cytokine induction, toxicity, and off—target s
associated with the use dified siRNA.
The modified siRNA generally comprises from about 1% to about 100% (e.g., about 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double—stranded region of the
siRNA duplex. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or
more of the nucleotides in the double-stranded region of the siRNA comprise d nucleotides.
In certain other embodiments, some or all of the modified nucleotides in the double—stranded region of
the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart from each other. In one preferred
embodiment, none of the modified nucleotides in the double—stranded region of the siRNA are
adjacent to each other (e.g., there is a gap of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified
nucleotides between each modified tide).
[00124] In some embodiments, less than about 50% (e. g., less than about 49%, 48%, 47%, 46%,
45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%, preferably less than about 35%, 34%,
33%, 32%, 31%, or 30%) of the nucleotides in the double-stranded region of the siRNA comprise
d (e.g., 2’OMe) nucleotides. In one aspect of these embodiments, less than about 50% of the
uridine and/or guanosine nucleotides in the double—stranded region of one or both strands of the
siRNA are selectively (e.g., only) d. In another aspect of these embodiments, less than about
50% of the nucleotides in the double-stranded region of the siRNA comprise 2’OMe nucleotides,
wherein the siRNA comprises 2’OMe nucleotides in both strands of the siRNA, wherein the siRNA
comprises at least one 2’OMe-guanosine nucleotide and at least one 2’OMe—uridine nucleotide, and
wherein 2’OMe—guanosine nucleotides and 2’OMe-uridine tides are the only 2’OMe
nucleotides present in the double-stranded region. In yet another aspect of these embodiments, less
than about 50% of the tides in the double-stranded region of the siRNA comprise 2’OMe
tides, wherein the siRNA comprises 2’OMe nucleotides in both strands of the modified siRNA,
wherein the siRNA comprises 2’OMe nucleotides selected from the group consisting of 2’OMe-
guanosine nucleotides, 2’OMe-uridine nucleotides, 2’OMe—adenosine nucleotides, and mixtures
thereof, and wherein the siRNA does not comprise cytosine nucleotides in the double-
WO 52677 PCT/U82012/058770
stranded . In a further aspect of these embodiments, less than about 50% of the nucleotides in
the double-stranded region of the siRNA comprise 2’OMe nucleotides, wherein the siRNA comprises
2’OMe nucleotides in both s of the siRNA, wherein the siRNA comprises at least one 2’OMe-
guanosine nucleotide and at least one 2’OMe-uridine nucleotide, and wherein the siRNA does not
comprise 2’OMe—cytosine nucleotides in the double-stranded region. In another aspect of these
embodiments, less than about 50% of the nucleotides in the double-stranded region of the siRNA
comprise 2’OMe nucleotides, wherein the siRNA comprises 2’OMe nucleotides in both strands of the
modified siRNA, wherein the siRNA comprises 2’OMe nucleotides ed from the group
consisting of 2’OMe-guanosine nucleotides, 2’OMe—uridine nucleotides, 2’OMe-adenosine
nucleotides, and mixtures thereof, and wherein the 2’OMe nucleotides in the double—stranded region
are not nt to each other.
In other embodiments, from about 1% to about 50% (e. g., from about 5%—50%, 10%-
50%, 15%—50%, 20%-50%, 25%-50%, %, 35%—50%, 40%—50%, 45%—50%, , 10%—
45%, 15%-45%, 20%-45%, 25%-45%, %, 35%—45%, 40%—45%, 5%-40%, lO%—40%, 15%-
40%, 20%—40%, 25%—40%, 25%—39%, 25%—38%, 25%—37%, 25%—36%, 26%—39%, 26%—38%, 26%—
37%, 26%-36%, 27%-39%, 27%—38%, 27%-37%, 27%—36%, 28%—39%, %, 28%-37%, 28%—
36%, 29%-39%, 29%-38%, 29%—37%, 29%—36%, 30%—40%, 30%—39%, 30%—38%, %, 30%-
36%, 31%-39%, 31%-38%, 3l%-37%, 31%-36%, 32%—39%, 32%-38%, 32%-37%, 32%-36%, 33%-
39%, 33%-38%, 33%—37%, 33%-36%, 34%—39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%, 5%-
35%, 10%-35%, 15%—35%, 20%—35%, 21%—35%, 22%—35%, 23%—35%, 24%—35%, 25%—35%, 26%-
%, 27%-35%, 28%—35%, 29%—35%, 30%—35%, 31%—35%, 32%—35%, 33%—35%, 34%—35%, 30%-
34%, 3l%-34%, 32%-34%, %, 30%-33%, 31%—33%, 32%—33%, 30%-32%, 3l%—32%, 25%-
34%, 25%-33%, 25%—32%, 25%—31%, 26%—34%, %, 26%—32%, %, 27%—34%, 27%-
33%, 27%—32%, 27%—31%, 28%—34%, 28%—33%, 28%—32%, 28%-3l%, 29%~34%, 29%—33%, 29%—
32%, %, 5%—30%, 10%-30%, 15%-30%, 20%—34%, 20%-33%, 20%—32%, 20%-3l%, 20%-
%, 21%-30%, 22%~30%, 23%-30%, 24%—30%, 25%—30%, 25%-29%, 25%-28%, 25%-27%, 25%-
26%, 26%—30%, 26%-29%, 26%-28%, 26%—27%, 27%-30%, 27%-29%, 27%—28%, 28%-30%, 28%—
29%, %, 5%—25%, 10%—25%, 15%~25%, 20%-29%, 20%-28%, 20%-27%, 20%—26%, 20%-
%, 5%—20%, lO%-20%, 15%-20%, 5%-15%, lO%—15%, or 5%—lO%) of the nucleotides in the
—stranded region of the siRNA comprise modified nucleotides. In one aspect of these
embodiments, from about 1% to about 50% of the uridine and/or ine nucleotides in the -
stranded region of one or both strands of the siRNA are selectively (e.g., only) d. In r
aspect of these embodiments, from about 1% to about 50% of the nucleotides in the double-stranded
region of the siRNA comprise 2’OMe tides, wherein the siRNA comprises 2’OMe nucleotides
in both strands of the siRNA, wherein the siRNA comprises at least one 2’OMe-guanosine nucleotide
and at least one 2’OMe-uridine nucleotide, and wherein 2’OMe—guanosine nucleotides and 2’OMe—
PCT/U82012/058770
uridine nucleotides are the only 2’OMe nucleotides present in the double—stranded region. In yet
r aspect of these embodiments, from about 1% to about 50% of the nucleotides in the double-
stranded region of the siRNA comprise 2’OMe nucleotides, wherein the siRNA ses 2’OMe
nucleotides in both strands of the modified siRNA, wherein the siRNA comprises 2’0Me nucleotides
selected from the group consisting of 2’OMe-guanosine nucleotides, 2’OMe-uridine nucleotides,
2’OMe-adenosine nucleotides, and mixtures thereof, and wherein the siRNA does not se
cytosine nucleotides in the double—stranded region. In a further aspect of these embodiments,
from about 1% to about 50% of the nucleotides in the double—stranded region of the siRNA comprise
2’OMe nucleotides, wherein the siRNA ses 2’OMe nucleotides in both s of the siRNA,
wherein the siRNA comprises at least one 2’OMe-guanosine nucleotide and at least one 2’OMe-
uridine nucleotide, and wherein the siRNA does not se 2’OMe—cytosine nucleotides in the
double—stranded region. In another aspect of these embodiments, from about 1% to about 50% of the
nucleotides in the -stranded region of the siRNA comprise 2’OMe nucleotides, wherein the
siRNA comprises 2’OMe nucleotides in both s of the modified siRNA, wherein the siRNA
comprises 2’0Me nucleotides selected from the group consisting of 2’OMe—guanosine nucleotides,
2’OMe-uridine nucleotides, 2’OMe-adenosine nucleotides, and mixtures thereof, and wherein the
2’OMe nucleotides in the double-stranded region are not adjacent to each other.
Additional ranges, percentages, and patterns of modifications that may be introduced into
siRNA are described in US. Patent Publication No. 20070135372, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
1. Selection of siRNA Sequences
Suitable siRNA sequences can be identified using any means known in the art.
Typically, the s described in Elbashir et al., Nature, 411:494—498 (2001) and Elbashir et al.,
EMBO J., 20:6877—6888 (2001) are combined with al design rules set forth in Reynolds 81 al.,
Nature Biotech, 22(3):326-330 (2004).
As a non-limiting example, the nucleotide sequence 3’ of the AUG start codon of a
transcript from the target gene of interest may be scanned for dinucleotide sequences (e.g., AA, NA,
CC, GG, or UU, wherein N = C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877—6888 (2001)).
The nucleotides immediately 3’ to the eotide sequences are fied as potential siRNA
sequences (i.e., a target sequence or a sense strand sequence). lly, the 19, 21, 23, 25, 27, 29,
31, 33, 35, or more nucleotides immediately 3’ to the dinucleotide sequences are identified as
potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA
ce and the 19 nucleotides immediately 3’ to the AA or NA dinucleotide are identified as
potential siRNA sequences. siRNA sequences are usually spaced at different positions along the
WO 52677
length of the target gene. To further enhance silencing efficiency of the siRNA sequences, potential
siRNA sequences may be analyzed to identify sites that do not contain regions of homology to other
coding sequences, e.g., in the target cell or organism. For example, a suitable siRNA sequence of
about 21 base pairs typically will not have more than 16-17 contiguous base pairs of homology to
coding sequences in the target cell or organism. If the siRNA sequences are to be expressed from an
RNA Pol III promoter, siRNA sequences lacking more than 4 contiguous A’s or T’s are ed.
Once a potential siRNA ce has been identified, a complementary sequence (i.e.,
an antisense strand sequence) can be designed. A potential siRNA sequence can also be analyzed
using a variety of criteria known in the art. For example, to enhance their silencing efficiency, the
siRNA sequences may be analyzed by a rational design algorithm to identify sequences that have one
or more of the following features: (1) G/C content of about 25% to about 60% G/C; (2) at least 3
A/Us at positions 15—19 of the sense ; (3) no internal repeats; (4) an A at position 19 of the
sense ; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7)
no G/C at position 19 of the sense strand; and (8) no G at position 13 of the sense strand. siRNA
design tools that incorporate algorithms that assign suitable values of each of these features and are
useful for selection of siRNA can be found at, e. g.
/ihome.ust.hl<f~bokcmho/siRNA/siRNA.html. One of skill in the art will appreciate that
sequences with one or more of the foregoing characteristics may be selected for further analysis and
testing as potential siRNA sequences.
[00130] Additionally, potential siRNA ces with one or more of the following criteria can
often be eliminated as siRNA: (1) sequences comprising a h of 4 or more of the same base in a
row; (2) sequences sing homopolymers of Gs (i.e., to reduce possible non—specific effects due
to structural teristics of these polymers; (3) sequences sing triple base motifs (e.g., GGG,
CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and (5)
sequences sing direct repeats of 4 or more bases within the ates ing in internal
ack structures. However, one of skill in the art will appreciate that sequences with one or more
of the foregoing characteristics may still be selected for further analysis and testing as potential
siRNA sequences.
In some embodiments, potential siRNA sequences may be further analyzed based on
siRNA duplex asymmetry as described in, e.g., Khvorova el al., Cell, 1152209-216 (2003); and
Schwarz et al., Cell, 115:199-208 . In other embodiments, potential siRNA sequences may be
further analyzed based on secondary structure at the target site as described in, e.g., Luo et al.,
Biophys. Res. Commun, 318:303-310 (2004). For example, secondary structure at the target site can
be modeled using the Mfold algorithm (available at http://mfold.burnet.edu.au/rna_form) to select
PCT/U82012/058770
siRNA ces which favor accessibility at the target site where less secondary structure in the
form of base—pairing and stem-loops is present.
Once a potential siRNA ce has been identified, the sequence can be analyzed for
the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo
animal model. Motifs in the sense and/or antisense strand of the siRNA sequence such as GU—rich
motifs (e. g., 5 ’—GU—3’, 5’-UGU—3’, 5’—GUGU—3’, 5’-UGUGU—3’, etc.) can also provide an indication
of whether the sequence may be immunostimulatory. Once an siRNA molecule is found to be
immunostimulatory, it can then be modified to decrease its immunostimulatory properties as
described herein. As a non—limiting example, an siRNA ce can be contacted with a mammalian
responder cell under conditions such that the cell produces a detectable immune response to determine
whether the siRNA is an immunostimulatory or a non—immunostimulatory siRNA. The mammalian
responder cell may be from a naive mammal (Le, a mammal that has not previously been in contact
with the gene product of the siRNA sequence). The mammalian der cell may be, e.g., a
peripheral blood mononuclear cell (PBMC), a macrophage, and the like. The detectable immune
response may comprise production of a cytokine or growth factor such as, e. g., TNF-a, IFN-a, lFN-B,
IFN—y, 1L-6, lL—12, or a combination thereof. An siRNA molecule identified as being
immunostimulatory can then be modified to decrease its immunostimulatory properties by replacing
at least one of the nucleotides on the sense and/or nse strand with modified nucleotides. For
example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the
nucleotides in the double—stranded region of the siRNA duplex can be replaced with modified
nucleotides such as 2’OMe nucleotides. The d siRNA can then be contacted with a
mammalian responder cell as described above to confirm that its stimulatory properties have
been d or abrogated.
Suitable in vitro assays for detecting an immune response include, but are not limited to,
the double monoclonal antibody sandwich immunoassay technique of David et al. (US. Patent No.
4,376,110); monoclonal—polyclonal antibody sandwich assays (Wide er al, in Kirkham and Hunter,
eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the “Western blot”
method of Gordon et al. (U.S. Patent No. 4,452,901); immunoprecipitation of labeled ligand (Brown
et al., J. Biol. Chem, 255:4980-4983 (1980)); enzyme-linked immunosorbent assays (ELISA) as
bed, for example, by Raines er al., J. Biol. Chem, 257:5154-5160 (1982); immunocytochemical
techniques, including the use of fluorochromes (Brooks er al., Clin. Exp. l, 39:477 (1980));
and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA, 81:2396-2400 (1984)).
In on to the immunoassays described above, a number of other assays are available,
including those bed in US. Patent Nos. 827; 3,850,752; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 074; and 4,098,876. The disclosures of these references are herein incorporated by
reference in their entirety for all purposes.
A non-limiting example of an in vivo model for detecting an immune response includes
an in viva mouse ne ion assay as described in, e.g., Judge er al., Moi. Then, 13:494-505
. (2006). In certain embodiments, the assay that can be performed as s: (1) siRNA can be
administered by standard intravenous. injection in the lateral tail vein; (2) blood can be collected by
cardiac puncture about 6 hours after administration and processed as plasma for cytokine analysis;
and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer’s
instructions (e.g., mouse and human IFN—a (PBL Biomedical; Piscataway, NJ); human IL—6 and TNF-
a (eBioscience; San Diego, CA); and mouse IL—6, TNF—(r, and IFN—y (BD Biosciences; San Diego,
CA)).
Monoclonal antibodies that specifically bind cytokines and growth factors are
cially available from multiple sources and can be generated using s known in the art
(see, e.g., Kohler e1 (11., Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A
LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Generation of
monoclonal antibodies has been previously bed and can be accomplished by any means known
in the art (Buhring er al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 ). In some methods, the
onal antibody is labeled (e.g., with any composition detectable by spectroscopic,
photochemical, biochemical, electrical, optical, or chemical means) to facilitate ion.
2. Generating siRNA Molecules
siRNA can be provided in several forms ing, e.g., as one or more isolated small-
interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA
transcribed from a transcriptional cassette in a DNA plasmid. In some ments, siRNA may be
produced enzymatically or by partial/total organic synthesis, and modified ribonucleotides can be
introduced by in vitro enzymatic or organic synthesis. In certain ces, each strand is prepared
chemically. s of synthesizing RNA molecules are known in the art, e.g., the chemical
synthesis methods as described in Verma and Eckstein (1998) or as described herein.
An RNA population can be used to provide long precursor RNAs, or long precursor
RNAs that have substantial or complete identity to a selected target sequence can be used to make the
siRNA. The RNAs can be isolated from cells or tissue, synthesized, and/or cloned according to
methods well known to those of skill in the art. The RNA can be a mixed population (obtained from
cells or tissue, transcribed from cDNA, cted, selected, etc), or can ent a single target
sequence. RNA can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in
vitro (e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA), or chemically
synthesized.
To form a long dsRNA, for synthetic RNAs, the ment is also transcribed in vitro
and hybridized to form a dsRNA. If a naturally occuring RNA tion is used, the RNA
complements are also provided (e.g., to form dsRNA for digestion by E. coli RNAse III or ,
e.g., by transcribing cDNAs corresponding to the RNA tion, or by using RNA polymerases.
The precursor RNAs are then hybridized to form double stranded RNAs for ion. The dsRNAs
can be directly administered to a subject or can be digested in vitro prior to administration.
Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and
ing cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and
Hoffman, Gene, 25:263-269 (1983); ok et al., supra; Ausubel et al., , as are PCR
methods (see, US. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and
Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those of skill in the
art. Additional basic texts disclosing the general methods of use in this invention include Sambrook
et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et
al., eds., 1994). The disclosures of these references are herein incorporated by reference in their
entirety for all purposes.
Preferably, siRNA are chemically synthesized. The ucleotides that comprise the
siRNA molecules of the invention can be synthesized using any of a variety of ques known in
the art, such as those described in Usrnan et al., J. Am. Chem. Soc, 10927845 (1987); Scaringe et al.,
Nucl. Acids Res, 1825433 (1990); Wincott et al., Nucl. Acids Res, 23:2677~2684 (1995); and t
et al., Methods Mol. Bio, 74:59 (1997). The synthesis of oligonucleotides makes use of common
nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5’—end and
phosphoramidites at the 3’—end. As a non-limiting example, small scale syntheses can be conducted
on an Applied Biosystems synthesizer using a 0.2 umol scale protocol. Alternatively, ses at the
0.2 umol scale can be performed on a 96—well plate synthesizer from Protogene (Palo Alto, CA).
However, a larger or smaller scale of synthesis is also within the scope of this invention. Suitable
ts for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA
purification are known to those of skill in the art.
siRNA molecules can also be sized via a tandem sis technique, wherein
both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a
cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to
form the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. The
tandem synthesis of siRNA can be readily adapted to both multiwell/multiplate synthesis
plaALDHorms as well as large scale synthesis plaALDHorms employing batch reactors, sis
columns, and the like. Alternatively, siRNA molecules can be assembled from two distinct
oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the
antisense strand of the siRNA. For example, each strand can be synthesized separately and joined
er by hybridization or on following sis and/or deprotection. In certain other
instances, siRNA molecules can be synthesized as a single continuous oligonucleotide fragment,
where the self—complementary sense and antisense regions ize to form an siRNA duplex having
hairpin secondary structure.
3. Modifying siRNA Sequences
] In certain aspects, siRNA molecules comprise a duplex having two strands and at least
one modified nucleotide in the double—stranded region, wherein each strand is about 15 to about 60
nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than a
corresponding fied siRNA sequence, but retains the capability of silencing the expression of a
target sequence. In preferred embodiments, the degree of chemical modifications introduced into the
siRNA molecule strikes a balance between reduction or abrogation of the immunostimulatory
ties of the siRNA and retention of RNAi activity. As a non-limiting example, an siRNA
molecule that targets a gene of interest can be minimally modified (e. g., less than about 30%, 25%,
%, 15%, 10%, or 5% modified) at ive uridine and/or guanosine nucleotides within the siRNA
duplex to eliminate the immune response generated by the siRNA while retaining its capability to
silence target gene expression.
