WO2022247817A1 - 一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物 - Google Patents
一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物 Download PDFInfo
- Publication number
- WO2022247817A1 WO2022247817A1 PCT/CN2022/094631 CN2022094631W WO2022247817A1 WO 2022247817 A1 WO2022247817 A1 WO 2022247817A1 CN 2022094631 W CN2022094631 W CN 2022094631W WO 2022247817 A1 WO2022247817 A1 WO 2022247817A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sirna
- hpv16
- hpv18
- hpv
- pharmaceutical composition
- Prior art date
Links
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 27
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 26
- 208000009608 Papillomavirus Infections Diseases 0.000 title claims abstract description 21
- 208000021145 human papilloma virus infection Diseases 0.000 title claims abstract description 4
- 108020004459 Small interfering RNA Proteins 0.000 claims abstract description 223
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 claims abstract description 21
- 239000004472 Lysine Substances 0.000 claims abstract description 21
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 21
- 239000003814 drug Substances 0.000 claims abstract description 20
- 239000000203 mixture Substances 0.000 claims abstract description 19
- 241000700605 Viruses Species 0.000 claims abstract description 18
- 229940079593 drug Drugs 0.000 claims abstract description 17
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 15
- 229920000642 polymer Polymers 0.000 claims abstract description 13
- -1 small molecule compound Chemical class 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 4
- 108010074708 B7-H1 Antigen Proteins 0.000 claims abstract description 3
- 102000008096 B7-H1 Antigen Human genes 0.000 claims abstract description 3
- 230000005764 inhibitory process Effects 0.000 claims abstract 4
- 241000701806 Human papillomavirus Species 0.000 claims description 62
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 101000767631 Human papillomavirus type 16 Protein E7 Proteins 0.000 claims description 32
- 150000007523 nucleic acids Chemical class 0.000 claims description 18
- 241000701646 Kappapapillomavirus 2 Species 0.000 claims description 17
- 238000012986 modification Methods 0.000 claims description 17
- 230000004048 modification Effects 0.000 claims description 17
- 108020004707 nucleic acids Proteins 0.000 claims description 17
- 102000039446 nucleic acids Human genes 0.000 claims description 17
- 241000341655 Human papillomavirus type 16 Species 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 14
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 14
- 201000010881 cervical cancer Diseases 0.000 claims description 14
- 230000008685 targeting Effects 0.000 claims description 13
- 101150013359 E7 gene Proteins 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 230000000692 anti-sense effect Effects 0.000 claims description 9
- 238000001727 in vivo Methods 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 239000003937 drug carrier Substances 0.000 claims description 7
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 7
- VWFCHDSQECPREK-LURJTMIESA-N Cidofovir Chemical compound NC=1C=CN(C[C@@H](CO)OCP(O)(O)=O)C(=O)N=1 VWFCHDSQECPREK-LURJTMIESA-N 0.000 claims description 6
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical class C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 claims description 6
- 229960000724 cidofovir Drugs 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 6
- WXJFKKQWPMNTIM-VWLOTQADSA-N [(2s)-1-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxypropan-2-yl]oxymethyl-(3-hexadecoxypropoxy)phosphinic acid Chemical compound CCCCCCCCCCCCCCCCOCCCOP(O)(=O)CO[C@H](CO)CN1C=CC(N)=NC1=O WXJFKKQWPMNTIM-VWLOTQADSA-N 0.000 claims description 5
- 229960002521 artenimol Drugs 0.000 claims description 5
- BJDCWCLMFKKGEE-ISOSDAIHSA-N artenimol Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@H](O)[C@@H]4C BJDCWCLMFKKGEE-ISOSDAIHSA-N 0.000 claims description 5
- 229950005107 brincidofovir Drugs 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 229930016266 dihydroartemisinin Natural products 0.000 claims description 5
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 5
- 229940126586 small molecule drug Drugs 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- FIHJKUPKCHIPAT-AHIGJZGOSA-N artesunate Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@@H](OC(=O)CCC(O)=O)[C@@H]4C FIHJKUPKCHIPAT-AHIGJZGOSA-N 0.000 claims description 4
- 229960004991 artesunate Drugs 0.000 claims description 4
- 230000003832 immune regulation Effects 0.000 claims description 4
- 230000028993 immune response Effects 0.000 claims description 4
- 108700021021 mRNA Vaccine Proteins 0.000 claims description 4
- 229940126582 mRNA vaccine Drugs 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- YIMATHOGWXZHFX-WCTZXXKLSA-N (2r,3r,4r,5r)-5-(hydroxymethyl)-3-(2-methoxyethoxy)oxolane-2,4-diol Chemical compound COCCO[C@H]1[C@H](O)O[C@H](CO)[C@H]1O YIMATHOGWXZHFX-WCTZXXKLSA-N 0.000 claims description 3
- 206010059313 Anogenital warts Diseases 0.000 claims description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 3
- 108020001507 fusion proteins Proteins 0.000 claims description 3
- 102000037865 fusion proteins Human genes 0.000 claims description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
- 230000009437 off-target effect Effects 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 230000010076 replication Effects 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 102000008203 CTLA-4 Antigen Human genes 0.000 claims description 2
- 108010021064 CTLA-4 Antigen Proteins 0.000 claims description 2
- 229940045513 CTLA4 antagonist Drugs 0.000 claims description 2
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 claims description 2
- 101710083479 Hepatitis A virus cellular receptor 2 homolog Proteins 0.000 claims description 2
- 101000831007 Homo sapiens T-cell immunoreceptor with Ig and ITIM domains Proteins 0.000 claims description 2