Examples of modified nucleotides le for use in the invention e, but are not
limited to, ribonucleotides having a 2’~O-methyl ), 2’—deoxy~2’—fluoro (2’F), 2’—deoxy, 5—C~
methyl, 2—methoxyethyl) (MOE), 4’—thio, 2’~amino, or 2’-C-allyl group. Modified nucleotides
having a Northern conformation such as those described in, e. g., Saenger, Principles ofNucleic Acid
Structure, Springer—Verlag Ed. (1984), are also suitable for use in siRNA molecules. Such modified
nucleotides include, without tion, locked nucleic acid (LNA) nucleotides (e.g., 2’—O, 4’-C-
methylene—(D-ribofuranosyl) nucleotides), 2’-O-(2-methoxyethyl) (MOE) nucleotides, 2’-methyl-
thyl nucleotides, 2’—deoxy—2’-fluoro (2’F) nucleotides, 2’—deoxy—2’-chloro (2’Cl) nucleotides,
and 2’-azido nucleotides. In n instances, the siRNA molecules described herein include one or
more G-clamp nucleotides. A G-clamp nucleotide refers to a modified cytosine analog wherein the
modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a
complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. $00., 120:8531-
8532 (1998)). In on, nucleotides having a nucleotide base analog such as, for example, C—
phenyl, C—naphthyl, other aromatic tives, inosine, azole carboxamides, and nitroazole
W0 2013f052677
derivatives such as 3-nitropyrrole, 4-nitroindole, S—nitroindole, and 6-nitroindole (see, e.g., Loakes,
Nucl. Acids Res, 29:2437-2447 (2001)) can be incorporated into siRNA molecules.
In certain embodiments, siRNA molecules may further se one or more chemical
modifications such as terminal cap moieties, phosphate backbone modifications, and the like,
Examples of terminal cap es include, without limitation, inverted deoxy abasic residues,
glyceryl modifications, methylene tides, 1-(B—D-erythrofuranosyl) nucleotides, 4’-thio
nucleotides, carbocyclic tides, 1,5—anhydrohexitol nucleotides, L-nucleotides, (it—nucleotides,
modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3’,4’—seco tides, acyclic
3,4—dihydroxybutyl nucleotides, acyclic 3,5—dihydroxypentyl nucleotides, 3’-3’-inverted nucleotide
moieties, 3’—3’—inverted abasic moieties, 3’—2’-inverted nucleotide es, 3’—2’—inverted abasic
moieties, 5’—5’—inverted nucleotide moieties, inverted abasic moieties, 3’—5’—inverted deoxy
abasic moieties, 5’-amino—alkyl phosphate, 1,3—diaminopropyl phosphate, 3-aminopropyl
phosphate, 6—aminohexyl ate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4—
butanediol phosphate, 3’-phosphoramidate, 5’-phosphoramidate, hosphate, aminohexyl
phosphate, 3’—phosphate, 5’—amino, 3’-phosphorothioate, 5 ’-phosphorothioate, phosphorodithioate,
and bridging or non-bridging methylphosphonate or capto moieties (see, e.g., US. Patent No.
,998,203; Beaucage el al., Tetrahedron 4921925 (1993)). Non-limiting examples of ate
backbone modifications (i.e., resulting in modified internucleotide linkages) include
orothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate,
carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfama’te, formacetal,
thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nuctet'c Acid ues:
Synthesis and Properties, in Modern Synthetic Methods, VCH, 331—417 (1995); Mesmaeker et al.,
Novel Backbone ements for Oligonucleotides, in Carbohydrate Modifications in Antisense
Research, ACS, 24—39 (1994)). Such chemical modifications can occur at the 5’—end and/or 3’—end of
the sense strand, antisense strand, or both strands of the siRNA. The disclosures of these references
are herein incorporated by reference in their entirety for all purposes.
In some ments, the sense and/or antisense strand of the siRNA molecule can
further comprise a 3’-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2’—deoxy
cleotides, modified (e. g., 2’OMe) and/or unmodified e ribonucleotides, and/or any other
combination ofmodified (e.g., 2’OMe) and unmodified nucleotides.
Additional examples of modified nucleotides and types of chemical modifications that
can be introduced into siRNA les are described, e. g., in UK Patent No. GB 2,397,818 B and
US. Patent Publication Nos. 20040192626, 20050282188, and 35372, the disclosures of
which are herein incorporated by reference in their entirety for all purposes.
] The siRNA molecules described herein can optionally comprise one or more non-
nucleotides in one or both strands of the siRNA. As used herein, the term “non-nucleotide” refers to
any group or compound that can be incorporated into a nucleic acid chain in the place of one or more
nucleotide units, including sugar and/or phosphate tutions, and allows the remaining bases to
exhibit their activity. The group or compound is abasic in that it does not contain a commonly
recognized tide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore
lacks a base at the l’—position.
In other embodiments, chemical modification of the siRNA comprises attaching a
conjugate to the siRNA molecule. The ate can be attached at the 5’ and/or 3’—end of the sense
and/or antisense strand of the siRNA via a covalent attachment such as, e.g., a biodegradable linker.
The conjugate can also be attached to the siRNA, e.g., through a carbamate group or other linking
group (see, e.g., US. Patent Publication Nos. 20050074771, 20050043219, and 20050158727). 1n
celtain instances, the conjugate is a molecule that facilitates the delivery of the siRNA into a cell.
Examples of conjugate molecules suitable for attachment to siRNA include, without limitation,
steroids such as terol, glycols such as hylene glycol (PEG), human serum albumin
(HSA), fatty acids, carotenoids, terpenes, bile acids, s (e.g., folic acid, folate analogs and
derivatives thereof), sugars (e.g., galactose, galactosamine, N—acetyl galactosamine, glucose,
mannose, fructose, fucose, eta), phospholipids, peptides, ligands for cellular receptors capable of
mediating ar uptake, and combinations thereof (see, e. g., US. Patent Publication Nos.
30186, 20040110296, and 20040249178; US. Patent No. 6,753,423). Other examples include
the lipophilic moiety, n, polymer, peptide, protein, nucleic acid, small molecule,
oligosaccharide, carbohydrate cluster, alator, minor groove , ng agent, and cross-
linking agent ate les described in US. Patent Publication Nos. 20050119470 and
20050107325. Yet other examples include the 2’—O-alkyl amine, 2’—O-alkoxyalkyl amine, polyamine,
C5—cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic
amino acid conjugate molecules described in US. Patent Publication No. 20050153337. Additional
examples include the hydrophobic group, membrane active compound, cell penetrating compound,
cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in US.
Patent ation No. 20040167090. Further examples include the conjugate molecules described in
US. Patent Publication No. 20050239739. The type of conjugate used and the extent of ation
to the siRNA molecule can be evaluated for improved pharmacokinetic profiles, bioavailability,
and/or stability of the siRNA while retaining RNAi activity. As such, one d in the art can screen
siRNA molecules having various conjugates attached thereto to identify ones having improved
properties and filll RNAi activity using any of a variety of well-known in vitro cell culture or in vivo
animal models. The disclosures of the above—described patent documents are herein incorporated by
reference in their entirety for all purposes.
PCT/U52012/058770
4. Exemplary siRNA Embodiments
In some embodiments, each strand of the siRNA molecule comprises from about 15 to
about 60 nucleotides in length (e.g., about 15-60, 15—50, 15-40, 15~30, 15-25, or 19-25 nucleotides in
, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particular
embodiment, the siRNA is chemically synthesized. The siRNA molecules of the ion are
capable of silencing the expression of a target sequence in vitro and/or in Viva.
In other embodiments, the siRNA comprises at least one d nucleotide. In n
embodiments, the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten, or more
modified nucleotides in the double-stranded region. In particular ments, less than about 50%
(e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in
the double—stranded region of the siRNA comprise modified nucleotides. In preferred embodiments,
from about 1% to about 50% (e. g., from about 5%-50%, 10%~50%, 15%-50%, 20%—50%, 25%—50%,
%-50%, 35%—50%, 40%—50%, 45%~50%, 5%—45%, 10%—45%, 15%—45%, 20%-45%, 25%-45%,
%—45%, 35%—45%, 40%-45%, 5%-40%, 10%—40%, 15%—40%, 20%—40%, %, 30%-40%,
35%—40%, 5%—35%, 10%-35%, 15%—35%, 20%-35%, 25%—35%, 30%-35%, 5%—30%, 10%-30%,
%-30%, 20%—30%, 25%-30%, 5%-25%, lO%-25%, 15%-25%, 20%-25%, 5%—20%, 10%-20%,
%-20%, 5%-15%, lO%—15%, or 5%-lO%) of the nucleotides in the -stranded region of the
siRNA comprise modified nucleotides.
In further embodiments, the siRNA ses modified tides including, but not
limited to, 2’-O-methyl (2’OMe) nucleotides, 2’-deoxy-2’—fluoro (2’F) nucleotides, 2’-deoxy
tides, 2’-O-(2—methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and
mixtures thereof. In preferred embodiments, the siRNA comprises 2’OMe nucleotides (e.g., 2’OMe
purine and/or pyrimidine nucleotides) such as, e.g., 2’OMe-guanosine nucleotides, 2’OMe—uridine
nucleotides, 2’OMe—adenosine nucleotides, 2’OMe—cytosine tides, or mixtures thereof. In one
particular embodiment, the siRNA comprises at least one 2’OMe-guanosine nucleotide, 2’OMe-
e nucleotide, or es thereof. In n instances, the siRNA does not comprise 2’OMe—
cytosine tides. In other embodiments, the siRNA ses a hairpin loop structure.
In certain embodiments, the siRNA comprises modified nucleotides in one strand (i.e.,
sense or antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably,
uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region
of the siRNA duplex. With regard to uridine nucleotide modifications, at least one, two, three, four,
five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified
uridine nucleotide such as a Z’OMe-uridine nucleotide. In some embodiments, every e
nucleotide in the sense and/or antisense strand is a 2’OMe—uridine nucleotide. With regard to
PCT/U52012/058770
guanosine nucleotide modifications, at least one, two, three, four, five, six, or more of the guanosine
tides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a
2’OMe—guanosine nucleotide. In some embodiments, every guanosine nucleotide in the sense and/or
antisense strand is a 2’OMe-guanosine tide.
In certain embodiments, at least one, two, three, four, five, six, seven, or more 5’-GU—3’
motifs in an siRNA ce may be modified, e. g., by introducing mismatches to eliminate the 5’—
GU—3’ motifs and/or by introducing modified nucleotides such as 2’OMe nucleotides. The 5’—GU—3’
motif can be in the sense , the antisense strand, or both s of the siRNA sequence. The 5’-
GU—3’ motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, or more nucleotides.
In some embodiments, a modified siRNA molecule is less immunostimulatory than a
corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule
with reduced immunostimulatory properties advantageously s RNAi activity against the target
sequence. In another ment, the immunostimulatory properties of the modified siRNA
molecule and its ability to silence target gene expression can be balanced or optimized by the
introduction of minimal and selective 2’OMe modifications within the siRNA sequence such as, e.g.,
within the double-stranded region of the siRNA duplex. In certain instances, the modified siRNA is
at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less
immunostimulatory than the corresponding unmodified siRNA. It will be readily nt to those of
skill in the art that the immunostimulatory properties of the modified siRNA molecule and the
corresponding unmodified siRNA molecule can be determined by, for example, measuring INF-0L
and/or IL—6 levels from about two to about twelve hours after systemic administration in a mammal or
transfection of a mammalian responder cell using an riate lipid-based delivery system (such as
the SNALP delivery system disclosed herein).
In other embodiments, a modified siRNA molecule has an ICso (i.e., half-maximal
inhibitory concentration) less than or equal to ten-fold that of the corresponding unmodified siRNA
(i.e., the modified siRNA has an IC50 that is less than or equal to ten-times the IC50 of the
corresponding fied siRNA). In other embodiments, the modified siRNA has an IC50 less than
or equal to three-fold that of the corresponding unmodified siRNA sequence. In yet other
ments, the modified siRNA has an IC5O less than or equal to two-fold that of the corresponding
unmodified siRNA. It will be readily apparent to those of skill in the art that a dose—response curve
can be ted and the ICSO values for the modified siRNA and the corresponding unmodified
siRNA can be y determined using methods known to those of skill in the art.
In another embodiment, an unmodified or modified siRNA molecule is capable of
ing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the expression of the target sequence
ve to a negative control (e. g., buffer only, an siRNA sequence that targets a different gene, a
scrambled siRNA sequence, eta).
In yet another embodiment, a modified siRNA molecule is capable of silencing at least
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%,
77%, 78%, 79%, 80%, 8l%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sion of the target sequence relative to the
corresponding unmodified siRNA ce.
In some embodiments, the siRNA molecule does not comprise phosphate backbone
modifications, e. g., in the sense and/or antisense strand of the double-stranded region. In other
embodiments, the siRNA comprises one, two, three, four, or more phosphate backbone modifications,
e. g., in the sense and/or antisense strand of the double—stranded region. In preferred embodiments, the
siRNA does not comprise phosphate backbone ations.
In further embodiments, the siRNA does not comprise 2’-deoxy nucleotides, e.g., in the
sense and/or antisense strand of the double-stranded region. In yet further embodiments, the siRNA
comprises one, two, three, four, or more 2’—deoxy nucleotides, e.g., in the sense and/0r antisense
strand of the double—stranded region. In preferred ments, the siRNA does not comprise 2’-
deoxy nucleotides.
] In certain instances, the nucleotide at the 3’-end of the double—stranded region in the
sense and/0r antisense strand is not a modified nucleotide. In n other instances, the nucleotides
near the 3’—end (e.g., within one, two, three, or four nucleotides of the 3’—end) of the double—stranded
region in the sense and/or antisense strand are not modified nucleotides.
The siRNA molecules described herein may have 3’ overhangs of one, two, three, four,
or more nucleotides on one or both sides of the double-stranded , or may lack overhangs (i.e.,
have blunt ends) on one or both sides of the double—stranded region. In certain ments, the 3’
overhang on the sense and/or antisense strand independently comprises one, two, three, four, or more
modified nucleotides such as 2’OMe nucleotides and/or any other modified nucleotide described
herein or known in the art.
In particular embodiments, siRNAs targeting ALDH RNA or ALDH mRNA are
administered using a carrier system such as a nucleic ipid particle. In a preferred embodiment,
the nucleic acid—lipid particle comprises: (a) one or more siRNA molecules targeting ALDH; (b) a
cationic lipid (e.g., DLinDMA, DLenDMA, DLin-K—CZ-DMA, and/or y-DLenDMA); and (c) a non-
cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, the c acid—
lipid particle may fiarther comprise a ated lipid that prevents aggregation of particles (e.g.,
PEG-DAA and/or POZ—DAA).
] In addition to its utility in silencing the expression of any of the above—described ALDH
genes for therapeutic purposes, the siRNA described herein are also useful in ch and
development applications as well as diagnostic, prophylactic, prognostic, clinical, and other healthcare
ations. As a non—limiting example, the siRNA can be used in target validation studies directed
at testing whether a specific member of the ALDH gene family has the ial to be a therapeutic
target.
B. Dicer-Substrate dsRNA
] As used herein, the term=“Dicer—substrate dsRNA” or “precursor RNAi molecule” is
intended to include any precursor molecule that is processed in vivo by Dicer to produce an active
siRNA which is incorporated into the RISC complex for RNA interference of a target gene.
In one embodiment, the Dicer-substrate dsRNA has a length sufficient such that it is
processed by Dicer to e an siRNA. According to this embodiment, the Dicer-substrate dsRNA
comprises (i) a first oligonucleotide sequence (also termed the sense ) that is between about 25
and about 60 nucleotides in length (e.g., about 25—60, 25-55, 25—50, 25—45, 25—40, 25-35, or 25-30
nucleotides in length), preferably between about 25 and about 30 nucleotides in length (e.g., 25, 26,
27, 28, 29, or 30 nucleotides in ), and (ii) a second oligonucleotide sequence (also termed the
antisense ) that anneals to the first sequence under biological conditions, such as the conditions
found in the cytoplasm of a cell. The second oligonucleotide sequence may be between about 25 and
about 60 nucleotides in length (e.g., about 25-60, 25-55, 25—50, 25—45, 25-40, 25—35, or 25-30
nucleotides in length), and is preferably between about 25 and about 30 nucleotides in length (e. g., 25,
26, 27, 28, 29, or 30 nucleotides in length). In addition, a region of one of the sequences, ularly
of the antisense strand, of the Dicer—substrate dsRNA has a sequence length of at least about 19
nucleotides, for e, from about 19 to about 60 nucleotides (e.g., about 19—60, 19-55, 19-50, 19-
45, 19-40, 19—35, 19-30, or 19—25 nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,
19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementary to a nucleotide sequence of the
RNA produced from the target gene to trigger an RNAi response.
] In a second embodiment, the Dicer-substrate dsRNA has several properties which
enhance its processing by Dicer. According to this embodiment, the dsRNA has a length sufficient
such that it is processed by Dicer to produce an siRNA and has at least one of the ing
properties: (i) the dsRNA is asymmetric, e.g., has a 3’—overhang on the antisense strand; and/or (ii)
the dsRNA has a d 3’—end on the sense strand to direct orientation of Dicer binding and
processing of the dsRNA to an active siRNA. According to this latter embodiment, the sense strand
ses from about 22 to about 28 nucleotides and the antisense strand comprises from about 24 to
about 30 nucleotides.
In one embodiment, the Dicer-substrate dsRNA has an overhang on the 3’—end of the
antisense . In another ment, the sense strand is modified for Dicer binding and
sing by suitable modifiers d at the 3’-end of the sense strand. Suitable modifiers include
nucleotides such as deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered
molecules such as fluorescent molecules and the like. When nucleotide modifiers are used, they
replace ribonucleotides in the dsRNA such that the length of the dsRNA does not change. In another
embodiment, the Dicer-substrate dsRNA has an overhang on the 3’—end of the antisense strand and the
sense strand is modified for Dicer processing. In another embodiment, the 5’-end of the sense strand
has a phosphate. In another embodiment, the 5’—end of the antisense strand has a phosphate. In
another embodiment, the antisense strand or the sense strand or both strands have one or more 2’—O-
methyl (2’0Me) modified nucleotides. In r embodiment, the antisense strand contains 2’OMe
modified nucleotides. In another embodiment, the antisense stand contains a 3’-overhang that is
comprised of 2’0Me modified nucleotides. The antisense strand could also e additional 2’OMe
modified nucleotides. The sense and nse strands anneal under biological conditions, such as the
conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly
of the antisense strand, of the Dicer-substrate dsRNA has a sequence length of at least about 19
nucleotides, n these nucleotides are in the 21-nucleotide region adjacent to the 3’—end of the
antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced
from the target gene. r, in accordance with this ment, the Dicer-substrate dsRNA may
also have one or more of the following additional properties: (a) the antisense strand has a right shift
from the typical 21—mer (11a, the antisense strand includes nucleotides on the right side of the
molecule when compared to the typical 21—mer); (b) the strands may not be tely
complementary, i.e., the strands may contain simple ch pairings; and (0) base modifications
such as locked nucleic acid(s) may be included in the 5’—end of the sense strand.
In a third embodiment, the sense strand comprises from about 25 to about 28 nucleotides
(e.g., 25, 26, 27, or 28 nucleotides), wherein the 2 nucleotides on the 3’~end of the sense strand are
deoxyribonucleotides. The sense strand contains a phosphate at the 5’-end. The antisense strand
comprises from about 26 to about 30 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides) and contains
a 3’—overhang of 1—4 tides. The nucleotides comprising the 3’—overhang are modified with
W0 2013;052677 PCT/U52012/058770
2’OMe modified ribonucleotides. The antisense strand contains alternating 2’0Me modified
nucleotides ing at the first monomer of the antisense strand adjacent to the 3’—overhang, and
extending 15-19 nucleotides from the first monomer adjacent to the 3’—overhang. For e, for a
27~nucleotide nse strand and counting the first base at the 5’-end of the nse strand as
position number 1, 2’OMe modifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23, 25,
26, and 27. In one embodiment, the Dicer—substrate dsRNA has the following structure:
’-pXXXXXXXXXXXXXXXXXXXXXXXDD-3’ (SEQ ID NO:1)
3’-YXXXXXXXXXXXXXXXXXXXXXXXXXp—5' (SEQ ID NO:2)
wherein “X” = RNA, “p” = a phosphate group, “2g” = 2’OMe RNA, “Y” is an overhang domain
comprised of l, 2, 3, or 4 RNA monomers that are optionally 2’OMe RNA monomers, and “D” =
DNA. The top strand is the sense strand, and the bottom strand is the antisense strand.
In a fourth embodiment, the Dicer—substrate dsRNA has l properties which
enhance its processing by Dicer. According to this ment, the dsRNA has a length sufficient
such that it is processed by Dicer to produce an siRNA and at least one of the following properties:
(i) the dsRNA is asymmetric, e. g., has a 3’~overhang on the sense ; and (ii) the dsRNA has a
modified 3’-end on the nse strand to direct orientation of Dicer binding and processing of the
dsRNA to an active siRNA. According to this embodiment, the sense strand comprises from about 24
to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30 nucleotides) and the antisense strand
comprises from about 22 to about 28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In
one embodiment, the Dicer—substrate dsRNA has an ng on the 3’—end of the sense strand. In
another embodiment, the antisense strand is modified for Dicer binding and processing by suitable
modifiers located at the 3’—end of the antisense strand. Suitable modifiers include nucleotides such as
deoxyribonucleotides, acyclonucleotides, and the like, and sterically hindered molecules such as
fluorescent molecules and the like. When nucleotide modifiers are used, they replace ribonucleotides
in the dsRNA such that the length of the dsRNA does not change. In another ment, the
dsRNA has an overhang on the 3’-end of the sense strand and the antisense strand is modified for
Dicer processing. In one embodiment, the antisense strand has a 5’-phosphate. The sense and
nse strands anneal under biological conditions, such as the conditions found in the asm of
a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the dsRNA
has a sequence length of at least 19 nucleotides, wherein these nucleotides are adjacent to the 3’—end
of antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA produced
from the target gene. Further, in accordance with this embodiment, the Dicer-substrate dsRNA may
also have one or more of the ing additional properties: (a) the nse strand has a left shift
from the typical 21—mer (i.e., the antisense strand includes nucleotides on the left side of the molecule
PCT/U82012/058770
when compared to the typical 21-mer); and (b) the strands may not be completely complementary,
i.e., the strands may contain simple mismatch pairings.
In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetric structure, with
the sense strand having a 25-base pair length, and the antisense strand having a 27-base pair length
with a 2 base 3’-overhang. In certain instances, this dsRNA having an asymmetric structure further
contains 2 deoxynucleotides at the 3’-end of the sense strand in place of two of the ribonucleotides.
In certain other instances, this dsRNA having an asymmetric structure further ns 2’OMe
modifications at positions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand (wherein the
first base at the 5’-end of the nse strand is position 1). In certain additional instances, this
dsRNA having an asymmetric structure further contains a 3’-overhang on the antisense strand
comprising 1, 2, 3, or 4 2’OMe nucleotides (e.g., a rhang of 2’OMe nucleotides at positions 26
and 27 on the antisense strand).
In another embodiment, Dicer—substrate dsRNAs may be designed by first selecting an
antisense strand siRNA ce having a length of at least 19 nucleotides. In some instances, the
antisense siRNA is modified to include about 5 to about 11 ribonucleotides on the 5’—end to provide a
length of about 24 to about 30 nucleotides. When the nse strand has a length of 21 nucleotides,
3-9, preferably 4-7, or more ably 6 nucleotides may be added on the 5’-end. gh the
added ribonucleotides may be complementary to the target gene sequence, full mentarity
between the target sequence and the antisense siRNA is not required. That is, the resultant antisense
siRNA is sufficiently complementary with the target sequence. A sense strand is then produced that
has about 22 to about 28 nucleotides. The sense strand is substantially mentary with the
antisense strand to anneal to the antisense strand under biological conditions. In one embodiment, the
sense strand is synthesized to contain a modified 3’-end to direct Dicer processing of the antisense
strand. In another embodiment, the antisense strand of the dsRNA has a 3’-overhang. In a further
embodiment, the sense strand is synthesized to contain a modified 3’—end for Dicer binding and
processing and the antisense strand of the dsRNA has a 3’-overhang.
In a related embodiment, the antisense siRNA may be d to e about 1 to
about 9 ribonucleotides on the 5’—end to provide a length of about 22 to about 28 nucleotides. When
the antisense strand has a length of 21 nucleotides, 1-7, preferably 2-5, or more preferably 4
ribonucleotides may be added on the 3’-end. The added ribonucleotides may have any sequence.
Although the added ribonucleotides may be complementary to the target gene sequence, full
complementarity between the target ce and the antisense siRNA is not required. That is, the
resultant antisense siRNA is ently complementary with the target sequence. A sense strand is
then produced that has about 24 to about 30 nucleotides. The sense strand is substantially
mentary with the antisense strand to anneal to the antisense strand under biological conditions.
W0 2013f052677 PCT/U52012/058770
In one embodiment, the antisense strand is synthesized to contain a modified 3’-end to direct Dicer
processing. In another ment, the sense strand of the dsRNA has a 3’—overhang. In a further
embodiment, the antisense strand is synthesized to contain a modified 3’—end for Dicer binding and
processing and the sense strand of the dsRNA has a 3’~overhang.
Suitable Dicer—substrate dsRNA sequences can be identified, synthesized, and modified
using any means known in the art for designing, synthesizing, and modifying siRNA sequences. In
certain embodiments, Dicer—substrate dsRNAs of the invention may silence ALDH gene expression.
In particular embodiments, Dicer-substrate dsRNAs targeting ALDH mRNA are administered using a
r system such as a nucleic acid—lipid particle. In a red embodiment, the nucleic ipid
particle comprises: (a) one or more Dicer-substrate dsRNA molecules targeting ALDH gene
expression; (b) a cationic lipid (6g, DLinDMA, DLenDMA, DLin—K—C2-DMA, and/0r y~
DLenDMA); and (c) a non-cationic lipid (e.g, DPPC, DSPC, DSPE, and/0r cholesterol). In n
instances, the nucleic acid-lipid particle may further comprise a conjugated lipid that prevents
aggregation of particles (e.g., PEG-DAA and/or POZ—DAA).
[00174] onal embodiments related to the Dicer—substrate dsRNAs of the invention, as well
as methods of ing and synthesizing such dsRNAs, are described in US. Patent Publication Nos.
20050244858, 20050277610, and 20070265220, 2011/0071208, the sures of which are herein
incorporated by reference in their entirety for all purposes.
C. shRNA
[00175] A “small hairpin RNA” or “short n RNA” or “shRNA” includes a short RNA
ce that makes a tight hairpin turn that can be used to silence gene expression via RNA
interference. The shRNAs of the invention may be chemically sized or transcribed from a
transcriptional cassette in a DNA plasmid. The shRNA hairpin structure is d by the ar
machinery into siRNA, which is then bound to the duced silencing complex (RISC).
[00176] The shRNAs of the invention are typically about 15-60, 15-50, or 15-40 (duplex)
nucleotides in length, more lly about 15-30, 15—25, or 19-25 (duplex) nucleotides in length, and
are preferably about 20-24, 21-22, or 21—23 (duplex) nucleotides in length (e.g., each complementary
sequence of the double—stranded shRNA is 15-60, 15-50, 15—40, 15—30, 15-25, or 19—25 nucleotides in
length, preferably about 20—24, 21—22, or 21-23 nucleotides in length, and the double-stranded shRNA
is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19—25 base pairs in length, ably about 18-22, 19-
, or 19-21 base pairs in length). shRNA duplexes may comprise 3’ overhangs of about 1 to about 4
nucleotides or about 2 to about 3 nucleotides on the antisense strand and/or 5’-phosphate termini on
the sense strand. In some embodiments, the shRNA comprises a sense strand and/or antisense strand
ce of from about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15—50, 15-45,
—40, 15-35, 15-30, or 15—25 tides in length), preferably from about 19 to about 40 nucleotides
in length (e.g., about 19-40, 19-35, l9-30, or 19-25 nucleotides in length), more preferably from about
19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23 nucleotides in length).
Non—limiting examples of shRNA include a double-stranded cleotide molecule
assembled from a single—stranded le, where the sense and antisense regions are linked by a
nucleic acid—based or non—nucleic acid—based linker; and a double-stranded polynucleotide molecule
with a hairpin secondary ure having self—complementary sense and antisense regions. In
preferred embodiments, the sense and antisense strands of the shRNA are linked by a loop structure
comprising from about 1 to about 25 nucleotides, from about 2 to about 20 nucleotides, from about 4
to about 15 nucleotides, from about 5 to about 12 nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.
Suitable shRNA sequences can be identified, synthesized, and modified using any means
known in the art for designing, synthesizing, and modifying siRNA sequences. In certain
embodiments, shRNAs of the invention may silence ALDH gene expression. In particular
embodiments, shRNAs ing ALDH mRNA are stered using a carrier system such as a
nucleic acid-lipid particle. In a preferred embodiment, the nucleic acid-lipid particle comprises: (a)
one or more shRNA les targeting ALDH gene expression; (b) a cationic lipid (e. g., DLinDMA,
A, DLin—K—C2-DMA, and/or y—DLenDMA); and (c) a non—cationic lipid (e.g., DPPC,
DSPC, DSPE, and/or cholesterol). In certain instances, the nucleic acid-lipid particle may further
comprise a conjugated lipid that prevents ation of particles (e.g., PEG-DAA and/or POZ—
DAA).
Additional embodiments related to the shRNAs of the invention, as well as methods of
designing and synthesizing such shRNAs, are described in US. patent application ation number
2011/0071208, the disclosure of which is herein incorporated by reference in its entirety for all
purposes.
D. aiRNA
Like siRNA, asymmetrical interfering RNA ) can recruit the RNA—induced
silencing complex (RISC) and lead to effective silencing of a variety of genes in mammalian cells by
mediating ce-specific cleavage of the target sequence between nucleotide 10 and 11 ve to
the 5’ end of the antisense strand (Sun et al., Nat. Biotech, 26:1379-1382 (2008)). Typically, an
aiRNA molecule comprises a short RNA duplex having a sense strand and an nse strand,
wherein the duplex contains overhangs at the 3’ and 5’ ends of the antisense strand. The aiRNA is
PCT/U82012/058770
generally asymmetric because the sense strand is shorter on both ends when ed to the
complementary antisense strand. In some aspects, aiRNA molecules may be designed, sized,
and annealed under conditions similar to those used for siRNA molecules. As a miting
example, aiRNA sequences may be selected and generated using the methods described above for
ing siRNA sequences.
In another embodiment, aiRNA duplexes of various s (e.g., about 10—25, 12—20,
12-19, 12—18, 13-17, or 14—17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 base
pairs) may be designed with ngs at the 3’ and 5’ ends of the nse strand to target an
mRNA of interest. In certain instances, the sense strand of the aiRNA molecule is about 10—25, 12—
20, 12-19, 12—18, 13—17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19,
or 20 nucleotides in length. In certain other instances, the nse strand of the aiRNA molecule is
about 15—60, 15—50, or 15-40 nucleotides in length, more typically about 15—30, 15-25, or 19—25
nucleotides in length, and is preferably about 20—24, 21-22, or 21-23 nucleotides in length.
] In some embodiments, the 5’ antisense overhang contains one, two, three, four, or more
geting nucleotides (e.g., “AA”, “UU”, “‘deT”, etc). In other embodiments, the 3’ antisense
overhang contains one, two, three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “deT”,
etc) In certain s, the aiRNA molecules described herein may comprise one or more modified
nucleotides, e.g., in the double-stranded (duplex) region and/or in the antisense overhangs. As a non-
limiting example, aiRNA sequences may comprise one or more of the modified nucleotides described
above for siRNA sequences. In a preferred embodiment, the aiRNA molecule comprises 2’OMe
nucleotides such as, for example, 2’OMe-guanosine nucleotides, 2’OMe—uridine nucleotides, or
mixtures thereof.
] In certain embodiments, aiRNA molecules may comprise an nse strand which
corresponds to the antisense strand of an siRNA molecule, e.g., one of the siRNA molecules bed
herein. In certain embodiments, aiRNAs of the invention may silence ALDH gene expression. In
particular embodiments, aiRNAs targeting ALDH mRNA are administered using a carrier system
such as a nucleic acid—lipid particle. In a preferred embodiment, the nucleic acid-lipid le
comprises: (a) one or more aiRNA molecules targeting ALDH gene expression; (b) a cationic lipid
(e.g., DLinDMA, DLenDMA, DLin-K-CZ-DMA, and/or y—DLenDMA); and (c) a non-cationic lipid
(e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, the nucleic acid-lipid particle
may further comprise a conjugated lipid that prevents aggregation of particles (e.g., PEG-DAA and/or
POZ-DAA).
Suitable aiRNA sequences can be identified, synthesized, and d using any means
known in the art for designing, synthesizing, and modifying siRNA sequences. Additional
2012/058770
embodiments related to the aiRNA molecules of the invention are described in US. Patent
Application No. 12/343,342, filed December 23, 2008, and US. Patent Application No. 12/424,367,
filed April 15, 2009, the disclosures of which are herein incorporated by reference in their entirety for
all purposes.
E. miRNA
Generally, microRNAs (miRNA) are single-stranded RNA molecules of about 21-23
nucleotides in length which regulate gene expression. miRNAs are encoded by genes from whose
DNA they are transcribed, but miRNAs are not ated into protein (non—coding RNA); instead,
each primary transcript (a pri-miRNA) is processed into a short stem—loop ure called a pre—
miRNA and finally into a functional mature miRNA. Mature miRNA molecules are either partially or
completely complementary to one or more messenger RNA (mRNA) les, and their main
fimction is to downregulate gene expression. The identification of miRNA molecules is described,
e. g., in Lagos-Quintana et al., Science, 3-858; Lau er (1]., Science, 2942858862; and Lee et al.,
Science, 294:862-864.
[00186] The genes encoding miRNA are much longer than the processed mature miRNA
le. miRNA are first transcribed as primary transcripts or pri-miRNA with a cap and poly—A
tail and processed to short, ~70-nucleotide oop structures known as pre-miRNA in the cell
nucleus. This sing is performed in animals by a protein complex known as the Microprocessor
complex, consisting of the nuclease Drosha and the —stranded RNA g protein Pasha
(Denli et al, Nature, 432:231—235 (2004)). These pre—miRNA are then processed to mature miRNA
in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the
RNA—induced silencing complex (RISC) (Bernstein el al., Nature, 409:363-366 (2001). Either the
sense strand or antisense strand ofDNA can function as templates to give rise to miRNA.
When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA
les are formed, but only one is integrated into the RISC complex. This strand is known as the
guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC
complex, on the basis of the stability of the 5’ end (Preall er (2]., Curr. Biol, 16:530-535 (2006)). The
remaining strand, known as the anti-guide or passenger , is degraded as a RISC complex
substrate (Gregory et (11., Cell, 1-640 (2005)). After integration into the active RISC complex,
miRNAs base pair with their complementary mRNA molecules and induce target mRNA degradation
and/or translational silencing.
] Mammalian miRNA molecules are usually complementary to a site in the 3’ UTR of the
target mRNA sequence. In certain instances, the annealing of the miRNA to the target mRNA
PCT/U82012/058770
inhibits protein ation by blocking the n translation machinery. In certain other instances,
the annealing of the miRNA to the target mRNA facilitates the ge and degradation of the target
mRNA through a process similar to RNA erence (RNAi). miRNA may also target methylation
of genomic sites which correspond to targeted mRNA. lly, miRNA function in ation
with a complement of proteins collectively termed the miRNP.
In certain s, the miRNA molecules bed herein are about 15—100, 15-90, 15—
80, 15-75, 15—70, 15—60, 15-50, or 15—40 nucleotides in length, more typically about 15—30, 15—25, or
19-25 nucleotides in length, and are preferably about 20-24, 21—22, or 21—23 nucleotides in length. In
certain other aspects, miRNA molecules may comprise one or more modified nucleotides. As a non-
limiting example, miRNA sequences may comprise one or more of the modified nucleotides
described above for siRNA sequences. In a preferred embodiment, the miRNA molecule comprises
2’OMe tides such as, for example, 2’OMe—guanosine nucleotides, 2’OMe-uridine nucleotides,
or mixtures thereof.
In some embodiments, miRNA molecules may be used to silence ALDH gene
expression. In particular embodiments, miRNAs targeting ALDH mRNA are administered using a
carrier system such as a nucleic acid—lipid particle. In a preferred embodiment, the nucleic acid-lipid
particle comprises: (a) one or more miRNA molecules targeting ALDH gene expression; (b) a
cationic lipid (e.g., DLinDMA, DLenDMA, DLin-K-CZ-DMA, and/or y-DLenDMA); and (c) a non-
cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol). In certain ces, the nucleic acid-
lipid particle may further comprise a conjugated lipid that prevents aggregation of particles (e.g.,
A and/or POZ—DAA).
In other embodiments, one or more agents that block the activity of an miRNA targeting
ALDH mRNA are administered using a lipid particle of the invention (e. g., a c acid-lipid
particle). es of blocking agents include, but are not limited to, steric blocking
oligonucleotides, locked nucleic acid oligonucleotides, and Morpholino oligonucleotides. Such
ng agents may bind directly to the miRNA or to the miRNA binding site on the target RNA.
V. Carrier Systems Containing Therapeutic Nucleic Acids
In one aspect, the present invention provides carrier systems containing one or more
therapeutic nucleic acids (e. g., interfering RNA such as dsRNA). In some embodiments, the carrier
system is a lipid-based carrier system such as a lipid particle (e.g., SNALP), a cationic lipid or
liposome nucleic acid complex (i.e., lipoplex), a liposome, a micelle, a virosome, or a e thereof.
In other embodiments, the carrier system is a polymer—based carrier system such as a ic
polymer—nucleic acid complex (i.e., polyplex). In additional embodiments, the carrier system is a
WO 2013052677 PCT/U82012/058770
cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex. In r
ments, the carrier system is a protein—based carrier system such as a cationic peptide—nucleic
acid complex. Preferably, the carrier system is a lipid particle such as a SNALP. One skilled in the
art will appreciate that the therapeutic nucleic acids of the present ion can also be red as a
naked molecule.
A. Lipid Particles
In n aspects, the present invention es lipid particles comprising one or more
therapeutic nucleic acids (e.g., interfering RNA such as dsRNA) and one or more of cationic (amino)
lipids or salts thereof. In some embodiments, the lipid particles of the invention further comprise one
or more non-cationic lipids. In other ments, the lipid particles further comprise one or more
conjugated lipids capable of reducing or inhibiting particle aggregation.
The lipid particles of the ion preferably comprise a therapeutic nucleic acid such as
an interfering RNA (e.g., siRNA), a cationic lipid, a non-cationic lipid, and a conjugated lipid that
ts aggregation of particles. In some embodiments, the therapeutic nucleic acid is fully
encapsulated within the lipid portion of the lipid particle such that the therapeutic nucleic acid in the
lipid particle is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid
particles described herein are substantially non-toxic to mammals such as . The lipid particles
of the invention typically have a mean diameter of from about 30 nm to about 150 nm, from about 40
nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from
about 70 nm to about 110 nm, or from about 70 to about 90 nm. The lipid particles of the invention
also typically have a lipidztherapeutic agent (e.g., lipidznucleic acid) ratio (mass/mass ratio) of from
about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1, from about 3:1
to about 20:1, from about 5:1 to about 15:], or from about 5:1 to about 10:1.
In preferred embodiments, the lipid particles of the ion are serum-stable nucleic
acid-lipid particles (SNALP) which comprise an interfering RNA (e.g., dsRNA such as siRNA, Dicer—
substrate dsRNA, shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or more cationic lipids
of Formula I—III or salts thereof as set forth herein), a non—cationic lipid (e.g., mixtures of one or more
phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles (e.g,
one or more PEG-lipid conjugates). The SNALP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more unmodified and/or modified interfering RNA (e.g., siRNA) that target one or more of the genes
described . Nucleic acid-lipid les and their method of preparation are described in, e. g.,
US. Patent Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017;
and PCT ation No. WO 96/40964, the disclosures of which are each herein incorporated by
reference in their entirety for all purposes.
In the nucleic ipid particles of the invention, the nucleic acid may be fiilly
encapsulated within the lipid portion of the particle, thereby ting the nucleic acid from nuclease
degradation. In preferred embodiments, a SNALP comprising a nucleic acid such as an interfering
RNA is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid
from se degradation. In certain instances, the nucleic acid in the SNALP is not substantially
ed after exposure of the particle to a nuclease at 37°C for at least about 20, 30, 45, or 60
minutes. In certain other instances, the c acid in the SNALP is not substantially degraded after
incubation of the particle in serum at 37°C for at least about 30, 45 or 60 minutes or at least about 2,
3, 4, 5, 6, 7, 8, 9, 10, 12, l4, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments,
the nucleic acid is complexed with the lipid portion of the particle. One of the benefits of the
ations of the present invention is that the nucleic acid-lipid particle compositions are
substantially non-toxic to mammals such as humans.