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 claims description 2
- 102000037982 Immune checkpoint proteins Human genes 0.000 claims description 2
- 108091008036 Immune checkpoint proteins Proteins 0.000 claims description 2
- 102000017578 LAG3 Human genes 0.000 claims description 2
- 101150030213 Lag3 gene Proteins 0.000 claims description 2
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 claims description 2
- 102100024834 T-cell immunoreceptor with Ig and ITIM domains Human genes 0.000 claims description 2
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 239000002082 metal nanoparticle Substances 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 108091081021 Sense strand Proteins 0.000 claims 7
- 241000124008 Mammalia Species 0.000 claims 6
- 239000000878 small molecule-drug conjugate Substances 0.000 claims 6
- 239000003795 chemical substances by application Substances 0.000 claims 3
- 239000000562 conjugate Substances 0.000 claims 2
- 206010017533 Fungal infection Diseases 0.000 claims 1
- 208000031888 Mycoses Diseases 0.000 claims 1
- 239000000427 antigen Substances 0.000 claims 1
- 102000036639 antigens Human genes 0.000 claims 1
- 108091007433 antigens Proteins 0.000 claims 1
- 239000012876 carrier material Substances 0.000 claims 1
- 238000007385 chemical modification Methods 0.000 claims 1
- 230000000295 complement effect Effects 0.000 claims 1
- 230000003902 lesion Effects 0.000 claims 1
- 231100000444 skin lesion Toxicity 0.000 claims 1
- 206010040882 skin lesion Diseases 0.000 claims 1
- 238000007910 systemic administration Methods 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 19
- 229960005486 vaccine Drugs 0.000 abstract description 8
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 230000002452 interceptive effect Effects 0.000 abstract description 3
- 230000009385 viral infection Effects 0.000 abstract description 3
- 208000036142 Viral infection Diseases 0.000 abstract description 2
- 101150044508 key gene Proteins 0.000 abstract 1
- 239000002539 nanocarrier Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 230000029812 viral genome replication Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 34
- 230000000694 effects Effects 0.000 description 21
- 241000283973 Oryctolagus cuniculus Species 0.000 description 14
- 238000000338 in vitro Methods 0.000 description 9
- 229940124897 Gardasil Drugs 0.000 description 8
- 238000011529 RT qPCR Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 208000031886 HIV Infections Diseases 0.000 description 7
- 208000037357 HIV infectious disease Diseases 0.000 description 7
- 208000009889 Herpes Simplex Diseases 0.000 description 7
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 7
- 229960002566 papillomavirus vaccine Drugs 0.000 description 7
- 238000004445 quantitative analysis Methods 0.000 description 7
- 230000002301 combined effect Effects 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 208000003154 papilloma Diseases 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000201835 Froelichia floridana Species 0.000 description 5
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 230000009368 gene silencing by RNA Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 108091030071 RNAI Proteins 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940102767 gardasil 9 Drugs 0.000 description 3
- 238000003197 gene knockdown Methods 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 150000008300 phosphoramidites Chemical class 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 229940124957 Cervarix Drugs 0.000 description 2
- 206010008263 Cervical dysplasia Diseases 0.000 description 2
- 208000000907 Condylomata Acuminata Diseases 0.000 description 2
- 101900175240 Cottontail rabbit papillomavirus Protein E7 Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 101100156155 Human papillomavirus type 16 E7 gene Proteins 0.000 description 2
- 241000883306 Huso huso Species 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 238000007259 addition reaction Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 208000025009 anogenital human papillomavirus infection Diseases 0.000 description 2
- 201000004201 anogenital venereal wart Diseases 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229940000425 combination drug Drugs 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 238000002715 modification method Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 206010040872 skin infection Diseases 0.000 description 2
- 241000894007 species Species 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 101150078635 18 gene Proteins 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 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
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 101100460203 Schizosaccharomyces pombe (strain 972 / ATCC 24843) new2 gene Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000000260 Warts Diseases 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940032077 cervical cancer vaccine Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 230000008696 hypoxemic pulmonary vasoconstriction Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012946 outsourcing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical group 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011555 rabbit model Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 208000013139 vaginal neoplasm Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000005186 women's health Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
-
- 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
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
-
- 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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the invention belongs to the technical field of new drugs, and in particular relates to a nucleic acid polypeptide nano-medicine composition used for the treatment and prevention of human papilloma virus infection.