The term “fully encapsulated” indicates that the nucleic acid in the nucleic acid-lipid
particle is not significantly degraded after exposure to serum or a se assay that would
significantly degrade free DNA or RNA. In a fully encapsulated system, preferably less than about
% of the c acid in the particle is degraded in a treatment that would normally degrade 100%
of free nucleic acid, more preferably less than about 10%, and most preferably less than about 5% of
the nucleic acid in the particle is degraded. “Fully encapsulated” also indicates that the nucleic acid-
lipid particles are stable, that is, that they do not y decompose into their component parts
upon in vivo administration.
In the context of nucleic acids, full encapsulation may be determined by performing a
membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced
fluorescence when associated with nucleic acid. Specific dyes such as OliGreen® and RiboGreen®
(Invitrogen Corp; Carlsbad, CA) are available for the quantitative determination of plasmid DNA,
single-stranded deoxyribonucleotides, and/or single- or double-stranded ribonucleotides.
Encapsulation is ined by adding the dye to a liposomal formulation, measuring the resulting
fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of
nonionic detergent. Detergent—mediated disruption of the mal bilayer releases the encapsulated
nucleic acid, allowing it to interact with the membrane—impermeable dye. Nucleic acid encapsulation
may be calculated as E = (I, — 0/10, where I and I, refer to the fluorescence intensities before and after
the on of detergent (see, Wheeler et al., Gene Then, 6:271-281 (1999)).
] In other embodiments, the t invention provides a nucleic acid-lipid particle (e. g.,
SNALP) composition comprising a plurality of nucleic acid—lipid particles.
WO 52677
In some instances, the SNALP composition ses nucleic acid that is fiilly
encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from
about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from
about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from
about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about
60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to
about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about
90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%,
from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof
or range therein) of the particles have the c acid encapsulated therein.
In other instances, the SNALP composition comprises nucleic acid that is fully
ulated within the lipid portion of the particles, such that from about 30% to about 100%, from
about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from
about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from
about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about
60% to about 95%, from about 70% to about 95%, from about 80% to about 95%, from about 85% to
about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about
90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%,
from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof
or range therein) of the input nucleic acid is encapsulated in the particles.
Depending on the intended use of the lipid particles of the invention, the proportions of
the components can be varied and the delivery efficiency of a particular formulation can be ed
using, e.g., an endosomal release parameter (ERP) assay.
1. Cationic Lipids
Any of a variety of cationic lipids or salts thereofmay be used in the lipid particles of the
present invention (e.g., SNALP), either alone or in combination with one or more other ic lipid
species or non—cationic lipid species. The cationic lipids include the (R) and/or (S) enantiomers
thereof.
] In one aspect, cationic lipids of Formula 1 having the following structure are useful in the
present invention:
PCT/U82012/058770
R1 R3
N—— OH( QWAO/R4
\R5 (I),
[00205] or salts thereof, wherein:
R1 and R2 are either the same or different and are independently hydrogen (H) or an
optionally substituted C1-C6 alkyl, C2—C6 alkenyl, or C2-C6 alkynyl, or R1 and R2 may join to form an
optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from
the group consisting of en (N), oxygen (0), and es thereof;
IO ] R3 is either absent or is en (H) or a C1—C6 alkyl to provide a quaternary amine;
R4 and R5 are either the same or different and are independently an ally substituted
C10-C24 alkyl, Clo-C24 alkenyl, Clo-C24 alkynyl, or Clo-C24 acyl, wherein at least one of R4 and R5
comprises at least two sites of unsaturation; and
n is 0, 1, 2, 3, or 4.
[00210] In some embodiments, R1 and R2 are independently an optionally substituted C1-C4 alkyl,
C2—C4 l, or C2-C4 alkynyl. In one preferred embodiment, R1 and R2 are both methyl . In
other preferred embodiments, n is l or 2. In other embodiments, R3 is absent when the pH is above
the pKa of the cationic lipid and R3 is hydrogen when the pH is below the pKa of the cationic lipid
such that the amino head group is protonated. In an alternative embodiment, R3 is an optionally
substituted C1—C4 alkyl to provide a quaternary amine. In further embodiments, R4 and R5 are
independently an optionally substituted Clg—Cgo or C14—C22 alkyl, C12~C20 or C14-C22 alkenyl, o or
C14-C22 alkynyl, or o or (314-ng acyl, wherein at least one of R4 and R5 comprises at least two
sites of unsaturation.
In certain embodiments, R4 and R5 are independently selected from the group consisting
of a dodecadienyl moiety, a tetradecadienyl moiety, a cadienyl moiety, an octadecadienyl
moiety, an icosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienyl
moiety, an catrienyl moiety, an icosatrienyl moiety, an arachidonyl moiety, and a
docosahexaenoyl moiety, as well as acyl derivatives thereof (e.g., linoleoyl, linolenoyl, y-linolenoyl,
etc). In some instances, one of R4 and R5 comprises a branched alkyl group (e.g., a yl moiety)
or an acyl derivative thereof (e.g., a phytanoyl moiety). In certain instances, the octadecadienyl
moiety is a linoleyl moiety. In certain other instances, the octadecatrienyl moiety is a linolenyl
moiety or a y—linolenyl moiety. In certain embodiments, R4 and R5 are both linoleyl moieties,
PCT/U82012/058770
nyl moieties, or y—linolenyl moieties. In particular embodiments, the cationic lipid of Formula I
is 1 ,2-dilin0leyloxy-N,N—dimethylaminopropane MA), l ,2—dilinolenyloxy—N,N—
dimethylaminopropane (DLenDMA), 1,2-dilinoleyloxy—(N,N—dimethyl)-butyl—4-amine (C2-
DLinDMA), 1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl—4-amine (CZ-DLinDAP), or mixtures f.
In some embodiments, the cationic lipid of Formula I forms a salt (preferably a
crystalline salt) with one or more anions. In one particular embodiment, the cationic lipid of Formula
I is the oxalate (e.g., hemioxalate) salt f, which is ably a crystalline salt.
The synthesis of ic lipids such as DLinDMA and DLenDMA, as well as additional
cationic lipids, is described in US. Patent ation No. 20060083780, the disclosure of which is
herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such
as C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids, is described in international
patent ation numberW020l1/000106 the disclosure of which is herein orated by
reference in its entirety for all purposes.
In r aspect, cationic lipids of Formula II having the following structure (or salts
thereof) are useful in the present invention:
R': R5“
\l/i *iCL] \ ’ 2:q
(11),
wherein R1 and R2 are either the same or different and are ndently an optionally
tuted C12"C24 alkyl, Clz—C24 alkenyl, C12—C24 alkynyl, or Cn-Cgr; acyl; R3 and R4 are either the
same or different and are independently an optionally substituted C1-C5 alkyl, C2-C5 alkenyl, or C2—C6
alkynyl, or R3 and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon
atoms and l or 2 atoms chosen from nitrogen and oxygen; R5 is either absent or is hydrogen (H)
or a C1-C6 alkyl to provide a quaternary amine; m, n, and p are either the same or different and are
independently either 0, l, or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, l, 2,
3, or 4; and Y and Z are either the same or different and are independently O, S, or NH. In a preferred
embodiment, q is 2.
In some embodiments, the cationic lipid of Formula II is 2,2-dilinoleyl(2-
dimethylaminoethyl)-[l,3]-dioxolane (DLin-K—CZ—DMA; “XTC2” or “C2K”), 2,2-dilinoleyl(3-
dimethylaminopropyl)-[l ,3 ] -dioxolane (DLin—K-C3-DMA; “C3K”), 2,2-dilinoleyl-4—(4—
dimethylaminobutyl)— [l ,3]-di0xolane (DLin—K—C4-DMA; “C4K”), 2,2-dilinoley1—5-
dimethylaminomethyl—[l ,3]-dioxane (DLin-K6—DMA), 2,2~dilinoleyl—4—N—methylpepiazino—[l ,3]-
W0 2013f052677 PCT/U82012/058770
dioxolane (DLin—K—MPZ), linoleyl—4-dimethylaminomethyl—[l,3]—dioxolane (DLin—K-DMA),
oleoyldimethylaminomethyl— [l ,3]-dioxolane (DO—K-DMA), 2,2-distearoyl
dimethylaminomethyl-[l oxolane (DS—K-DMA), 2,2-dilinoleyl—4-N—morpholino-[1,3]—dioxolane
(DLin-K-MA), 2,2-Dilinoleyltrimethylamino-[l,3]-dioxolane chloride (DLin-K-TMACI), 2,2-
dilinoleyl—4,5-bis(dimethylaminomethyl)— [l oxolane (DLin-Kz-DMA), 2,2—dilinoleyl—4—
methylpiperzine-[l,3]-dioxolane (D—Lin—K-N—methylpiperzine), or mixtures thereof. In preferred
ments, the cationic lipid of Formula II is DLin—K-CZ-DMA.
In some embodiments, the cationic lipid of Formula II forms a salt (preferably a
crystalline salt) with one or more anions. In one particular ment, the cationic lipid of Formula
II is the oxalate (e.g., alate) salt f, which is preferably a crystalline salt.
The synthesis of cationic lipids such as DLin—K-DMA, as well as additional cationic
lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein
incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as
DLin—K—CZ—DMA, DLin-K-C3—DMA, DLin—K—C4—DMA, DLin-K6—DMA, DLin—K-MPZ, DO-K—
DMA, DS—K~DMA, DLin—K-MA, -TMA.C1, z-DMA, and D—Lin-K—N-
methylpiperzine, as well as additional cationic lipids, is described in PCT Application No.
PCT/USZOO9/060251, entitled “Improved Amino Lipids and Methods for the Delivery of Nucleic
Acids,” filed October 9, 2009, the disclosure of which is incorporated herein by reference in its
entirety for all purposes.
[00219] In a r aspect, cationic lipids of Formula III having the following structure are
useful in the present invention:
R1 R3
N—(CH2)n
/ YO '— — “" R4
O _ ____ __ R5
(III)
or salts thereof, wherein: R1 and R2 are either the same or different and are
ndently an optionally substituted C1-C6 alkyl, C2—C5 alkenyl, or C2—C6 alkynyl, or R1 and R2
may join to form an optionally tuted heterocyclic ring of 4 to 6 carbon atoms and l or 2
heteroatoms selected from the group consisting of nitrogen (N), oxygen (0), and mixtures thereof; R3
is either absent or is hydrogen (H) or a C1—C5 alkyl to provide a quaternary amine; R4 and R5 are either
absent or present and when present are either the same or different and are independently an
optionally substituted C1—C10 alkyl or C2-C10 alkenyl; and n is O, 1, 2, 3, or 4.
In some embodiments, R1 and R2 are independently an optionally substituted C1-C4 alkyl,
C2-C4 alkenyl, or C2-C4 alkynyl. In a preferred embodiment, R1 and R2 are both methyl groups. In
another preferred embodiment, R4 and Rs are both butyl groups. In yet another preferred
embodiment, n is 1. In other embodiments, R3 is absent when the pH is above the pKa of the cationic
lipid and R3 is hydrogen when the pH is below the pK, of the cationic lipid such that the amino head
group is protonated. In an alternative embodiment, R3 is an optionally substituted C1-C4 alkyl to
provide a quaternary amine. In further ments, R4 and R5 are independently an optionally
substituted C2—C6 or C2—C4 alkyl or C2—C6 or C2-C4 alkenyl.
] In an alternative embodiment, the cationic lipid of Formula 111 comprises ester linkages
n the amino head group and one or both of the alkyl chains. In some embodiments, the
cationic lipid of Formula III forms a salt (preferably a crystalline salt) with one or more anions. In
one particular embodiment, the cationic lipid of Formula III is the oxalate (e.g., hemioxalate) salt
thereof, which is preferably a crystalline salt.
Although each of the alkyl chains in Formula III ns cis double bonds at ons 6,
9, and 12 (116., CiS,CiS,CiS-A6,A9,A12), in an alternative embodiment, one, two, or three of these double
bonds in one or both alkyl chains may be in the trans configuration.
In a ularly preferred embodiment, the cationic lipid of Formula III has the
Structure:
\TAp
7~DLenDMA
The sis of cationic lipids such as y-DLenDMA, as well as additional cationic
lipids, is described in US. Provisional Application No. 61/222,462, entitled “Improved Cationic
Lipids and s for the Delivery of c Acids,” filed July 1, 2009, the disclosure of which is
herein incorporated by reference in its entirety for all purposes.
In particular ments, a cationic lipid having the following structure is useful in the
present invention:
0 _ _
l o _ _
DLin-M~C3—DMA (“MC3”)
W0 2013/052677
The synthesis of cationic lipids such as DLin-M-C3-DMA (“MC3”), as well as
additional cationic lipids (e.g., certain analogs of MC3), is described in US. Provisional Application
No. ,800, entitled “Novel Lipids and Compositions for the Delivery of Therapeutics,” filed
June 10, 2009, and US. Provisional Application No. 61/287,995, entitled “Methods and Compositions
for Delivery of Nucleic Acids,” filed December 18, 2009, the disclosures of which are herein
incorporated by reference in their entirety for all purposes.
Examples of other cationic lipids or salts thereof which may be included in the lipid
particles of the present invention include, but are not limited to, cationic lipids such as those described
in W02011/000106, the sure of which is herein incorporated by reference in its entirety for all
purposes, as well as cationic lipids such as N,N—dioleyl-N,N—dimethylammonium de (DODAC),
1,2-dioleyloxy-N,N—dimethylaminopropane (DODMA), 1,2-distearyloxy—N,N-dimethylaminopropane
(DSDMA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N—trimethylammonium chloride (DOTMA), N,N—
distearyl—N,N-dimethylammonium bromide (DDAB), N-(1—(2,3-dioleoyloxy)propyl)-N,N,N-
hylammonium chloride (DOTAP), 3 ’,N’-dimethylaminoethane)—carbamoyl)cholesterol
(DC—C1101), N—(l,2—dimyristyloxypropy1)-N,N—dimethyl-N-hydroxyethyl ammonium bromide
(DMRIE), 2,3—dioleyloxy-N— [2(spermine-carboxamido)ethyl]-N,N—dimethyl- l —
propanaminiumtrifluoroacetate ), dioctadecylamidoglycyl spermine (DOGS), 3—
dimethylamino(cholest—5 -en—3 —beta-oxybutanoxy)(cis,cis-9, 1 2-octadecadienoxy)propane
(CLinDMA), 2~[5 ’ -(cholestenbeta-oxy)-3 ’-oxapentoxy)dimethy—1-(cis,cis-9’ ,1-2’ -
octadecadienoxy)propane (CpLinDMA), N,N—dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-
N,N’~dioleylcarbamyl—3~dimethylaminopropane (DOcarbDAP), N’~dilinoleylcarbamyl
dimethylaminopropane (DLincarbDAP), 1 ,2—dilinoleylcarbamoyloxy—3-dimethylaminopropane
(DLin—C—DAP), 1,2-dilinoleyoxy—3—(dimethylamino)acetoxypropane DAC), 1,2-dilinoleyoxy—
3~morpholinopropane (DLin—MA), 1,2-dilinoleoyl-3—dimethylaminopropane (DLinDAP), 1,2-
dilinoleylthio—3—dimethylaminopr0pane (DLin—S—DMA), 1-linoleoyl—2-linoleyloxy
dimethylaminopropane Z—DMAP), 1,2—dilinoleyloxy—3~trimethylaminopropane chloride salt
(DLin-TMA.C1), l,2—dilinoleoyl—3-trimethylaminopropane chloride salt (DLin-TAPCI), 1,2—
dilinoleyloxy—3-(N-methylpiperazino)propane (DLin—MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol
(DLinAP), 3—(N,N-dioleylamino)-1,2-propanedio , l,2—dilinoleyloxo—3-(2-N,N-
dimethylamino)ethoxypropane (DLin—EG-DMA), l,2-dioeylcarbamoyloxydimethylaminopropane
(DO-C-DAP), l,2—dimyristoleoyl-3—dimethylaminopropane (DMDAP), 1,2-dioleoyl—3 -
trimethylaminopropane chloride (DOTAP.C1), leylmethyldimethylaminopropionate (DLin~
M-C2-DMA; also known as DLin—M-K-DMA or DLin-M-DMA), and mixtures thereof. Additional
cationic lipids or salts thereof which may be ed in the lipid les of the present invention are
described in US. Patent Publication No. 20090023673, the disclosure of which is herein incorporated
by reference in its entirety for all purposes.
The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids,
is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein
incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as
DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLinDMAP, DLinTMA.Cl,
DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin—EG—DMA, as well as additional ic lipids,
is described in PCT Publication No. WO 09/086558, the sure of which is herein incorporated by
reference in its entirety for all purposes. The synthesis of cationic lipids such as AP,
DMDAP, DOTAP.C1, DLin—M-CZ—DMA, as well as additional cationic lipids, is described in PCT
Application No. 2009/060251, entitled “Improved Amino Lipids and Methods for the
Delivery of Nucleic ” filed October 9, 2009, the disclosure of which is incorporated herein by
reference in its entirety for all purposes. The sis of a number of other cationic lipids and related
analogs has been described in U.S. Patent Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185;
,753,613; and 5,785,992; and PCT Publication No. W0 96/ 10390, the sures of which are each
herein incorporated by reference in their entirety for all purposes. onally, a number of
commercial preparations of cationic lipids can be used, such as, e.g., LIPOFECTIN® (including
DOTMA and DOPE, available from Invitrogen); LIPOFECTAMINE® (including DOSPA and DOPE,
available from Invitrogen); and TRANSFECTAM® (including DOGS, available from Promega
Corp.).
In some embodiments, the cationic lipid comprises from about 50 mol % to about 90 mol
%, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50
mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65
mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from
about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid t
in the particle. In ular ments, the cationic lipid comprises about 50 mol %, 51 mol %, 52
mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61
mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fraction thereof) of the total lipid present
in the particle.
In other ments, the cationic lipid comprises from about 2 mol % to about 60 mol
%, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20
mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40
mol %, or about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the
particle.
] Additional percentages and ranges of cationic lipids suitable for use in the lipid particles
of the present invention are bed in PCT Publication No. W0 09/127060, U.S. Published
Application No. US 2011/0071208, PCT Publication No. WOZOll/000106, and U.S. Published
PCT/U82012/058770
Application No. US 2011/0076335, the disclosures of which are herein incorporated by reference in
their entirety for all purposes.
It should be understood that the percentage of cationic lipid present in the lipid particles
of the invention is a target amount, and that the actual amount of cationic lipid present in the
formulation may vary, for example, by i 5 mol %. For example, in the 1:57 lipid particle (e.g.,
SNALP) formulation, the target amount of cationic lipid is 57.1 mol %, but the actual amount of
cationic lipid may be i 5 mol %, d: 4 mol %, :1: 3 mol %, i 2 mol %, :t 1 mol %, i 0.75 mol%, i 0.5
mol %, :E 0.25 mol %, or :1: 0.1 mol % of that target amount, with the balance of the formulation being
made up of other lipid components (adding up to 100 mol % of total lipids present in the particle).
2. Non-cationic Lipids
The non-cationic lipids used in the lipid particles of the invention (e.g., SNALP) can be
any of a variety of neutral uncharged, rionic, or anionic lipids capable of producing a stable
complex.
] Non—limiting examples of tionic lipids include phospholipids such as lecithin,
phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic
acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoy1phosphatidylcholine (DPPC),
dioleoylphosphatidylglycercl (DOPG), dipalmitoylphosphatidylglycerol ,
dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC),
palmitoyloleoyl—phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG),
dioleoylphosphatidylethanolamine 4-(N—maleimidomethyl)-cyclohexane-l-carboxylate mal),
dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl~phosphatidylethanolamine (DMPE),
distearoyl-phosphatidylethanolamine (DSPE), monomethyl—phosphatidylethanolamine, dimethyl-
atidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-
atidylethanolamine (SOPE), osphatidylcholine, dilinoleoylphosphatidylcholine, and
mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine
olipids can also be used. The acyl groups in these lipids are preferably acyl groups derived
from fatty acids having Clo-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, yl, or oleoyl.
[00236] Additional examples of non-cationic lipids include sterols such as cholesterol and
derivatives thereof. Non—limiting examples of cholesterol derivatives include polar analogues such as
Set-cholestanol, rostanol, cholesteryl-(Z’—hydroxy)—ethyl ether, cholesteryl—(4’—hydr0xy)—butyl
ether, and cholestanol; non-polar ues such as Su—cholestane, cholestenone, Soc-
W0 2013I’052677
cholestanone, SB-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred
embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4’-hydroxy)-butyl
ether. The synthesis of cholesteryl-(2’-hydroxy)-ethy1 ether is described in PCT ation No. W0
09/ 127060, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
In some embodiments, the non—cationic lipid present in the lipid particles (e.g., SNALP)
comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative
f. In other embodiments, the non—cationic lipid present in the lipid particles (e.g., SNALP)
comprises or consists of one or more phospholipids, e. g., a cholesterol—free lipid particle formulation.
In yet other embodiments, the non—cationic lipid present in the lipid particles (e.g., SNALP) comprises
or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation.
Other es of non-cationic lipids suitable for use in the present invention include
nonphosphorous ning lipids such as, e.g., stearylamine, lamine, hexadecylamine, acetyl
palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl ate, amphoteric acrylic polymers,
triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides,
decyldimethyl um bromide, de, sphingomyelin, and the like.
In some embodiments, the non—cationic lipid ses from about 10 mol % to about 60
mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about
mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about
45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from
about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to
about 42 mol %, or about 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %,
42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein) of the total
lipid present in the particle.
In embodiments where the lipid particles contain a mixture of phospholipid and
cholesterol or a cholesterol derivative, the e may comprise up to about 40 mol %, 45 mol %, 50
mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
In some embodiments, the phospholipid component in the mixture may comprise from
about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to
about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol %
(or any fraction thereof or range therein) of the total lipid t in the particle. In certain preferred
ments, the phospholipid component in the mixture comprises from about 5 mol % to about 10
mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6
mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %,
2012/058770
8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in
the particle. As a non—limiting example, a 1:57 lipid particle formulation sing a mixture of
phospholipid and cholesterol may comprise a phospholipid such as DPPC or DSPC at about 7 mol %
(or any fraction thereof), e. g., in a mixture with cholesterol or a terol derivative at about 34 mol
% (or any fraction thereof) of the total lipid present in the particle. As another non—limiting example,
a 7:54 lipid particle formulation comprising a mixture of phospholipid and cholesterol may comprise
a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof), e.g., in a mixture
with cholesterol or a cholesterol derivative at about 32 mol % (or any on thereof) of the total
lipid present in the particle.
] In other embodiments, the cholesterol component in the mixture may comprise from
about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to
about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %,
from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction
thereof or range therein) of the total lipid present in the particle. In certain preferred embodiments,
the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from
about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to
about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %,
or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof
or range therein) of the total lipid present in the particle. Typically, a 1:57 lipid particle formulation
comprising a mixture of phospholipid and terol may se terol or a cholesterol
derivative at about 34 mol % (or any on thereof), e.g., in a mixture with a phospholipid such as
DPPC or DSPC at about 7 mol % (or any fraction thereof) of the total lipid present in the particle.
Typically, a 7:54 lipid particle formulation comprising a mixture of olipid and cholesterol may
comprise cholesterol or a cholesterol derivative at about 32 mol % (or any fraction thereof), e. g., in a
mixture with a phospholipid such as DPPC or DSPC at about 7 mol % (or any fraction thereof) of the
total lipid present in the particle.
In embodiments where the lipid particles are phospholipid-free, the cholesterol or
derivative thereof may se up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50
mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
3O [00244] In some embodiments, the cholesterol or derivative thereof in the phospholipid—free lipid
particle formulation may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to
about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,
from about 31 mol % to about 39 mol %, from about 32 mol % to about 38 mol %, from about 33 mol
% to about 37 mol %, from about 35 mol % to about 45 mol %, from about 30 mol % to about 35 mol
%, from about 35 mol % to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34
mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof or
range therein) of the total lipid present in the particle. As a non—limiting example, a 1:62 lipid particle
formulation may comprise cholesterol at about 37 mol % (or any fraction thereof) of the total lipid
present in the particle. As another non-limiting example, a 7:58 lipid particle formulation may
comprise cholesterol at about 35 mol % (or any fraction thereof) of the total lipid present in the
particle.
In other embodiments, the non-cationic lipid comprises from about 5 mol % to about 90
mol %, from about 10 mol % to about 85 mol %, from about 20 mol % to about 80 mol %, about 10
mol % (e.g., phospholipid only), or about 60 mol % (e.g., phospholipid and cholesterol or derivative
thereof) (or any fraction thereof or range n) of the total lipid t in the particle.
Additional percentages and ranges of non-cationic lipids suitable for use in the lipid
particles of the present invention are described in PCT Publication No. W0 09/127060, U.S.
Published Application No. US 2011/0071208, PCT Publication No. W02011/000106, and US.
Published Application No. US 2011/0076335, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.
It should be understood that the percentage of non-cationic lipid t in the lipid
les of the invention is a target , and that the actual amount of tionic lipid present in
the formulation may vary, for example, by i 5 mol %. For example, in the 1:57 lipid particle (e.g.,
SNALP) formulation, the target amount of phospholipid is 7.1 mol % and the target amount of
cholesterol is 34.3 mol %, but the actual amount of olipid may be :t 2 mol %, i 1.5 mol %, i 1
mol %, i 0.75 mol %, i 0.5 mol %, i- 0.25 mol %, or i 0.1 mol % of that target amount, and the
actual amount of cholesterol may be i 3 mol %, i 2 mol %, i 1 mol %, :1: 0.75 mol %, i 0.5 mol %, :l:
0.25 mol %, or i 0.1 mol % of that target amount, with the balance of the formulation being made up
of other lipid components (adding up to 100 mol % of total lipids present in the particle). Similarly,
in the 7:54 lipid particle (e.g., SNALP) formulation, the target amount of phospholipid is 6.75 mol %
and the target amount of cholesterol is 32.43 mol %, but the actual amount of phospholipid may be i
2 mol %, 5: 1.5 mol %, d: 1 mol %, :t 0.75 mol %, d: 0.5 mol %, i 0.25 mol %, or i 0.1 mol % ofthat
target amount, and the actual amount of cholesterol may be i 3 mol %, :t 2 mol %, i 1 mol %, :i: 0.75
mol %, i 0.5 mol %, :t 0.25 mol %, or 3: 0.1 mol % of that target amount, with the balance of the
formulation being made up of other lipid components (adding up to 100 mol % of total lipids t
in the particle).
W0 20131052677
3. Lipid Conjugates
In addition to cationic and non—cationic lipids, the lipid particles of the invention (e. g.,
SNALP) may further comprise a lipid conjugate. The conjugated lipid is useful in that it prevents the
aggregation of particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid
conjugates, POZ—lipid conjugates, ATTA—lipid conjugates, cationic-polymer-lipid conjugates (CPLs),
and mixtures thereof. In certain embodiments, the particles comprise either a PEG-lipid conjugate or
an ATTA—lipid conjugate together with a CPL.
In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids
include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-BAA) as described in, e.g.,
PCT Publication No. W0 05/026372, PEG coupled to glycerol (PEG-DAG) as described in,
e.g., US. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such
as phosphatidylethanolamine (PEG—PE), PEG conjugated to des as described in, e.g., US
Patent No. 5,885,613, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
The sures of these patent documents are herein orated by reference in their entirety for all
purposes. [0001] Additional PEG-lipids suitable for use in the invention include, without
limitation, mPEGZOOO—l,2-di-O—alkyl—sn3-carbomoylglyceride -DOMG). The synthesis of
DOMG is described in PCT Publication No. WO 09/086558, the sure of which is herein
incorporated by reference in its entirety for all purposes. Yet additional suitable PEG-lipid conjugates
e, without limitation, l—[8 ”-(l ,2-dimyristoyl-3—propanoxy)—carboxamido-3 ’ ,6’—
2O dioxaoctanyl]carbamoyl—co-methyl—poly(ethylene ) (2KPEG-DMG). The synthesis of ZKPEG-
DMG is described in US. Patent No. 7,404,969, the disclosure of which is herein incorporated by
reference in its entirety for all purposes.
PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two
terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000
has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular
weight of about 5,000 daltons. PEGs are cially available from Sigma Chemical Co. and other
ies and include, but are not limited to, the following: monomethoxypolyethylene glycol
(MePEG—OH), monomethoxypolyethylene —succinate (MePEG-S), monomethoxypolyethylene
glycol—succinimidyl succinate (MePEG—S—NHS), thoxypolyethylene glycol-amine (MePEG—
NHg), monomethoxypolyethylene glycol—tresylate (MePEG—TRES), monomethoxypolyethylene
glycol-imidazolyl-carbonyl (MePEG-IM), as well as such compounds containing a al yl
group instead of a terminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NHg, etc).
Other PEGs such as those described in US. Patent Nos. 6,774,180 and 150 (e.g., mPEG (20
KDa) amine) are also useful for ing the PEG-lipid conjugates of the present invention. The
disclosures of these patents are herein orated by reference in their entirety for all purposes. In
W0 2013f052677 PCT/U82012/058770
addition, monomethoxypolyethyleneglycol—acetic acid (MePEG-CHZCOOH) is particularly useful for
preparing PEG-lipid conjugates including, e. g., A conjugates.
The PEG moiety of the PEG—lipid conjugates described herein may comprise an average
molecular weight g from about 550 daltons to about 10,000 daltons. In certain instances, the
PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e. g.,
from about 1,000 daltons to about 5,000 daltons, from about 1,500 s to about 3,000 daltons,
from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc).
In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or
about 750 daltons.
[00252] In n instances, the PEG can be optionally substituted by an alkyl, , acyl, or
aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker
. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e. g., non—
ester ning linker moieties and ester—containing linker moieties. In a preferred embodiment, the
linker moiety is a non-ester containing linker moiety. As used herein, the term “non-ester containing
linker moiety” refers to a linker moiety that does not contain a carboxylic ester bond (—OC(O)—).
Suitable non-ester ning linker moieties include, but are not limited to, amido (-C(O)NH-),
amino (-NR-), carbonyl (-C(O)-), carbamate O)O-), urea (—NHC(O)NH-), disulphide (-S-S-),
ether (-O-), succinyl (-(O)CCH2CH2C(O)—), amidyl (—NHC(O)CH2CH2C(O)NH-), ether,
disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker
moiety and an amido linker moiety). In a preferred embodiment, a carbamate linker is used to couple
the PEG to the lipid.
In other embodiments, an ester containing linker moiety is used to couple the PEG to the
lipid. Suitable ester containing linker moieties include, e. g., carbonate (~OC(O)O-), succinoyl,
phosphate esters (—O-(O)POH—O-), sulfonate esters, and combinations thereof.
[00254] atidylethanolamines having a y of acyl chain groups of varying chain
lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such
phosphatidylethanolamines are commercially available, or can be isolated or Synthesized using
conventional techniques known to those of skilled in the art. Phosphatidyl—ethanolamines containing
saturated or unsaturated fatty acids with carbon chain s in the range of C10 to C20 are preferred.
Phosphatidylethanolamines with mono— or diunsaturated fatty acids and mixtures of saturated and
unsaturated fatty acids can also be used. le phosphatidylethanolamines include, but are not
limited to, dimyristoyl—phosphatidylethanolamine (DMPE), dipa1mitoyl—phosphatidylethanolamine
(DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine
(DSPE).
2012/058770
The term “ATTA” or “polyamide” includes, without limitation, compounds described in
US Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by
reference in their entirety for all purposes. These compounds include a compound having the
formula:
If t t2
R N—(CHZCHZO);(CH2)5"'-C-(NH—%—E)q R3
n (IV),
wherein R is a member selected from the group consisting of hydrogen, alkyl and acyl;
R1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R1 and
the nitrogen to which they are bound form an azido moiety; R2 is a member of the group selected from
hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid;
R3 is a member selected from the group consisting of hydrogen, n, hydroxy, alkoxy, to,
hydrazine, amino and NR4R5, wherein R4 and R5 are independently en or alkyl; n is 4 to 80; m
is 2 to 6; p is 1 to 4; and q is 0 or 1. It will be apparent to those of skill in the art that other
polyamides can be used in the compounds of the present invention.
] The term “diacylglycerol” or “DAG” includes a compound having 2 fatty acyl chains, R1
and R2, both of which have independently between 2 and 30 carbons bonded to the 1- and 2-position
of glycerol by ester linkages. The acyl groups can be saturated or have varying degrees of
unsaturation. le acyl groups include, but are not limited to, l (C12), myristoyl (C14),
palmitoyl (C16), yl (C18), and icosoyl (C20). In preferred embodiments, R1 and R2 are the same,
i.e., R1 and R2 are both myristoyl (118., dimyristoyl), R1 and R2 are both stearoyl (128., distearoyl), etc.
Diacylglycerols have the following general formula:
CH20— (V)
The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkyl chains, R1
and R2, both of which have independently between 2 and 30 carbons. The alkyl groups can be
saturated or have varying degrees of unsaturation. Dialkyloxypropyls have the following general
formula:
CHZO-R‘
| 2
(IDH O— R
CHz‘ (VI).
In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate having the following
formula:
ergo -R‘
CHO— R2
2-1—4353 (W),
wherein R1 and R2 are independently selected and are long-chain alkyl groups having
from about 10 to about 22 carbon atoms; PEG is a polyethyleneglycol; and L is a ter containing
linker moiety or an ester containing linker moiety as described above. The long-chain alkyl groups
can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, decyl (C10),
lauryl (C12), myristyl (C14), palmityl (C16), l (C18), and icosyl (C20). In preferred embodiments,
R1 and R2 are the same, 126., R1 and R2 are both yl (21a, dimyristyl), R1 and R2 are both stearyl
(i.e., distearyl), etc.
In Formula VII above, the PEG has an average molecular weight ranging from about 550
s to about 10,000 daltons. In certain instances, the PEG has an e molecular weight of
from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons,
from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from
about 750 daltons to about 2,000 s, etc). In preferred embodiments, the PEG has an average
molecular weight of about 2,000 daltons or about 750 daltons. The PEG can be optionally substituted
with alkyl, alkoxy, acyl, or aryl groups. In certain embodiments, the terminal hydroxyl group is
substituted with a methoxy or methyl group.
In a preferred embodiment, “L” is a non-ester ning linker moiety. Suitable non-
ester containing linkers e, but are not limited to, an amido linker moiety, an amino linker
moiety, a carbonyl linker moiety, a carbamate linker , a urea linker moiety, an ether linker
, a disulphide linker moiety, a succinamidyl linker moiety, and combinations thereof. In a
W0 2013;052677 PCT/U82012/058770
red embodiment, the non-ester containing linker moiety is a carbamate linker moiety (i.e., a
PEG-C—DAA conjugate). In another preferred embodiment, the non—ester containing linker moiety is
an amido linker moiety (i.e., a PEG—A—DAA conjugate). In yet r preferred embodiment, the
non-ester containing linker moiety is a succinamidyl linker moiety (129., a PEG-S—DAA conjugate).
In ular embodiments, the PEG-lipid conjugate is selected from:
\ \ \ \ x "\ \ , \ ’\. six 2“
/ ' xO
‘ ” ‘ ‘ O“ ‘ ’ ~N‘ O‘ \ ‘
\ ~ ~ ~ ~ « A O
‘ ’ ‘ ’ \‘ “ \ ‘ N" i ‘ n (PEG-C—DMA); and
“\\ “\v, \\v \\ x \x. \xV “\Ofl'ix'N’ffi‘ ,O\\
\O. ‘\:’ 9-”
E H
\\ \'\‘_I r‘ ’A\ \
x \ x '\’\V O — n
(PEG-C-DOMG).
The PEG-DAA conjugates are synthesized using standard techniques and reagents
known to those of skill in the art. It will be recognized that the PEG-DAA conjugates will contain
various amide, amine, ether, thio, carbamate, and urea linkages. Those of skill in the art will
recognize that methods and reagents for forming these bonds are well known and readily available.
See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock,
COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Fumiss, S
TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989). It will also be
appreciated that any functional groups present may require protection and deprotection at different
points in the sis of the PEG—DAA ates. Those of skill in the art will recognize that such
techniques are well known. See, e.g., Green and Wuts, PROTECTIVE GROUPS IN ORGANIC
SYNTHESIS (Wiley 1991).
Preferably, the PEG—DAA conjugate is a decyloxypropyl (C10) conjugate, a PEG—
dilauryloxypropyl (C12) conjugate, a PEG—dimyristyloxypropyl (C14) conjugate, a PEG—
dipalmityloxypropyl (C15) conjugate, or a PEG—distearyloxypropyl (C18) ate. In these
embodiments, the PEG preferably has an e molecular weight of about 750 or about 2,000
daltons. In one particularly preferred embodiment, the PEG—lipid conjugate comprises PEGZOOO-C—
DMA, wherein the “2000” denotes the average molecular weight of the PEG, the “C” denotes a
carbamate linker moiety, and the “DMA” s dimyristyloxypropyl. In another particularly
preferred embodiment, the PEG-lipid conjugate comprises PEG750-C-DMA, wherein the “750”
denotes the average lar weight of the PEG, the “C” denotes a carbamate linker moiety, and the
“DMA” s dimyristyloxypropyl. In ular embodiments, the terminal hydroxyl group of the
PCT/U52012/058770
PEG is substituted with a methyl group. Those of skill in the art will readily appreciate that other
dialkyloxypropyls can be used in the PEG—DAA conjugates of the present invention.
In addition to the foregoing, it will be readily apparent to those of skill in the art that
other hydrophilic polymers can be used in place of PEG. Examples of le polymers that can be
used in place of PEG include, but are not limited to, polyvinylpyrrolidone, thyloxazoline,
polyethyloxazoline, polyhydroxypropyl methacrylamide, thacrylamide and
polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as
hydroxymethylcellulose or hydroxyethylcellulose.
In addition to the foregoing components, the lipid particles (e.g., SNALP) of the present
invention can further se cationic thylene glycol) (PEG) lipids 0r CPLs (see, e.g., Chen et
(11., Bioconj. Chem, -437 (2000); US Patent No. 6,852,334; PCT Publication No. WO
00/62813, the disclosures of which are herein incorporated by reference in their entirety for all
purposes).
Suitable CPLs include compounds of Formula VIII:
A—W—Y (VIII),
wherein A, W, and Y are as described below.
With reference to Formula VIII, “A” is a lipid moiety such as an amphipathic lipid, a
neutral lipid, or a hydrophobic lipid that acts as a lipid anchor. le lipid examples include, but
are not limited to, diacylglycerolyls, dialkylglycerolyls, N—N—dialkylaminos, 1,2—diacyloxy—3-
aminopropanes, and l,2-dialkyl-3—aminopropanes.
“W” is a polymer or an oligomer such as a hydrophilic r or oligomer. Preferably,
the hydrophilic polymer is a patable polymer that is nonimmunogenic or possesses low
inherent immunogenicity. Alternatively, the hydrophilic polymer can be weakly antigenic if used
with appropriate nts. Suitable nonimmunogenic polymers include, but are not limited to, PEG,
polyamides, polylactic acid, polyglycolic acid, polylactic acid/polyglycolic acid copolymers, and
combinations thereof. In a red embodiment, the polymer has a molecular weight of from about
250 to about 7,000 daltons.
“Y” is a polycationic moiety. The term polycationic moiety refers to a nd,
derivative, or functional group having a positive charge, preferably at least 2 positive charges at a
selected pH, preferably physiological pH. Suitable polycationic es include basic amino acids
and their derivatives such as ne, asparagine, glutamine, lysine, and histidine; spermine;
spermidine; cationic dendrimers; polyamines; polyamine sugars; and amino polysaccharides. The
PCT/U52012/058770
polycationic es can be linear, such as linear ysine, branched or meric in structure.
Polycationic moieties have between about 2 to about 15 positive charges, preferably between about 2
to about 12 ve charges, and more preferably between about 2 to about 8 positive charges at
selected pH values. The selection of which polycationic moiety to employ may be determined by the
type of particle application which is desired.