- Human papillomaviruses human papillomaviruses, HPV
- HPV human papillomaviruses
- ST sexually transmitted
- HPV has an 8 kb circular genome with three major genomic domains, the early genes (E6, E7, E1, E2, E4, and ES), the late genes (L1 and L2), and the longer gene between L1 and L6.
- Figure 1 presents a typical HPV genome structure, using the medically important HPV-16 as a model. The early transcription terminates at position 4215, encoding six early genes, and the late transcription terminates at position 7221, encoding two late genes.
- E6 and E7 are early transcribed cancer transforming proteins because they can inactivate the tumor suppressor proteins p53 (inactivated by E6) and pRb (inactivated by E7) [8] .
- the present invention describes a complex of siRNA and a histidine-lysine polymer for the treatment of HPV.
- Gardasil an HPV vaccine produced by Merck, consisting of a hollow virus-like particle (VLP) assembled from recombinant HPV coat protein.
- VLP hollow virus-like particle
- Gardasil targets HPV 16, 18, 6 and 11, intended for women and girls. Later, according to extended studies, Gardasil was also effective in preventing male genital warts.
- the FDA also approved a second HPV vaccine, Cervarix, targeting HPV 16 and HPV 18, produced by G1axoSmithKline [10] .
- Merck & Co Merck & Co (Merck&Co) received great news in terms of European supervision.
- Gardasil 9 (9-valent HPV vaccine) was approved by the European Commission.
- the vaccine is Gardasil 4 (4-valent HPV vaccine) Vaccine), covering 9 genotypes of HPV, has the potential to prevent about 90% of cervical, vulvar, vaginal, and anal cancers.
- the FDA had approved Gardasil 9 in December 2014.
- the industry predicts that Gardasil 9 will replace Gardasil 4 as the world's best-selling HPV vaccine, with sales peaking at $1.9 billion.
- RNA interference was originally discovered in plants but quickly proved to be a general process covering both lower and higher biological species. This is an efficient process that induces double-stranded RNA formation and leads to the recognition, binding, and degradation of specific target messenger RNAs [15] .
- RNAi has not only been used in various biological research, but also in the development of various therapies [16] . So far, at least 15 RNAi therapies have been developed, and these therapies are in different stages of clinical trials or have completed clinical trials [17] , and four new RNAi drugs have been approved to enter the market in the United States and Europe.
- RNAi offers a very attractive technology to develop innovative therapeutics, multiple projects have not been successful. The failure of most projects is attributed to the stability of siRNA [16] . Naked siRNA must be modified to avoid degradation or packaged with other molecules (pack) to facilitate siRNA entry into cells or to functionalize siRNA to reduce target gene expression [18] . Therefore, the development of delivery methods has been one of the most important areas of siRNA therapy research and development [19] .
- Histidine-Lysine branched polypeptide is a positively charged branched polymer ( Figure 2), which has been successfully used for in vivo delivery of plasmid DNA and siRNA .
- HKP Histidine-Lysine Co-polymer
- Figure 2 Histidine-Lysine Co-polymer
- the technical problem to be solved by the present invention is to provide a pharmaceutical composition that can be used to prepare targeted drugs for treating HPV infection.
- the present invention takes the following technical solutions:
- a nucleic acid polypeptide nanomedicine composition including HPV16-E7 and HPV18-E7 siRNAs, conjugates of these siRNAs and small molecule drugs, and pharmaceutically acceptable carriers suitable for delivering drugs in vivo, as well as nanometers composed of carriers and nucleic acids. drug.
- siRNA targeting HPV16 E7 and HPV18 E7 and a carrier suitable for in vivo introduction, and the carrier is a histidine-lysine branched polypeptide (HKP) or a modification thereof.
- HTP histidine-lysine branched polypeptide
- the HPV16 E7 siRNA comprises HPV16 E7 siRNA-45#, and the sequence of the HPV16 E7 siRNA-45# is 5'-GCACCCUGGGCAUCCUGUGCCCCAU-3'.
- the HPV16 E7-45#siRNA is double-stranded and easy to degrade. It needs to be dissolved in RNase-free treated water, and can be encapsulated by adding a positively charged carrier to improve stability.
- composition comprises a pharmaceutically acceptable carrier, which may be selected from carriers including but not limited to: physiological saline (saline), sugar solution, polypeptide, polymer, lipid, cream gel , micellar materials, metal nanoparticles, dendrimers and HK polymers.