The charges on the polycationic moieties can be either distributed around the entire
particle moiety, or alternatively, they can be a discrete concentration of charge density in one
particular area of the particle moiety e.g., a charge spike. If the charge y is distributed on the
le, the charge density can be equally distributed or lly distributed. All variations of
charge distribution of the polycationic moiety are encompassed by the present invention.
] The lipid “A” and the nonimmunogenic polymer “W” can be attached by various
s and preferably by covalent attachment. Methods known to those of skill in the art can be
used for the covalent attachment of “A” and “W.” Suitable linkages include, but are not limited to,
amide, amine, carboxyl, carbonate, ate, ester, and hydrazone linkages. It will be apparent to
those skilled in the art that “A” and “W” must have complementary functional groups to effectuate the
e. The reaction of these two groups, one on the lipid and the other on the polymer, will provide
the desired linkage. For example, when the lipid is a diacylglycerol and the terminal hydroxyl is
activated, for instance with NHS and DCC, to form an active ester, and is then reacted with a polymer
which contains an amino group, such as with a polyamide (see, e.g., US. Patent Nos. 6,320,017 and
6,586,559, the sures of which are herein incorporated by reference in their entirety for all
purposes), an amide bond will form between the two groups.
In certain instances, the polycationic moiety can have a ligand attached, such as a
targeting ligand or a chelating moiety for complexing calcium. ably, after the ligand is
attached, the cationic moiety maintains a positive . In certain instances, the ligand that is
attached has a positive charge. Suitable ligands include, but are not limited to, a compound or device
with a reactive functional group and include lipids, amphipathic lipids, carrier compounds, bioaffinity
compounds, biomaterials, biopolymers, biomedical devices, analytically able compounds,
therapeutically active compounds, enzymes, peptides, proteins, antibodies, immune stimulators,
abels, fluorogens, biotin, drugs, haptens, DNA, RNA, polysaccharides, liposomes, virosomes,
micelles, immunoglobulins, fianctional groups, other targeting moieties, or toxins.
In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1
mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol
%, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about
0.8 mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9 mol % to
W0 2013(052677
about 1.8 mol %, from about 1 mol % to about 1.8 mol %, from about 1 mol % to about 1.7 mol %,
from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3
mol % to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % (or any fraction thereof or
range therein) of the total lipid present in the particle.
In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 0 mol
% to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol
%, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4
mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5 mol % to about 12
mol %, or about 2 mol % (or any fraction thereof or range n) of the total lipid present in the
particle.
In fiirther embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from about 4
mol % to about 10 mol %, from about 5 mol % to about 10 mol %, from about 5 mol % to about 9
mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6
mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or
any fraction thereof or range therein) of the total lipid present in the particle.
Additional percentages and ranges of lipid conjugates suitable for use in the lipid
particles of the present invention are described in PCT ation No. W0 09/127060, U.S.
Published Application No. US 2011/0071208, PCT Publication No. /000106, and US.
Published ation No. US 2011/0076335, the disclosures of which are herein incorporated by
reference in their entirety for all purposes.
] It should be understood that the percentage of lipid conjugate (e.g., PEG—lipid) t in
the lipid particles of the ion is a target amount, and that the actual amount of lipid conjugate
present in the formulation may vary, for example, by i 2 mol %. For example, in the 1:57 lipid
particle (e.g., SNALP) formulation, the target amount of lipid conjugate is 1.4 mol %, but the actual
amount of lipid conjugate may be i 0.5 mol %, i 0.4 mol %, i 0.3 mol %, :t 0.2 mol %, i 0.1 mol %,
or d: 0.05 mol % of that target , with the balance of the formulation being made up of other
lipid components (adding up to 100 mol % of total lipids present in the particle). Similarly, in the
7:54 lipid particle (e.g., SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %, but
the actual amount of lipid conjugate may be i 2 mol %, i: 1.5 mol %, i 1 mol %, i 0.75 mol %, i 0.5
mol %, 3c 0.25 mol %, or i 0.1 mol % of that target amount, with the balance of the formulation being
made up of other lipid components (adding up to 100 mol % of total lipids present in the particle).
WO 52677
One of ordinary skill in the art will appreciate that the concentration of the lipid
conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid
particle is to become fusogenic.
By controlling the composition and concentration of the lipid conjugate, one can control
the rate at which the lipid conjugate exchanges out of the lipid particle and, in turn, the rate at which
the lipid particle becomes fusogenic. For instance, when a PEG-DAA conjugate is used as the lipid
conjugate, the rate at which the lipid particle becomes fusogenic can be , for e, by
varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by
varying the chain length and degree of saturation of the alkyl groups on the A conjugate. In
addition, other variables ing, for example, pH, temperature, ionic strength, etc. can be used to
vary and/0r control the rate at which the lipid particle becomes fusogenic. Other methods which can
be used to l the rate at which the lipid particle s fusogenic will become apparent to those
of skill in the art upon reading this disclosure. Also, by controlling the ition and concentration
of the lipid conjugate, one can control the lipid particle (e.g., SNALP) size.
B. Additional Carrier Systems
Non-limiting examples of additional based carrier systems suitable for use in the
present invention include lipoplexes (see, e.g., US. Patent Publication No. 20030203865; and Zhang
et al., J. Control Release, 100:165—180 (2004)), pH—sensitive exes (see, e.g., US. Patent
ation No. 20020192275), reversibly masked lipoplexes (see, e.g., US. Patent Publication Nos.
20030180950), cationic lipid—based compositions (see, e.g., US. Patent No. 6,756,054; and US.
Patent Publication No. 20050234232), cationic liposomes (see, e.g., US. Patent Publication Nos.
20030229040, 60038, and 20020012998; US. Patent No. 5,908,635; and PCT Publication No.
WO 01/72283), anionic liposomes (see, e.g., US. Patent Publication No. 20030026831), pH—sensitive
liposomes (see, e.g., US. Patent Publication No. 20020192274; and AU 2003210303), antibody—
coated liposomes (see, e. g., US. Patent Publication No. 20030108597; and PCT Publication No. WO
00/50008), cell—type specific liposomes (see, e.g., US. Patent ation No. 20030198664),
liposomes containing nucleic acid and peptides (see, e.g., US. Patent No. 6,207,456), liposomes
containing lipids derivatized with releasable hydrophilic rs (see, e. g., US. Patent Publication
No. 20030031704), lipid-entrapped nucleic acid (see, e.g., PCT Publication Nos. WO 03/057190 and
WO 03/059322), lipid-encapsulated nucleic acid (see, e.g, US. Patent Publication No. 20030129221;
and US. Patent No. 5,756,122), other liposomal compositions (see, e.g., US. Patent Publication Nos.
20030035829 and 72794; and US. Patent No. 6,200,599), stabilized mixtures of liposomes
and emulsions (see, e.g., EP1304160), emulsion itions (see, e. g., US. Patent No. 6,747,014),
and nucleic acid micro-emulsions (see, e.g., US. Patent Publication No. 20050037086).
W0 2013l052677
Examples of polymer-based carrier systems suitable for use in the t invention
include, but are not limited to, cationic polymer—nucleic acid complexes (i.e., polyplexes). To form a
ex, a c acid (e.g., interfering RNA) is typically complexed with a ic polymer having
a linear, branched, star, or dendritic polymeric structure that condenses the nucleic acid into positively
charged particles capable of interacting with anionic proteoglycans at the cell surface and ng
cells by endocytosis. In some embodiments, the polyplex comprises nucleic acid (e.g., interfering
RNA) complexed with a ic polymer such as polyethylenimine (PEI) (see, e.g., U.S. Patent No.
240; commercially available from Qbiogene, Inc. (Carlsbad, CA) as In vivo jetPEITM, a linear
form of PEI), polypropylenimine (PPI), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL),
diethylaminoethyl -dextran, polyGB—amino ester) (PAE) polymers (see, e.g., Lynn et (11., J.
Am. Chem. Soc., 123:8155—8156 (2001)), chitosan, polyamidoamine (PAMAM) dendrimers (see, e. g.,
Kukowska—Latallo et al., Proc. Natl. Acad. Sci. USA, 93:4897-4902 (1996)), porphyrin (see, e.g., U.S.
Patent No. 6,620,805), polyvinylether (see, e. g., U.S. Patent Publication No. 20040156909),
polycyclic amidinium (see, e.g., U.S. Patent ation No. 20030220289), other polymers
comprising y amine, imine, guanidine, and/or imidazole groups (see, e.g., U.S. Patent No.
6,013,240; PCT Publication No. WO/9602655; PCT Publication No. WO95/21931; Zhang et al., J.
Control Refease, 1002165—180 (2004); and Tiera et (1]., Curr. Gene Ther., 1 (2006)), and a
mixture thereof. In other embodiments, the polyplex comprises cationic polymer-nucleic acid
complexes as described in U.S. Patent Publication Nos. 20060211643, 20050222064, 20030125281,
and 20030185890, and PCT Publication No. WO 03/066069; biodegradable poly(B-amino ester)
polymer—nucleic acid xes as described in U.S. Patent Publication No. 20040071654;
articles containing polymeric es as described in U.S. Patent Publication No.
20040142475; other microparticle compositions as described in U.S. Patent Publication No.
20030157030; condensed nucleic acid complexes as described in U.S. Patent Publication No.
20050123600; and nanocapsule and microcapsule itions as described in AU 2002358514 and
PCT Publication No. WO 02/096551.
In certain instances, the interfering RNA may be complexed with cyclodextrin or a
polymer thereof. Non-limiting examples of cyclodextrin—based carrier systems include the
cyclodextrin—modified polymer—nucleic acid complexes described in U.S. Patent Publication No.
20040087024; the linear cyclodextrin copolymer—nucleic acid complexes described in U.S. Patent
Nos. 6,509,323, 6,884,789, and 7,091,192; and the extrin polymer-complexing nucleic
acid complexes described in U.S. Patent No. 7,018,609. In n other instances, the interfering
RNA may be complexed with a peptide or polypeptide. An e of a protein-based carrier system
includes, but is not limited to, the cationic oligopeptide-nucleic acid complex described in PCT
Publication No. WO95/2193 l.
VI. Preparation of Lipid Particles
The lipid particles of the present ion, e. g., SNALP, in which a nucleic acid such as
an interfering RNA (e.g., siRNA) is entrapped within the lipid portion of the particle and is protected
from degradation, can be formed by any method known in the art including, but not limited to, a
continuous mixing method, a direct dilution process, and an in—line dilution process.
In particular embodiments, the cationic lipids may comprise lipids of Formula I-III or
salts thereof, alone or in combination with other cationic lipids. In other ments, the non—
cationic lipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), l -palmitoyl-2—oleoyl—phosphatidylcholine (POPC),
dipalmitoyl—phosphatidylcholine (DPPC), monomethyl—phosphatidylethanolamine, dimethyl—
phosphatidylethanolamine, 14:0 PE (l,2-dimyristoyl—phosphatidylethanolamine ), 16:0 PE
(1 ,2-dipalmitoyl—phosphatidylethanolamine (DPPE)), 1 8:0 PE (1 ,2-distearoyl—
phosphatidylethanolamine (DSPE)), 18:1 PE (l,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1
trans PE (l,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0—1821 PE (l-stearoyl-Z-oleoyl—
phosphatidylethanolamine ), 16:0-18:1 PE (1—palmitoyl-2—oleoyl-phosphatidylethanolamine
(POPE)), polyethylene glycol—based polymers (e.g., PEG 2000, PEG 5000, PEG—modified
diacylglycerols, or dified dialkyloxypropyls), cholesterol, derivatives thereof, or
combinations thereof.
In certain ments, the present invention provides nucleic ipid particles (e. g.,
SNALP) produced via a continuous mixing method, e.g., a process that includes providing an aqueous
on comprising a nucleic acid (e.g., ering RNA) in a first reservoir, providing an organic
lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are
solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous
solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous
solution so as to substantially instantaneously produce a lipid vesicle (e.g., liposome) encapsulating
the nucleic acid within the lipid vesicle. This process and the apparatus for carrying out this process
are described in detail in US. Patent ation No. 20040142025, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
The action of continuously introducing lipid and buffer solutions into a mixing
nment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the
buffer solution, y producing a lipid vesicle substantially instantaneously upon mixing. As used
herein, the phrase “continuously diluting a lipid on with a buffer solution” (and variations)
lly means that the lipid solution is diluted sufficiently rapidly in a hydration process with
ent force to effectuate vesicle generation. By mixing the aqueous solution comprising a nucleic
2012/058770
acid with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise
dilution in the presence of the buffer solution (116., aqueous solution) to produce a nucleic acid—lipid
particle.
The nucleic acid-lipid particles formed using the continuous mixing method lly
have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50
nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from
about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm,
from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,
less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50
nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm,
115 nm, 120 nm, 125 nm, 130 mm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or
range therein). The particles thus formed do not aggregate and are optionally sized to achieve a
uniform particle size.
In another embodiment, the present invention provides nucleic acid-lipid particles (e.g.,
SNALP) produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome)
solution and immediately and directly introducing the lipid vesicle solution into a collection vessel
containing a controlled amount of dilution buffer. In preferred aspects, the collection vessel includes
one or more elements configured to stir the contents of the collection vessel to facilitate dilution. In
one aspect, the amount of dilution buffer present in the collection vessel is substantially equal to the
volume of lipid e solution introduced thereto. As a non-limiting example, a lipid vesicle
on in 45% l when uced into the collection vessel containing an equal volume of
dilution buffer will ageously yield smaller particles.
In yet another embodiment, the present ion provides nucleic ipid particles
(cg, SNALP) produced via an in—line dilution process in which a third reservoir containing dilution
buffer is fluidly d to a second mixing region. In this ment, the lipid vesicle (e.g.,
liposome) solution formed in a first mixing region is immediately and directly mixed with on
buffer in the second mixing region. In preferred aspects, the second mixing region includes a T—
connector arranged so that the lipid vesicle on and the dilution buffer flows meet as opposing
180° flows; however, connectors providing shallower angles can be used, e.g., from about 27° to about
180° (e.g., about 90"). A pump mechanism delivers a controllable flow of buffer to the second mixing
region. In one aspect, the flow rate of dilution buffer provided to the second mixing region is
controlled to be ntially equal to the flow rate of lipid vesicle solution introduced thereto from
the first mixing region. This ment advantageously allows for more control of the flow of
dilution buffer mixing with the lipid vesicle solution in the second mixing region, and therefore also
the concentration of lipid vesicle solution in buffer throughout the second mixing process. Such
W0 2013/052677 PCT/U52012/058770
control of the dilution buffer flow rate advantageously allows for small particle size formation at
reduced concentrations.
These processes and the apparatuses for carrying out these direct dilution and in-line
dilution processes are described in detail in US. Patent Publication No. 20070042031, the disclosure
of which is herein incorporated by reference in its entirety for all purposes.
The nucleic acid-lipid particles formed using the direct on and in—line dilution
processes typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150
nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to
about 110 nm, from about 70 nm to about 100 nm, from about 80 mn to about 100 nm, from about 90
nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70
nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35
nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100
nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any
fraction thereof or range therein). The particles thus formed do not aggregate and are optionally sized
to achieve a uniform particle size.
If needed, the lipid particles of the invention (e.g., SNALP) can be sized by any of the
methods available for sizing liposomes. The sizing may be conducted in order to achieve a desired
size range and relatively narrow distribution of particle sizes.
Several ques are available for sizing the particles to a desired size. One sizing
, used for liposomes and equally able to the present particles, is described in US. Patent
No. 4,737,323, the disclosure of which is herein incorporated by reference in its entirety for all
purposes. ting a le suspension either by bath or probe sonication produces a progressive
size reduction down to particles of less than about 50 nm in size. Homogenization is r method
which relies on shearing energy to fragment larger particles into smaller ones. In a typical
nization procedure, les are recirculated through a standard emulsion homogenizer until
selected particle sizes, typically between about 60 and about 80 nm, are observed. In both methods,
the le size distribution can be monitored by conventional laser-beam particle size discrimination,
or QELS.
Extrusion of the particles through a small—pore polycarbonate membrane or an
asymmetric ceramic membrane is also an effective method for reducing le sizes to a relatively
well-defined size distribution. Typically, the suspension is cycled through the membrane one or more
times until the desired particle size distribution is achieved. The particles may be extruded through
successively smaller—pore membranes, to e a gradual reduction in size.
In some embodiments, the nucleic acids present in the particles are densed as
described in, e.g., US. Patent Application No. 09/744,103, the disclosure of which is herein
incorporated by reference in its entirety for all purposes.
] In other embodiments, the methods may further comprise adding pid polycations
which are useful to effect the lipofection of cells using the present compositions. Examples of
suitable non—lipid polycations include, hexadimethrine bromide (sold under the brand name
POLYBRENE®, from Aldrich Chemical Co., Milwaukee, sin, USA) or other salts of
hexadimethrine. Other suitable polycations e, for example, salts of poly—L-ornithine, poly—L-
ne, poly—L—lysine, poly—D-lysine, polyallylamine, and polyethyleneimine. Addition of these
salts is preferably after the particles have been formed.
In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios) in a formed
nucleic acid—lipid particle (e.g., SNALP) will range from about 0.01 to about 0.2, from about 0.05 to
about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about
0.08. The ratio of the starting materials (input) also falls within this range. In other embodiments, the
particle preparation uses about 400 pg nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass
ratio of about 0.01 to about 0.08 and, more preferably, about 0.04, which corresponds to 1.25 mg of
total lipid per 50 pg of nucleic acid. In other red embodiments, the particle has a nucleic
acid:lipid mass ratio of about 0.08.
In other ments, the lipid to nucleic acid ratios (mass/mass ratios) in a formed
nucleic acid—lipid particle (e.g., SNALP) will range from about 1 (1:1) to about 100 (100:1), from
about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about
50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5
(5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),
from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to
about 25 (25:1), from about 5 (5:1) to about 20 , from about 5 (5:1) to about 15 (15:1), from
about 5 (5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:l),10(10:1),11(1121),
12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1), 16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21
(21:1), 22 (22:1), 23 (23:1), 24 (24:1), or 25 (25: 1), or any fraction thereof or range therein. The ratio
of the starting materials (input) also falls within this range.
[00302] As previously discussed, the conjugated lipid may r include a CPL. A variety of
general methods for making SNALP-CPLs (CPL-containing SNALP) are discussed herein. Two
general techniques include the “post—insertion” technique, that is, insertion of a CPL into, for
example, a pre—formed SNALP, and the “standard” que, wherein the CPL is included in the
lipid e during, for e, the SNALP formation steps. The nsertion technique results in
SNALP having CPLs mainly in the external face of the SNALP r membrane, whereas standard
techniques provide SNALP having CPLs on both internal and external faces. The method is
especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for
vesicles containing PEG—lipids (such as PEG-DAAs and PEG-DAGs). Methods of making SNALP-
CPLs are taught, for example, in US. Patent Nos. 5,705,385; 6,586,410; 501; 6,534,484; and
6,852,334; US. Patent Publication No. 72121; and PCT Publication No. WO 13, the
disclosures ofwhich are herein incorporated by reference in their entirety for all purposes.
VII. Kits
The present invention also provides lipid particles (e.g., SNALP) in kit form. In some
embodiments, the kit ses a container which is compartmentalized for holding the various
elements of the lipid particles (e.g., the active agents or therapeutic agents such as nucleic acids and
the individual lipid components of the particles). Preferably, the kit comprises a container (e.g., a vial
or ampoule) which holds the lipid particles of the ion (e.g., SNALP), wherein the particles are
produced by one of the processes set forth herein. In certain embodiments, the kit may further
comprise an endosomal membrane destabilizer (e.g., calcium ions). The kit typically contains the
particle compositions of the invention, either as a suspension in a pharmaceutically acceptable carrier
or in dehydrated form, with instructions for their rehydration (if lyophilized) and administration.
The SNALP formulations of the present invention can be tailored to preferentially target
ular cells, tissues, or organs of interest. ential targeting of SNALP may be d out by
controlling the composition of the SNALP itself. In particular embodiments, the kits of the invention
comprise these lipid particles, wherein the les are present in a container as a suspension or in
dehydrated form.