- a pharmaceutically acceptable carrier including but not limited to: physiological saline (saline), sugar solution, polypeptide, polymer, lipid, cream gel , micellar materials, metal nanoparticles, dendrimers and HK polymers.
- the positively charged carrier is histidine-lysine branched polypeptide (HKP).
- the HKP carrier is H3K4b, H3K(+H)4b, H2K4b or H3K(+N)4b, these HKP have a lysine backbone, and its four branches contain multiple repeated histidine, lysine or asparagine.
- the HPV16 E7 siRNA includes HPV16-CRPV E7 siRNA-43#, and the sequence of the HPV16-CRPV E7 siRNA-43# is 5'-GGAAGACCUGCUGAUGGGCACCCU-3'.
- HPV16-CRPV E7 siRNA-43# is an siRNA sequence designed according to the mRNA homologous sequence of CRPVE7 (ie cotton wool rabbit papillomavirus) and HPV16 E7.
- HPV18 E7 siRNA includes HPV18 E7 siRNA-44#, and the sequence of the HPV18 E7 siRNA-44# is 5'-GCUCAGCAGACGACCUUCGAGCAUU-3'.
- HPV18 E7 siRNA includes HPV18 E7 siRNA-46#, and the sequence of the HPV18 E7 siRNA-46# is 5'-GCUGUUUCUGAACACCCUGUCCUUU-3'.
- the siRNA molecules include HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-44#, and the HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-44# are mixed into a dual-target siRNA cocktail It can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- the siRNA molecules include HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-46#, and the HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-46# are mixed into a double-target siRNA cocktail It can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- the siRNA molecules include HPV16 E7 siRNA-45# and HPV18 E7 siRNA-44#, and the HPV16 E7 siRNA-45# and HPV18 E7 siRNA-44# mixed into a double-target siRNA cocktail can be used to enhance It acts against infection by HPV, HIV and/or HSV.
- the siRNA molecules include HPV16 E7 siRNA-45# and HPV18 E7 siRNA-46#, and the HPV16 E7 siRNA-45# and HPV18 E7 siRNA-46# mixed into a double-target siRNA cocktail can be used to enhance It acts against infection by HPV, HIV and/or HSV.
- the siRNA molecules include HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-44#, HPV18 E7 siRNA-46#, the HPV16-CRPV E7 siRNA-43# and HPV18 E7 siRNA-44# , HPV18 E7 siRNA-46# mixed into a three-target multi-effect siRNA cocktail can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- the siRNA molecules include HPV16 E7 siRNA-45# and HPV18 E7 siRNA-44#, HPV18 E7 siRNA-46#, and the HPV16 E7 siRNA-45# and HPV18 E7 siRNA-44#, HPV18 E7 siRNA -46# mixed into a three-target multi-point siRNA cocktail can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- the siRNA molecules include HPV18 E7 siRNA-44# and HPV16-CRPV E7 siRNA-43#, HPV16 E7 siRNA-45#, the HPV18 E7 siRNA-44# and HPV16-CRPV E7 siRNA-43# , HPV16 E7 siRNA-45# mixed into a three-target multi-point siRNA cocktail can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- the siRNA molecules include HPV18 E7 siRNA-46# and HPV16-CRPV E7 siRNA-43#, HPV16 E7 siRNA-45#, and the HPV18 E7 siRNA-46# and HPV16-CRPV E7 siRNA-43# , HPV16 E7 siRNA-45# mixed into a three-target multi-point siRNA cocktail can be used to enhance its anti-HPV, HIV and/or HSV infection effect.
- a pharmaceutical composition for preventing or treating HPV infection the active ingredients of the pharmaceutical composition include an siRNA molecule that inhibits HPV replication and another molecule that includes an siRNA that inhibits genes related to human immune regulation One or more of molecules, anti-HPV small molecule compounds, cervical cancer mRNA vaccines, or anti-HPV monoclonal antibodies.
- siRNA molecules that inhibit human immune regulation-related genes are siRNA molecules that inhibit immune checkpoints, including but not limited to: siRNA molecules that inhibit PD-1, siRNA molecules that inhibit PD-L1, and siRNA molecules that inhibit LAG-3, siRNA molecules that inhibit TIM-3, siRNA molecules that inhibit VISTA, siRNA molecules that inhibit TIGIT, and siRNA molecules that inhibit CTLA-4/B7.
- anti-HPV small molecule compounds are selected from one or more of cidofovir and brincidofovir, or artesunate, dihydroartemisinin ( one or more of dihydroartemisinin).
- the above cervical cancer mRNA vaccine is a messenger ribonucleic acid vaccine that uses HPV gene fragments to encode specific proteins to induce human body to form a protective effect against HPV infection.
- the above-mentioned anti-HPV monoclonal antibody is a therapeutic antibody drug for treating various diseases caused by HPV infection.
- the histidine-lysine branched polypeptide is a positively charged branched histidine-lysine polymer, and in various tissue types For nucleic acid delivery.
- the modification of the histidine-lysine branched polypeptide is a branched histidine-lysine polymer (HKP+H) with a histidine added, which can be used in various tissue types It is used in nucleic acid delivery and induces extremely low immune and inflammatory responses.
- HTP+H branched histidine-lysine polymer
- histidine-lysine branched polypeptide adopts H3K4b, which consists of three lysine cores and four branches, each of which includes a large number of repeated histidine, lysine Amino acid, its specific structure is shown in Figure 2.
- the modification of the histidine-lysine branched polypeptide adopts H3K(+H)4b, and its specific structure is to add a histidine on the branch of H3K4b, that is, H3K(+H)4b
- a nucleic acid polypeptide nanomedicine composition comprises a pharmaceutically acceptable carrier, and the carrier mixes siRNA molecules at a specific nitrogen-to-phosphorus ratio (N:P) to form a nanomedicine of a specific size.
- N:P nitrogen-to-phosphorus ratio
- the average diameter of the nanoparticles is in the range of 50-300 nanometers, further preferably, the size of the nanoparticles is 80-150 nanometers.
- the N:P mass ratio of the carrier to the small nucleic acid molecule siRNA is between 16:1 and 1:8.
- the N:P mass ratio of the carrier to the small nucleic acid molecule siRNA is greater than or equal to 4:1.
- siRNA molecule binds the mRNA encoded by one HPV gene.
- the 20-40 nucleotide pairs of HPV16 or HPV18 are inserted into the end of the E7 gene of cottontail rabbit Papilloma Virus in the same "reading frame" to form a fusion protein, and the 20-40 nucleotide pairs can be used as the attack sequence site of siRNA .
- the fusion virus formed by this fusion protein can infect the skin of cottontail rabbits and form normal infection spots. Changes in this pattern of infection will be indicative of the effectiveness of the siRNA therapy.
- the HKP of the present invention is entrusted to an outsourcing company to synthesize it according to the patented technology owned by the inventor.
- Figure 3 details the specific steps of HKP synthesis.
- the second aspect of the present invention provides a pharmaceutical composition for preventing or treating HPV virus infection
- the active ingredients of the pharmaceutical composition include siRNA molecules targeting HPV virus and small molecular compounds against HPV virus.
- the HPV virus-inhibiting nucleotide analogue is selected from one or more of cidofovir and brincidofovir.
- the artemisinin derivative is selected from one or more of artesunate and dihydroartemisinin.
- small nucleic acid siRNA includes special 2'-OMe, 2'-F, 2'-MOE, sulfur-modified phosphate backbone, base modification, antisense and sense 5' end modification and other chemically modified small nucleic acids to improve
- the stability of small nucleic acid siRNA can reduce the off-target effect and immune response of small nucleic acid siRNA.
- the modified small nucleic acid siRNA includes 19+2 double strands, 21+23 double strands and the like with a special asymmetric structure.
- Figure 1A is the HPV genome. Bottom is the amplification of the E7 gene, and the three sequences to be inserted into the genome of the Cotton tail Rabbit are marked in black.
- Figure 1B shows siRNA targeting the wild-type E7 gene of HPV 16 and the heterozygous E7 gene from HPV 16 and CRPV. Red indicates the CRPV sequence used to replace the corresponding HPV 16 fragment. The yellow area in CRPV E7 siRNA indicates the result of codon optimization.
- Figure 1C is the gene structure of chimeric human rabbit papillomavirus (cH-RPV). Three epitope sequences A, B and C from the HPV 16 E7 gene were inserted into the same reading frame at the end of the CRPV E7 gene.
- Fig. 2 is a schematic diagram of the structure of a histidine-lysine branched polypeptide and a histidine modification added to the side chain, wherein, R represents the amino acid sequence of the four branched side chains.
- Figure 3 is a schematic diagram of the synthesis steps of HKP.
- Fig. 4 is the complex formation process of siRNA and HKP (left figure) and cottontail rabbit skin infection papillomavirus model (SIRAM) (right figure).
- Figure 5 is the screening results of HPV 16 E7 siRNA in SiHa cells (real-time fluorescent quantitative method to detect the mRNA expression of target genes).
- Figure 6 shows the screening results of HPV-CRPV16 E7 siRNA in SiHa cells (real-time fluorescence quantitative method to detect the mRNA expression of target genes).
- Figure 7 is the screening results of HPV 18 E7 siRNA in Hela cells (real-time fluorescent quantitative method to detect the mRNA expression of target genes).
- Fig. 8 shows the inhibitory effect of siRNA on the protein expression level of HPV16E7 gene in SiHa cells detected by Western method.