In certain instances, it may be desirable to have a targeting moiety attached to the surface
of the lipid particle to further enhance the ing of the particle. Methods of attaching targeting
moieties (e.g., antibodies, ns, etc.) to lipids (such as those used in the t particles) are
known to those of skill in the art.
VIII. Administration of Lipid Particles
Once formed, the lipid particles of the invention (e. g., SNALP) are particularly useful for
the introduction of nucleic acids (e.g., interfering RNA such as dsRNA) into cells. Accordingly, the
present invention also provides methods for ucing a nucleic acid (e.g., interfering RNA) into a
cell. In particular embodiments, the nucleic acid (e.g., interfering RNA) is introduced into an infected
cell such as reticuloendothelial cells (e.g., macrophages, monocytes, etc.) as well as other cell types,
including fibroblasts, endothelial cells (such as those fining the interior surface of blood vessels),
2012/058770
and/or platelet cells. The methods may be carried out in vitro or in vivo by first forming the particles
as described above and then contacting the particles with the cells for a period of time sufficient for
delivery ofthe interfering RNA to the cells to occur.
The lipid particles of the invention (e.g., SNALP) can be adsorbed to almost any cell
type with which they are mixed or contacted. Once adsorbed, the particles can either be endocytosed
by a portion of the cells, exchange lipids with cell membranes, or fuse with the cells. er or
incorporation of the nucleic acid (e.g., interfering RNA) portion of the le can take place via any
one of these ys. In particular, when fusion takes place, the particle membrane is integrated into
the cell membrane and the contents of the particle combine with the intracellular fluid.
[00308] The lipid particles of the invention (e.g., SNALP) can be administered either alone or in
a mixture with a ceutically acceptable carrier (e.g., physiological saline or phosphate buffer)
selected in ance with the route of administration and standard pharmaceutical practice.
Generally, normal buffered saline (rag, 135—150 mM NaCl) will be employed as the pharmaceutically
acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3%
glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein,
globulin, etc. Additional suitable carriers are described in, e.g., REMINGTON’S
PHARMACEUTICAL SCIENCES, Mack Publishing Company, elphia, PA, 17th ed. (1985).
As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents,
antibacterial and ngal agents, isotonic and absorption delaying agents, buffers, carrier solutions,
suspensions, colloids, and the like. The phrase aceutically acceptable” refers to molecular
entities and compositions that do not produce an allergic or r untoward on when
administered to a human.
The pharmaceutically acceptable carrier is lly added following lipid particle
formation. Thus, after the lipid particle (e.g., SNALP) is formed, the particle can be diluted into
pharmaceutically acceptable carriers such as normal buffered saline.
The tration of particles in the pharmaceutical formulations can vary widely, i.e.,
from less than about 0.05%, usually at or at least about 2 to 5%, to as much as about 10 to 90% by
weight, and will be selected ily by fluid volumes, ities, etc, in accordance with the
particular mode of stration selected. For example, the concentration may be increased to lower
the fluid load associated with treatment. This may be particularly desirable in patients having
atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, particles
composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of
administration.
The pharmaceutical itions of the present ion may be sterilized by
conventional, well—known sterilization techniques. Aqueous solutions can be packaged for use or
filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a
sterile aqueous solution prior to administration. The compositions can contain pharmaceutically
acceptable auxiliary substances as required to imate physiological conditions, such as pH
adjusting and ing , tonicity adjusting agents and the like, for example, sodium acetate,
sodium lactate, sodium chloride, potassium chloride, and calcium chloride. onally, the particle
suspension may include lipid—protective agents which protect lipids against free-radical and lipid-
peroxidative damages on storage. ilic free-radical quenchers, such as alphatocopherol, and
water—soluble iron—specific chelators, such as ferrioxamine, are suitable.
In some embodiments, the lipid particles of the invention (e.g., SNALP) are particularly
useful in methods for the eutic delivery of one or more nucleic acids comprising an interfering
RNA sequence (e.g., siRNA). In particular, it is an object of this invention to provide in viva methods
for treatment of alcoholism in humans by domegulating or silencing the transcription and/or
translation of one or more ALDH isozyrnes.
A. In vivo Administration
] Systemic delivery for in vivo therapy, e.g., ry of a therapeutic c acid to a
distal target cell via body systems such as the circulation, has been achieved using c acid—lipid
particles such as those described in PCT Publication Nos. WO 196, W0 05/ 121348, W0
05/ 120152, and WO 04/002453, the disclosures of which are herein incorporated by reference in their
entirety for all purposes. The present invention also es fully encapsulated lipid particles that
protect the nucleic acid from nuclease ation in serum, are non—immunogenic, are small in size,
and are suitable for repeat dosing.
For in vivo administration, administration can be in any manner known in the art, e. g., by
injection, oral administration, inhalation (e.g., intransal or intratracheal), transdermal application, or
rectal administration. Administration can be accomplished via single or divided doses. The
pharmaceutical compositions can be administered parenterally, i.e., intraarticularly, intravenously,
intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical
itions are administered intravenously or intraperitoneally by a bolus injection (see, e.g., US.
Patent No. 5,286,634). Intracellular c acid delivery has also been discussed in Straubringer et
(11., Methods Enzymol., 1012512 (1983); Mannino et al., Biotechniques, 6:682 ; Nicolau et (1].,
Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res, 26:274 (1993). Still
other methods of administering lipid—based therapeutics are described in, for example, US. Patent
Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles can
2012/058770
be administered by direct injection at the site of disease or by injection at a site distal from the site of
disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc, Publishers, New
York. pp.70—71(l994)). The disclosures of the above—described references are herein incorporated by
reference in their entirety for all purposes.
In embodiments Where the lipid particles of the present invention (e.g., SNALP) are
stered intravenously, at least about 5%, 10%, 15%, 20%, or 25% of the total injected dose of
the particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection. In other
embodiments, more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% of the total
injected dose of the lipid particles is present in plasma about 8, 12, 24, 36, or 48 hours after injection.
In certain instances, more than about 10% of a plurality of the particles is present in the plasma of a
mammal about 1 hour after administration. In certain other instances, the ce of the lipid
particles is detectable at least about 1 hour after stration of the particle. In some embodiments,
the presence of a eutic nucleic acid such as an interfering RNA molecule is detectable in cells at
about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In other embodiments,
downregulation of expression of a target sequence, such as a viral or host sequence, by an interfering
RNA (e.g., siRNA) is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In
yet other embodiments, downregulation of expression of a target sequence, such as a viral or host
sequence, by an interfering RNA (e.g., siRNA) occurs entially in infected cells and/or cells
capable of being infected. In further embodiments, the presence or effect of an interfering RNA (e.g.,
siRNA) in cells at a site al or distal to the site of administration is detectable at about 12, 24,
48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after
administration. In additional embodiments, the lipid particles (e.g., SNALP) of the ion are
administered parenterally or intraperitoneally.
The compositions of the present invention, either alone or in combination with other
le components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be
administered via inhalation (e.g., intranasally or intratracheally) (see, Brigham et al., Am. J. Sci.,
298:278 (1989)). Aerosol formulations can be placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, e, nitrogen, and the like.
In n embodiments, the pharmaceutical itions may be delivered by intranasal
sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering nucleic acid
compositions directly to the lungs via nasal aerosol sprays have been described, e.g., in US. Patent
Nos. 5,756,353 and 5,804,212. Likewise, the ry of drugs using intranasal microparticle resins
and lysophosphatidyl-glycerol compounds (US. Patent 5,725,871) are also well—known in the
pharmaceutical arts. Similarly, transmucosal drug delivery in the form of a polytetrafluoroetheylene
W0 2013(052677 PCT/U52012/058770
support matrix is described in US. Patent No. 5,780,045. The disclosures of the above-described
patents are herein incorporated by nce in their entirety for all purposes.
Formulations suitable for parenteral administration, such as, for example, by
intraarticular (in the ), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can n
antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending
, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention,
compositions are preferably administered, for example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically, or intrathecally.
Generally, when administered intravenously, the lipid le formulations are
formulated with a suitable pharmaceutical r. Many pharmaceutically acceptable rs may be
ed in the compositions and methods of the present ion. le formulations for use in
the present invention are found, for example, in REMINGTON’S PHARMACEUTICAL SCIENCES,
Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). A variety of aqueous carriers may be
used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include
glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. Generally, normal
ed saline (135-150 mM NaCl) will be employed as the pharmaceutically acceptable carrier, but
other suitable carriers will suffice. These compositions can be sterilized by tional liposomal
sterilization techniques, such as filtration. The compositions may n pharmaceutically
acceptable auxiliary substances as required to approximate logical conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example,
sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, an
monolaurate, anolamine oleate, etc. These compositions can be sterilized using the techniques
referred to above or, alternatively, they can be produced under e conditions. The resulting
aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the
lyophilized preparation being ed with a sterile aqueous solution prior to administration.
In certain applications, the lipid particles disclosed herein may be red via oral
administration to the individual, The les may be incorporated with excipients and used in the
form of ingestible tablets, buccal s, troches, capsules, pills, lozenges, elixirs, mouthwash,
suspensions, oral sprays, syrups, wafers, and the like (see, e. g., US. Patent Nos. 5,641,515, 5,580,579,
and 5,792,451, the disclosures of which are herein incorporated by reference in their entirety for all
purposes). These oral dosage forms may also contain the following: binders, gelatin; excipients,
lubricants, and/or flavoring agents. When the unit dosage form is a capsule, it may contain, in
addition to the materials described above, a liquid carrier. Various other materials may be present as
WO 52677
coatings or to otherwise modify the physical form of the dosage unit. Of course, any material used in
preparing any unit dosage form should be ceutically pure and substantially non-toxic in the
amounts employed.
Typically, these oral formulations may contain at least about 0.1% of the lipid particles
or more, although the percentage of the particles may, of course, be varied and may conveniently be
between about 1% or 2% and about 60% or 70% or more of the weight or volume of the total
formulation. Naturally, the amount of les in each therapeutically useful composition may be
prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half—life, route of administration, product shelf
life, as well as other pharmacological considerations will be contemplated by one skilled in the art of
preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens
may be ble.
Formulations suitable for oral administration can consist of: (a) liquid solutions, such as
an effective amount of a packaged therapeutic nucleic acid (e.g., ering RNA) ded in
diluents such as water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a
predetermined amount of a therapeutic nucleic acid (e.g., interfering RNA), as s, solids,
granules, or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms
can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,
potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate,
stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, ning
agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharrnaceutically compatible
carriers. Lozenge forms can comprise a therapeutic nucleic acid (e. g., interfering RNA) in a flavor,
e. g., sucrose, as well as pastilles comprising the therapeutic nucleic acid in an inert base, such as
gelatin and glycerin or e and acacia emulsions, gels, and the like containing, in addition to the
therapeutic nucleic acid, carriers known in the art.
In r example of their use, lipid particles can be incorporated into a broad range of
topical dosage forms. For instance, a suspension containing c acid-lipid particles such as
SNALP can be formulated and administered as gels, oils, emulsions, topical , ,
ointrnents, s, foams, mousses, and the like.
[00324] When preparing pharmaceutical preparations of the lipid particles of the invention, it is
preferable to use quantities of the particles which have been d to reduce or eliminate empty
les or particles with therapeutic agents such as nucleic acid associated with the al surface.
2012/058770
The methods of the present invention may be practiced in a variety of hosts. red
hosts include mammalian species, such as primates (e.g., humans and chimpanzees as well as other
nonhuman primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice),
lagomorphs, and swine.
The amount of particles administered will depend upon the ratio of therapeutic c
acid (e.g., interfering RNA) to lipid, the ular therapeutic nucleic acid used, the disease or
disorder being treated, the age, weight, and condition of the patient, and the judgment of the clinician,
but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably
between about 0.1 and about 5 mg/kg of body weight, or about 108—1010 les per administration
(e.g., injection).
B. In vitro Administration
For in vitro applications, the delivery of therapeutic nucleic acids (e.g., interfering RNA)
can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and
of any tissue or type. In preferred embodiments, the cells are animal cells, more preferably
mammalian cells, and most preferably human cells.
] Contact between the cells and the lipid particles, when d out in vitro, takes place in
a biologically compatible medium. The tration of particles varies widely depending on the
particular application, but is generally between about 1 umol and about 10 mmol. Treatment of the
cells with the lipid particles is generally carried out at physiological temperatures (about 37°C) for
2O periods oftime of from about 1 to 48 hours, preferably of from about 2 to 4 hours.
In one group of preferred embodiments, a lipid particle suspension is added to 60~80%
confluent plated cells having a cell density of from about 103 to about 105 cells/ml, more preferably
about 2 x 104 ml. The tration of the suspension added to the cells is preferably of from
about 0.01 to 0.2 ug/ml, more preferably about 0.1 ug/ml.
[00330] To the extent that tissue culture of cells may be required, it is well-known in the art. For
example, Freshney, e of Animal Cells, a Manual of Basic Technique, 3rd Ed., Wiley—Liss, New
York (1994), Kuchler er al., Biochemical Methods in Cell Culture and gy, Dowden, Hutchinson
and Ross, Inc. (1977), and the references cited therein provide a general guide to the culture of cells.
Cultured cell s often will be in the form of monolayers of cells, although cell suspensions are
also used.
Using an Endosomal Release Parameter (ERP) assay, the delivery efficiency of the
SNALP or other lipid particle of the invention can be optimized. An ERP assay is described in detail
in US Patent Publication No. 20030077829, the disclosure of which is herein incorporated by
nce in its entirety for all purposes. More particularly, the e of an ERP assay is to
distinguish the effect of various cationic lipids and helper lipid components of SNALP or other lipid
particle based on their relative effect on binding/uptake or fusion with/destabilization of the
endosomal membrane. This assay allows one to ine quantitatively how each component of the
SNALP or other lipid particle affects delivery efficiency, thereby zing the SNALP or other lipid
particle. Usually, an ERP assay measures expression of a reporter protein (e.g., luciferase, [3—
galactosidase, green fluorescent n (GFP), etc), and in some instances, a SNALP formulation
optimized for an sion plasmid will also be appropriate for encapsulating an interfering RNA. In
other instances, an ERP assay can be adapted to measure downregulation of transcription or
translation of a target sequence in the presence or absence of an interfering RNA (e.g., siRNA). By
ing the ERPs for each of the various SNALP or other lipid particles, one can readily determine
the zed system, e.g., the SNALP or other lipid particle that has the greatest uptake in the cell.
C. Cells for Delivery of Lipid Particles
] The compositions and methods of the present invention are particularly well suited for
treating lism by targeting ALDH gene expression in vivo. The present invention can be
practiced on a wide variety of cell types from any vertebrate species, ing mammals, such as, e.g,
canines, s, equines, s, ovines, caprines, rodents (e.g., mice, rats, and guinea pigs),
lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).
D. Detection of Lipid Particles
In some embodiments, the lipid particles of the present invention (e.g., SNALP) are
detectable in the subject at about l, 2, 3, 4, 5, 6, 7, 8 or more hours. In other embodiments, the lipid
particles of the present invention (e.g., SNALP) are detectable in the subject at about 8, 12, 24, 48, 60,
72, or 96 hours, or about 6, 8, 10, 12, l4, 16, 18, 19, 22, 24, 25, or 28 days after administration of the
particles. The presence of the particles can be detected in the cells, tissues, or other biological
samples from the subject. The particles may be detected, e.g., by direct detection of the particles,
detection of a therapeutic nucleic acid such as an interfering RNA (e.g., siRNA) sequence, detection
of the target sequence of interest (Len, by detecting expression or reduced expression of the ce
of interest), detection of a compound ted by an EBOV protein (e. g., interferon), detection of
viral load in the t, or a combination thereof.
1. Detection of Particles
Lipid particles of the invention such as SNALP can be detected using any method known
in the art. For example, a label can be coupled directly or indirectly to a component of the lipid
W0 2013;052677 2012/058770
particle using methods well-known in the art. A wide variety of labels can be used, with the choice of
label depending on sensitivity required, ease of conjugation with the lipid particle component,
stability requirements, and available instrumentation and disposal provisions. Suitable labels e,
but are not limited to, spectral labels such as fluorescent dyes (e.g., fluorescein and derivatives, such
as fluorescein isothiocyanate (FITC) and Oregon GreenTM; rhodamine and derivatives such Texas red,
hodimine isothiocynate ), eta, digoxigenin, biotin, phycoerythrin, AMCA, CyDyesTM,
and the like; radiolabels such as 3H, 125I, 358, MC, 321’, 33P, eta; enzymes such as horse radish
peroxidase, alkaline phosphatase, eta; spectral colorimetric labels such as colloidal gold or colored
glass or plastic beads such as polystyrene, polypropylene, latex, etc. The label can be detected using
any means known in the art.
2. Detection of Nucleic Acids
Nucleic acids (e.g., interfering RNA) are detected and quantified herein by any of a
number of means well-known to those of skill in the art. The detection of nucleic acids may proceed
by well-known methods such as Southern analysis, rn analysis, gel electrophoresis, PCR,
radiolabeling, scintillation counting, and affinity chromatography. Additional analytic biochemical
methods such as spectrophotometry, raphy, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffiision
tography may also be employed.
The selection of a c acid hybridization format is not critical. A variety of nucleic
acid hybridization s are known to those skilled in the art. For example, common formats
include sandwich assays and competition or displacement . Hybridization techniques are
generally described in, e.g., ic Acid Hybridization, A Practical Approach,” Eds. Hames and
Higgins, IRL Press (1985).
The sensitivity of the hybridization assays may be enhanced through the use of a nucleic
acid amplification system which multiplies the target nucleic acid being detected. In vitro
amplification techniques le for amplifying sequences for use as molecular probes or for
generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques
sufficient to direct persons of skill through such in vitro cation methods, including the
rase chain reaction (PCR), the ligase chain reaction (LCR), QB-replicase amplification, and
other RNA polymerase mediated techniques (e.g., NASBATM) are found in ok el al, In
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2000); and Ausubel
et al, SHORT PROTOCOLS 1N LAR BIOLOGY, eds., Current Protocols, Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc. (2002); as well as US. Patent No. 4,683,202; PCR
Protocols, A Guide to Methods and Applications (Innis et al. eds.) Academic Press Inc. San Diego,
PCT/U52012/058770
CA (1990); m & Levinson (October 1, 1990), C&EN 36; The Journal OfNIH Research, 3:81
(1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 8621173 (1989); li et (21., Proc. Natl. Acad.
Sci. USA, 87:1874 (1990); Lomell et al., J. Clin. Chem, 35:1826 (1989); Landegren et al., Science,
241:1077 (1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene, 4:560 (1989);
Barringer et al., Gene, 89:117 (1990); and Sooknanan and Malek, Biotechnology, 132563 (1995).
Improved methods of cloning in vitro amplified nucleic acids are described in US. Pat. No.
,426,039. Other methods described in the art are the nucleic acid sequence based amplification
(NASBATM, Cangene, Mississauga, Ontario) and QB—replicase systems. These systems can be used to
directly fy mutants where the PCR or LCR primers are designed to be extended or ligated only
when a select sequence is present. Alternatively, the select sequences can be generally amplified
using, for e, nonspecific PCR primers and the amplified target region later probed for a
specific sequence indicative of a mutation. The disclosures of the above—described references are
herein incorporated by reference in their entirety for all purposes.
Nucleic acids for use as probes, e. g., in in vitro amplification methods, for use as gene
probes, or as inhibitor components are typically synthesized chemically according to the solid phase
phosphoramidite triester method described by Beaucage et al., Tetrahedron Lens, 2221859 1862
(1981), e.g., using an automated synthesizer, as described in Needham VanDevanter et al., Nucleic
Acids Res, 1226159 (1984). Purification of polynucleotides, where necessary, is typically performed
by either native acrylamide gel electrophoresis or by anion exchange HPLC as described in Pearson et
al., J. Chrom, 2552137 149 (1983). The sequence of the tic polynucleotides can be d
using the chemical degradation method of Maxam and Gilbert (1980) in Grossman and Moldave
(eds) Academic Press, New York, s in Enzymology, 65:499.
] An ative means for determining the level of ription is in situ hybridization.
In situ hybridization assays are nown and are generally described in r et al., Methods
Enzymol., 1522649 (1987). In an in situ hybridization assay, cells are fixed to a solid support,
typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells
are then contacted with a hybridization solution at a moderate temperature to permit annealing of
specific probes that are labeled. The probes are preferably labeled with radioisotopes or cent
reporters.