- Western blot (left) and quantitative data (right) show that siRNA can effectively reduce the expression of E7 protein, and the order of knockdown effect is -45>-43>-44>-37, which is consistent with the results of real-time fluorescence quantitative experiments.
- Fig. 9 The therapeutic effect of siRNA (CRPV-43) in the cH-RPV cottontail rabbit model, the results show that the siRNA has a good inhibitory effect on the growth of rabbit skin warts (L), and the data is shown on the right (R).
- Figure 10 is a summary of the data of different siRNAs treating cH-RPV, and the effective siRNAs are highlighted.
- Figure 11 is the in vitro real-time fluorescence quantitative method to detect the effect of HPV16 and HPV18 siRNA combination drug 1.
- Figure 12 is the in vitro real-time fluorescence quantitative method to detect the effect of HPV16 and HPV18 siRNA combination drug 2.
- Figure 13 is the in vitro real-time fluorescence quantitative method to detect the effect of HPV16 and HPV18 siRNA combined medication 3.
- Figure 14 is the in vitro real-time fluorescence quantitative method to detect the effect of HPV16 and HPV18 siRNA combined medication 4.
- Figure 15 shows the coupling method of siRNA molecules and nucleotide analogues.
- the molecules all contain amino groups, hydroxyl groups, and phosphoric acid active groups. Molecular modification is carried out on them to make phosphoramidite monomers suitable for solid-phase synthesis, which can be directly For siRNA ligation.
- Figure 16 shows the coupling mode of siRNA molecules and artemisinin derivatives, the molecules contain carboxylic acid or hydroxyl active groups, and siRNA can be connected by addition reaction.
- Figure 17 shows a general way to conjugate other drug molecules at one end of siRNA. Through the phosphate group, specific small molecules that treat HPV infection can be linked to siRNA molecules that inhibit HPV replication.
- Figure 18 shows the modification method of siRNA.
- A is a schematic diagram of the modification method of the phosphate backbone or base, and B shows the modification at different positions of siRNA to form a special asymmetric structure of 19+2 double strands, 21+23 double strands, etc.
- Example 1 Preparation of effective siRNA duplexes targeting HPV 16-E7, HPV 18-E7 and cH-RPV-E7
- siRNAs are most effective in inhibiting the expression of specific genes.
- several key features of the siRNA should be considered during in silico design and subsequent in vitro and in vivo testing:
- siRNAs targeting conserved gene sequences which are shared by as many HPV species as possible, so as to increase the wide applicability of siRNAs. Furthermore, preliminary results obtained by us have demonstrated that 25mer siRNA is more effective than 21mer siRNA. We used 25mer siRNA to design siRNA targeting the early gene E7.
- the specific siRNA sequence is as follows:
- HPV18E7-31 GCAUGGACCUAAGGCAACAUUGCAA
- HPV18E7-34 GGUUGACCUUCUAUGUCACGAGCAA
- HPV18E7-36 GCAAUUAAGCGACUCAGAGGAAGAA
- HPV18E7-38 CGAUGAAAUAGAUGGAGUUAAUCAU
- HPV18E7-39 CGAGCCGAACCACAACGUCACACAA
- HPV18E7-44 GCUCAGCAGACGACCUUCGAGCAUU
- HPV16E7-34 GCAUGGAGAUACACCUACAUUGCAU
- HPV16E7-35 GGAGAUACACCUACAUUGCAUGAAU
- HPV16E7-36 GCAUGAAUAUAUGUUAGAUUUGCAA
- HPV16E7-37 GGACAGAGCCCAUUACAAUAUUGUA
- HPV16E7-38 GCCCAUUACAAUAUUGUAACCUUUU
- HPV16E7-39 GCAAGUGUGACUCUACGCUUCGGUU
- HPV16E7-40 GCGUACAAAGCACACACGUAGACAU
- HPV16E7-41 CGUACAAAGCACACACGUAGACAUU
- HPV16E7-42 GCACACACGUAGACAUUCGUACUUU
- HPV16E7-43 GGAAGACCUGUUAAUGGGCACACUA
- HPV16E7-44 CCUGUUAAUGGGCACACUAGGAAUU
- HPV16E7-45 GCACACUAGGAAUUGUGUGCCCCAU
- siRNA design sequence for the E7 gene in cH-RPV (chimeric human papillomavirus):
- CRPE7-36 5'-GCAUGAAUAUAUAUGUUGGAUCUGCA-3'
- CRPE7-37 5'-GGACAGAGCCCACUACAACAUCGU-3'
- CRPE7-38 5'-GCCCACUACAACAUCGUGACCUUUU-3'
- CRPE7-43 5'-GGAAGACCUGCUGAUGGGCACCCU-3'
- CRPE7-44 5'-CCUGCUGAUGGGCACCCUGGGCAU-3'
- CRPE7-45 5'-GCACCCUGGGCAUCCUGUGCCCCAU-3'
- SiHa is a cervical cancer cell line that contains the HPV 16 genome and expresses the oncogene protein E7.
- the SiHa cell line was used to screen the function of siRNAs targeting the E7 gene in HPV 16 and cH-RPV strains. Cultivate SiHa cells with RPMI 1640 medium containing 10% FBS, 100U/ml penicillin, 100 ⁇ g/ml streptomycin at 37°C in an incubator containing 10% CO2. siRNA was transfected into cells using LipofectAmine 2000 following the manufacturer's instructions. Cells were harvested and E7 gene expression levels were assessed by qRT-PCR. In addition, the same cell samples were also used for ELISA and Western analysis.
- HPV 18 genome was fused to the cellular genome of HeLa cervical cancer cells to screen for siRNA targeting HPV 18 gene expression.
- Cells were cultured in a matrix similar to that described above.
- siRNA transfection, qRT-PCR, ELISA and Western follow the same procedure.
- the result in Fig. 7 shows, the effect of HPV18E7 siRNA-39#-44#-46# is better.
- Embodiment 3.Western method confirms the siRNA sequence that can effectively knock down E7 protein expression
- the cottontail rabbit breed used in the experiment was CRPV/NZW.
- siRNA siRNA
- FIG. 9 6 different wild-type and hybrid viruses were inoculated in the skin of NZW rabbits. Each animal wore an Elizabeth collar to avoid disturbance of the treatment site by other animals.
- Each animal was infected with 6 different viruses, as shown in FIG. 9 .
- the left side of the papilloma was treated with the corresponding test siRNA, N.C. siRNA and Cidovofir (a small molecule inhibitor of viral infection) locally for 5 consecutive days.
- Papilloma growth was monitored from week 3 until the end of the experiment at the end of week 5. Take photos and record at the same time. On the right is a non-treated control of the treated site on the left. If the siRNA worked, we would have seen smaller or no papillomas at all on the left.
- L1-R1 infected with wild-type CRPV served as a specificity control for siRNA. Therefore, if an epitope-specific siRNA is effective, it should not affect the L1-R1 site, but will affect sites that infect viruses containing the epitope, such as L5-R5.
- L5-R5 L5-R5, CRPV, HPV 16 E7/82-90 fusion virus containing L2;
- siRNAs Two weeks after papillomas appeared on the skin or infected with virus, we used different siRNAs to treat papillomas to evaluate the curative effect of these siRNAs.
- the following siRNAs were used in animals:
- siRNAs to inhibit the growth of heterozygous human rabbit papillomavirus (cH-RPV) is summarized in FIG. 10 .
- siRNAs with relatively good transfection effects were selected from the siRNAs of HPV16 and HPV18 respectively, and then HPV16-18 siRNAs were mixed according to different ratios, and simultaneously transfected into Siha cells (specifically expressing HPV16) and Hela cells (specifically expressing HPV16). HPV18), and then use real-time quantitative PCR to detect the mRNA expression of the corresponding target genes (HPV16E7 and HPV18E7), so as to determine the combined effect of the two siRNAs.
- Cell preparation prepare Hela cells and siha cells the day before, in a 12-well cell culture plate, 2 ⁇ 10 5 cells/well.
- RT Reverse transcription
- Realtime quantitative
- Gene knockout experiments can be evaluated by detecting changes in mRNA in siRNA-treated cells, using RT-PCR to amplify RNA isolated from the corresponding cells. Selection of appropriate upstream and downstream primers is an initial step in evaluating target gene knockdown and selecting appropriate cell lines.
- the primer sequences used for RT-PCR analysis are:
- HPV16 PCR primers The sequences of HPV16 PCR primers are as follows:
- HPV18 PCR primers The sequences of HPV18 PCR primers are as follows:
- 18E6-1B (241-260):CCATACACAGAGTCTGAATA
- RT-PCR first strand cDNA was synthesized using a cDNA synthesis kit (GE Healthcare, Chicago, IL) according to the manufacturer's instructions. PCR reactions were started with lower cycle numbers, from 25, 30 to 35, to avoid possible amplification plateaus. PCR analysis was performed using a Geneamp 9700 thermal cycler and Taqman (ABI, CA). Amplified products were analyzed by gel electrophoresis.
- E7 gene in HPV-16 is expressed in SiHa cell line PCR primer sequence is as follows:
- the E7 gene in HPV-18 is expressed in the HeLa cell line.
- the PCR primer sequences are as follows:
- Figure 15 shows the coupling mode and structure of siRNA molecules and small molecule drugs such as nucleotide analogs.
- Both cidofovir and brincidofovir are nucleotide analogs, and the molecules contain amino, hydroxyl and phosphoric acid active groups. It can be molecularly modified by general nucleic acid chemistry professionals to make phosphoramidite monomers suitable for solid-phase synthesis. The phosphoramidite monomer obtained through such modification can be directly used in the solid-phase synthesis of siRNA and insert one or more cidofovir or brincidofovir at any position of the siRNA.