IX. es
The present invention will be described in r detail by way of specific examples.
The following examples are offered for illustrative es, and are not intended to limit the
invention in any manner. Those of skill in the art will readily recognize a variety of noncritical
parameters which can be changed or modified to yield essentially the same results.
2012/058770
Example 1.
This examples demonstrates that serum stable nucleic acid-lipid particles (SNALP)
containing an siRNA that targets ALDHZ reduces aldehyde dehydrogenase 2 gene expression in a
murine hepatocyte cell line in vitro.
Materials:
All siRNA molecules used in these studies were chemically synthesized and annealed
using standard procedures.
Aldehyde dehydrogenase 2 (ALDH2)~targeting siRNA sequences used in this study:
SiRNA
Sense Strand (5 , , - to 3 ) Antisense Strand (5 , to 3 ,)
rUrUrUrCrUrCrAmGrUrArUrArUrCmUrUrGrUmUrGrG
(SE0 ID N023 SE0 ID NO:4
(SE0 ID N0:5 (SE0 ID N026
rUrUrCrGrUrCrAmUrAICrAICrAIUmUrCrUrCrCmUrG
(SE0 ID N027) (SE0 ID N028)
(SE0 ID N029) (SE0 ID NO:10)
Where ‘r’ indicates a ribonucleotide, ‘m’ indicates a 2’-O-methylated ribonucleotide, and
lack of either prefix tes a deoxynucleotide.
] Additionally, a non-targeting siRNA was ed in the study as a control. This siRNA
s firefly luciferase gene and is not intended to have any specific gene silencing activity in
mammalian cells.
Methods:
[00346] The nucleic acid-lipid particles were composed of the following “1 :57”
formulation: 1.4% PEGQOOO~C-DMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3%
cholesterol. Typically, in the 1:57 formulation, the amount of DLinDMA was 57.1 mol % i 5
mol %, and the amount of lipid conjugate was 1.4 mol % i 0.5 mol %, with the e of the
1:57 formulation being made up of non—cationic lipid (e.g., phospholipid, cholesterol, or a
mixture of the two) with final siRNA/lipid ratio of approximately 0.11 to 0.15 (wt/wt). Upon
formation of the siRNA-loaded particles, mean particle sizes were 85 — 100 nm in diameter.
ian Cell ents and mRNA Analysis:
y hepatocytes were isolated from a mouse and maintained as adherent cultures in
96-well plates. ALDH—Z targeting or non—targeting control SNALP were added to the culture medium
PCT/U82012/058770
at siRNA concentrations of 0.03125 to 0.5 ug/mL; each treatment ion was performed in
triplicate wells. After approximately 24 h incubation in the presence of SNALP, cells were harvested
and lysed for mRNA analysis. Mouse ALDH2 mRNA in cell lysate was measured using a branched
DNA assay (QuantiGene®, Affymetrix) with normalization against mouse glyceraldehyde 3~
phosphate dehydrogenase (GAPDH).
Results:
Table A shows the gene ing activity of nucleic acid treatments (mean of
triplicate wells, 3: standard deviation) relative to the amount of GAPDH-normalized ALDH2 mRNA
measured in untreated culture wells arbitrarily set at “100.0%” (mean of nine replicate wells, is 6.9%
standard deviation).
Table A
0.03125 _mL 0.125 mL
Conclusion:
[00349] All four siRNA formulations that target mouse ALDH2 demonstrated gene silencing
activity in isolated y mouse hepatocytes, in a dose—responsive manner. Of the four tested,
duplex l was the most active and duplex 4 was the least active.
Example 2
This example shows that SNALP that include an siRNA that targets ALDH2 reduce
aldehyde dehydrogenase 2 gene expression in a whole—animal system via an enous route of
administration.
Materials:
Mouse ALDH—2 siRNA duplex 1 and non—targeting control siRNA used in this study are
described in e 1.
Methods:
SNALP formulations were prepared as described in Example 1.
2012/058770
Animal Treatments:
] Female BALB/c mice were obtained from Harlan Labs. Animals were administered a
single dose of SNALP-formulated siRNA, or phosphate-buffered saline, via 10 mL/kg intravenous
injection in the lateral tail vein. The administered siRNA dosage was either 0.025, 0.050 or 0.25 mg
per kg body . Approximately 48 h after SNALP injection, animals were euthanized and liver
tissue was collected into RNAlater® RNA stabilizing solution.
Tissue Analysis:
Liver tissues were analyzed for mouse ALDH2 mRNA levels normalized against against
GAPDH mRNA levels using the QuantiGene® branched DNA assay (Panomics, Freemont, CA; now
part ofAffymetrix) essentially as bed in Judge et (11., 2006, Molecular Therapy 13:494.
Results:
Table B shows the gene silencing activity of SNALP treatments (mean of 3 animals, i
rd deviation) relative to the amount of normalized liver ALDH2 mRNA measured in
control PBS-treated animals arbitrarily set at “100.0%” (mean of six animals, i 6.3% standard
deviation).
Table B
ent Liver ALDH2zGAPD mRNA Ratio
Relative to PBS Control Grou Mean
Formulated siRNA 1 0 025 m/k 70.2 i 18.4 %
Formulated siRNA 1, 0.050 m k 35.9 :1: 3.7 %
Formulated siRNA l, 0.25 m k 10.8 :1: 0.9 %
Non-tar_etin control siRNA, 0.25 mH/k 86.8 d: 6.3)%
Conclusion:
[00356] A single intravenous administration of SNALP—formulated siRNA 1 targeting mouse
ALDH2 resulted in ALDH2 gene silencing activity in mice, in a dose—responsive manner.
Example 3.
] This Example describes a method for making stable nucleic acid-lipid particles of
the present invention.
[00358] siRNA molecules are chemically synthesized and annealed using standard procedures.
In some embodiments, siRNA les are encapsulated into serum-stable nucleic
acid-lipid particles composed of the following lipids: (1) the lipid conjugate O—C—DMA (3-N-
[(-methoxypoly(ethylene glycol)2000)carbam0yl]-l,2-dimyristyloxypropylamine); (2) a cationic lipid,
e.g. DLinDMA; (3) the phospholipid DPPC (1,2-dipalmitoyl-sn-glyceroph0sphocholine) (Avanti
Polar Lipids; Alabaster, AL); and (4) synthetic cholesterol (Sigma—Aldrich Corp; St. Louis, MO) in
the molar ratio l.4:57.1:7.1:34.3, respectively. In other words, siRNA molecules are encapsulated
into nucleic acid—lipid particles of the following “1 :57” formulation: 1.4% PEG2000-C-DMA; 57.1%
cationic lipid; 7.1% DPPC; and 34.3% cholesterol. It should be understood that the 1:57 formulation
is a target formulation, and that the amount of lipid (both cationic and tionic) present and the
amount of lipid conjugate present in the formulation may vary. Typically, in the 1:57 formulation, the
amount of cationic lipid is 57.1 mol % i 5 mol %, and the amount of lipid ate is 1.4 mol % :t
0.5 mol %, with the balance of the 1:57 ation being made up of non—cationic lipid (e.g.,
phospholipid, cholesterol, or a mixture of the two). ations are made using the process
described in United States Patent Application /0042031, which is incorporated herein by
reference in its entirety. Upon formation the nucleic acid—lipid particles are dialyzed against PBS and
filter sterilized h a 0.2 pm filter before use. Mean particle sizes can be in the range of 75 — 90
nm, with final siRNA/lipid ratio of 0.15 (wt/wt).
Example 4.
This Example describes mediated reduction of ALDH2 gene expression
in human cells in vitro.
Materials:
All siRNA molecules used in these studies were chemically synthesized and
ed using standard procedures.
Table A in this Example shows the aldehyde dehydrogenase 2 (ALDH2)-
targeting siRNA sequences used in the experiments described in this Example:
PCT/U82012/058770
siRNA
Sense Strand (5 , , ) Antisense Strand (5‘ ,
to 3 to 3 ),
erUIG IGrAmU rGrArA rArCmU rCrArG mUrUrU rAxArG rUrArA rArCrU mGrAmG rUrUrU rCrAmU rCrCrA rCrCrU
(SE0 ID NO:11) (SE0 ID NO:12)
mGrUmG l‘Gl‘AIU mGrArA IAICI'U rCrAmG rUrUrU rArArG rUrAIA rArCrU mGrAmG rUrUrU rCrAmU rCrCrA l‘Cl‘CIU
(SEQ ID NO:13 (SE. ID NO:12
erGrA rUmGrA rArArC rUrCrA mGrUmU rUrAIA rGrArA rCrUrU rAIArA rCmUrG rArGrU mUrUrC rAmUrC rCrArC
(SE0 ID N0214 SE0 ID No:15
mGrGrA rUmGrA rArArC rUrCrA erUrU mUrAIA rGrArA rCrUrU rArArA ICmUrG rAIGrU mUrUrC rAmUrC xCrAIC
(SEO ID N0216) (SEO ID NO:15)
rCmUrG rUrCmU IUrCrA rCrArA rAmGrG rAmUrU rUrGrG
(SE0 ID N0217) (SE0 ID NO:18)
rCmUrG mUrCrU mUrCrA rCrArA rAerG rAIUrU rUrGrG
SE0 ID NO:19 SE0 ID N0218
Where ‘r’ indicates a ribonucleotide and ‘m’ indicates a ethylated
ribonucleotide
Methods:
The nucleic acid-lipid particles were composed of the following “1:57”
formulation: 1.4% O—C—DMA; 57.1% DLinDMA; 7.1% DPPC; and 34.3%
cholesterol. Typically, in the 1:57 formulation, the amount of A was 57.1 mol % d: 5
mol %, and the amount of lipid conjugate was 1.4 mol % d: 0.5 mol %, with the balance of the
1:57 formulation being made up of tionic lipid (e.g., olipid, cholesterol, or a
mixture of the two) with final siRNA/lipid ratio of approximately 0.11 to 0.15 (wt/wt). Upon
formation of the siRNA-loaded particles, mean particle sizes were 85 — 100 nm in diameter.
Mammalian Cell Treatment and mRNA Analysis: HepG2 (human
hepatocellular carcinoma) cells were maintained as adherent cultures in 96—well plates.
ALDHZ-targeting or non—targeting control nucleic acid—lipid particles were added to the
culture medium at siRNA concentrations of 3.91 and 15.63 ng/mL; each treatment condition
was performed in duplicate wells. After approximately 48 hours nucleic acid—lipid le
incubation, cells were harvested and lysed for mRNA analysis. Human ALDHZ mRNA in
cell lysate was measured using a branched DNA assay (Panomics, Freemont, CA; now part of
Affymetrix) with normalization against human glyceraldehyde 3-phosphate dehydrogenase
(GAPDH).
Results:
] Table B in this Example shows the gene silencing activity of nucleic acid-lipid
particle treatments (mean of duplicate wells, i- standard deviation) are described as
percentage GAPDH-normalized ALDHZ mRNA relative to the amount of GAPDH—
normalized ALDH2 mRNA measured in untreated culture wells arbitrarily set at %" (mean of 2
replicate wells, 3: 7.4% standard deviation).
Table B
3.9 n mL 15.63 n_/mL
19.8 i: 3.4)% 7.5 i 0.6)%
(18.9 d: 0.8)% 6.6 d: 0.6)%
(34.2 i 0.0)% 11.2 i 1.4 %
(22.6 i: 0.5)% 7.6 :t 0.9)%
21.9 i 3.2)% 6.8 i 0.7)%
(24.1 :1: 2.2)% (7.7 :t 0.6)%
Non—taretin; control 85.5 :1: 7.1 % (90.2 i 3.8)%
Conclusions:
] All six siRNAs, formulated within lipid particles, silenced ALDH2 activity in human
HepG2 cells, in a dose—responsive manner. Of the six siRNA that were tested, siRNA 2 was the most
active and siRNA 3 was the least active.
] All of the US. patents, US. patent application publications, US. patent applications,
foreign patents, foreign patent applications and non-patent publications referred to in this cation
are incorporated herein by reference, in their entirety to the extent not inconsistent with the present
description.
From the foregoing it will be appreciated that, although specific embodiments of the
invention have been described herein for purposes of illustration, various modifications may be made
without ing from the range and scope of the invention.
Claims (40)
1. A composition comprising an interfering RNA that silences aldehyde dehydrogenase (ALDH) gene expression, wherein the interfering RNA ses a sense strand and a complementary antisense strand, and wherein the interfering RNA comprises a double-stranded region of about 15 to about 60 nucleotides in length, wherein the interfering RNA is ed from the group consisting of siRNA Identifier l (SEQ ID NOs:11 & 12), siRNA fier 2 (SEQ ID NOszl3 & 12), siRNA Identifier 3 (SEQ ID NOszl4 & 15), siRNA Identifier 4 (SEQ ID NOs:16 & 15), siRNA Identifier S (SEQ ID NOszl7 & l8) and siRNA Identifier 6 (SEQ ID NOszl9 & 18).
2. The composition of claim 1, wherein the interfering RNA comprises at least one modified nucleotide in the double—stranded region.
3. The composition of claim 2, wherein less than about 30% of the nucleotides in the double-stranded region comprise modified tides.
4. The composition of claim 2, wherein the modified nucleotide is a nethyl (2'OMe) nucleotide.
5. The composition of claim 4, n the 2'OMe nucleotide comprises a 2'OMe- guanosine nucleotide, a uridine nucleotide, or mixtures thereof.
6. The composition of claim 1, r comprising a pharmaceutically acceptable carrier.
7. The composition of claim 1 wherein the interfering RNA silences aldehyde dehydrogenaseZ (ALDHZ) gene expression.
8. A nucleic acid— lipid particle comprising: (a) an ering RNA of claim 1 ; (b) a cationic lipid; and (c) a non-cationic lipid.
9. The nucleic acid- lipid particle of claim 8, n the cationic lipid is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2—dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), l,2-di-y-linolenyloxy—N,N-dimethylaminopropaneI (y- DLenDMA), a salt thereof, or a mixture thereof.
10. The nucleic acid- lipid particle of claim 8, wherein the cationic lipid is 2,2-dilinoleyl- 4-(2-dimethylamin0ethyl)-[1 ,3]—dioxolane (DLin—K—CZ—DMA), 2,2—dilinoleyl-4— dimethylaminomethyl—[l,3]—dioxolane (DLin—K-DMA), a salt thereof, or a mixture thereof.
11. The nucleic acid- lipid particle of claim 8, wherein the cationic lipid is (6Z,9Z,2SZ,31Z)-heptatriaconta—6,9,28,31-tetraen-l9-yl 4-(dimethylamino) butanoate (DLin-M-C3~ DMA), dilinoleylmethyl—3—dimethylaminopropionate (DLin—M—CZ-DMA), a salt thereof, or a mixture thereof.
12. The nucleic acid- lipid particle of claim 8, wherein the non—cationic lipid is a phospholipid.
13. The nucleic acid—lipid particle of claim 8, wherein the non-cationic lipid is cholesterol or a tive thereof.
14. The nucleic acid-lipid particle of claim 8, wherein the non-cationic lipid is a mixture of a olipid and cholesterol or a derivative f.
15. The c acid- lipid particle of claim 12, wherein the phospholipid comprises dipalmitoylphosphatidylcholine , roylphosphatidylcholine (DSPC), or a mixture thereof.
16. The nucleic acid-lipid particle of claim 8, wherein the non-cationic lipid is a mixture of DPPC and cholesterol.
17. The nucleic acid-lipid particle of claim 8, further comprising a conjugated lipid that inhibits aggregation of particles.
18. The nucleic acid- lipid particle of claim 17, wherein the conjugated lipid that inhibits aggregation of particles is a polyethyleneglycol (PEG)—lipid conjugate.
19. The nucleic acid~ lipid particle of claim 18, wherein the PEG-lipid conjugate is selected from the group consisting of a PEG—diacylglycerol (PEG—DAG) conjugate, a PEG- dialkyloxypropyl (PEG-BAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG—Cer) conjugate, and a mixture thereof.
20. The nucleic ipid particle of claim 18, wherein the PEG-lipid conjugate is a PEG-DAA conjugate.
21. The c ipid particle of claim 20, wherein the PEG-DAA ate is selected from the group consisting of a PEG-didecyloxypropyl (C10) conjugate, a PEG— yloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG- dipalmityloxypropyl (C16) conjugate, a PEG-distearyloxypropyl (C18) conjugate, and a mixture thereof.
22. The nucleic acid-lipid particle of claim 8, wherein the interfering RNA is fully encapsulated in the particle.
23. The nucleic acid-lipid particle of claim 8, wherein the particle has a 1ipid:interfering RNA mass ratio of from about 5:1 to about 15:1.
24. The c acid—lipid particle of claim 8, wherein the particle has a median diameter of from about 30 nm to about 150 nm.
25. The nucleic acid-lipid particle of claim 8, wherein the cationic lipid comprises from about 50 mol % to about 65 mol % of the total lipid present in the particle.
26. The nucleic acid-lipid particle of claim 8, wherein the non-cationic lipid comprises a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the olipid comprises from about 4 mol % to about 10 mol % of the total lipid t in the le and the cholesterol or derivative thereof comprises from about 30 mol % to about 40 mol % of the total lipid present in the particle.
27. The nucleic acid-lipid particle of claim 26, wherein the phospholipid comprises from about 5 mol % to about 9 mol % of the total lipid t in the particle and the cholesterol or derivative f comprises from about 32 mol °/o to about 37 mol % of the total lipid present in the particle.
28. The nucleic acid- lipid particle of claim 17, wherein the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 2 mol % of the total lipid present in the particle.
29. A pharmaceutical composition comprising a nucleic acid- lipid particle of claim 8 and a pharmaceutically acceptable r.
30. Use of a nucleic acid— lipid particle of claim 8 in the manufacture of a medicament for silencing ALDH gene expression in a mammal.
3 l. The use of claim 30, wherein the particle is stered Via a systemic route.
32. The use of claim 30, wherein the mammal is a human.
33. The use of claim 32, n the human is suffering from alcoholism.
34. The use of claim 30, wherein administration of the particle reduces ALDH RNA levels in the mammal by at least about 50% relative to ALDH RNA levels in the absence of the particle.
35. The use of claim 30, wherein the nucleic ipid particle es ALDH2 gene expression.
36. The composition of claim 1, substantially as herein described with reference to any one of the Examples thereof.
37. The composition of any one of claims 1 to 7, substantially as herein described.
38. The nucleic acid-lipid particle of any one of claims 8 to 28, substantially as herein described.
39. The pharmaceutical composition of claim 29, substantially as herein described.
40. The use of any one of claims 30 to 35, ntially as herein described.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161543700P | 2011-10-05 | 2011-10-05 | |
US61/543,700 | 2011-10-05 | ||
US201261599238P | 2012-02-15 | 2012-02-15 | |
US61/599,238 | 2012-02-15 | ||
PCT/US2012/058770 WO2013052677A1 (en) | 2011-10-05 | 2012-10-04 | Compositions and methods for silencing aldehyde dehydrogenase |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ623181A NZ623181A (en) | 2015-11-27 |
NZ623181B2 true NZ623181B2 (en) | 2016-03-01 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2012318249B2 (en) | Compositions and methods for silencing aldehyde dehydrogenase | |
EP3201338B1 (en) | Compositions and methods for silencing hepatitis b virus gene expression | |
US9428751B2 (en) | Compositions and methods for silencing apolipoprotein C-III expression | |
US20230212578A1 (en) | Compositions and methods for treating hypertriglyceridemia | |
US8236943B2 (en) | Compositions and methods for silencing apolipoprotein B | |
US9222086B2 (en) | Compositions and methods for silencing genes expressed in cancer | |
US9035039B2 (en) | Compositions and methods for silencing SMAD4 | |
WO2016183366A2 (en) | Compositions and methods for silencing expression of hepatitis d virus rna | |
CA2767127A1 (en) | Novel lipid formulations for delivery of therapeutic agents to solid tumors | |
US8455455B1 (en) | Compositions and methods for silencing genes involved in hemorrhagic fever | |
NZ623181B2 (en) | Compositions and methods for silencing aldehyde dehydrogenase | |
US9765333B2 (en) | Compositions and methods for silencing marburg virus gene expression |