- Figure 16 shows the coupling mode and structure of siRNA molecules and artemisinin derivatives.
- artesunate and dihydroartemisinin are derivatives of artemisinin, and their molecules contain carboxylic acid or hydroxyl active groups. It can be connected to the end or side chain of siRNA by common methods such as addition reaction and condensation reaction. In addition, through the phosphate group, different molecules can be efficiently attached to one end of the siRNA (Figure 17).
- nucleic acid siRNA can reduce the off-target effect and immune response of small nucleic acid siRNA.
- the modified small nucleic acid siRNA includes 19+2 double strands, 21+23 double strands, etc. with a special asymmetric structure ( FIG. 18B ).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种用于治疗和预防人***状瘤病毒感染的核酸多肽纳米药物组合物。用于抑制、治疗HPV感染引起的各类疾病的小干扰核酸siRNA分子通过靶向抑制HP16/18关键基因的表达而阻断病毒复制生命周期,降低病毒感染并最终清除病毒。基于该siRNA分子的药物组合物包括siRNA分子和另一种分子,具体为抑制PD-1/PD-L1的siRNA分子、对抗HPV感染的小分子化合物、治疗性mRNA/neoantigen疫苗等。siRNA分子与其它抗HPV药物通过特定的化学键偶联,形成新的偶联分子,该组合物还包含一种药学上可接受的多肽聚合物纳米导入载体,载体优选组氨酸-赖氨酸多肽聚合物类纳米载体。
Description
本发明属于新药技术领域,具体涉及一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物。
HPV和***
人***状瘤病毒(人***瘤病毒,HPV)是一组包膜DNA病毒,目前有超过100个不同的种类,是世界上成人中最常见的性传播(ST)感染病毒。来自德国的Harald zur Hausen在1976年发表的假定中认为,人***瘤病毒在诱导***组织中发挥重要作用
[1]。在1983年和1984年,zur Hausen和他的合作者鉴定出了***中的HPV 16和HPV 18
[2-4]。由此,zur Hausen获得了2008年的诺贝尔生理学和医学奖。据估计,年龄达到50岁的美国女性,超过80%会感染至少一种HPV病毒株
[5]。另外,全球每年会新增49万***病例,导致27万人死亡。在美国,每年有25万-100万妇女产生宫颈非典型增生,这会导致1.1万人进一步发展为***,并导致4000人死亡
[6]。在19个会导致***的“高风险”HPV中,HPV 16和HPV 18导致***的概率约为70%
[7]。
HPV有一个8kb的圆形基因组,含三个主要基因组域,即早期基因(E6、E7、E1、E2、E4和ES)、晚期基因(L1和L2),以及L1和L6之间的较长控制区域(LCR)。图1给出了典型的HPV基因组结构,将医学上重要的HPV-16作为模型。早期的转录终止于4215位,编码6个早期基因,晚期的转录终止于7221位,编码两个晚期基因。E6和E7为早期转录的癌症转化蛋白(cancer transforming proteins),因为它们能使肿瘤抑制蛋白p53(由E6灭活)和pRb(由E7灭活)失活
[8]。
尽管美国FDA已经批准了两个HPV疫苗(后文将详细描述),但HPV的治疗仍然有巨大的需求。尽管如此,市面上还缺乏有效的治疗措施
[9]。本发明描述了siRNA和组氨酸-赖氨酸聚合物组成的复合物治疗HPV的疗法。
HPV疫苗
2006年,美国FDA批准了Gardasil,一个由默克生产的HPV疫苗,由一中空的病毒样颗粒(VLP)组成,该病毒样颗粒由重组HPV外壳蛋白组装而来,Gardasil针对HPV 16、18、6和11,旨在用于妇女和女童。后来,根据扩展研究,Gardasil还能有效预防***疣。FDA于2009年10月16日批准了Gardasil在男性和男童中的应用。在2009年10月,FDA还批准了第二个HPV疫苗,Cervarix,针对HPV 16和HPV 18,由G1axoSmithKline生产
[10]。2015年6月,默沙东(Merck&Co)又在在欧洲监管方面收获重大喜讯,其超级人***瘤病毒(HPV)疫苗Gardasil 9(9价HPV疫苗)获得欧盟委员会批准,该疫苗是Gardasil 4(4价疫苗)的接班人,覆盖了9种基因型HPV,具有预防大约90%的宫颈、外阴、***、***癌症的潜力。此前,FDA已于2014年12月批准Gardasil 9。业界预测,Gardasil 9将取代Gardasil 4成为全球最畅销的HPV疫苗,销售峰值将达到19亿美元。
发达国家和地区(如澳大利亚、加拿大、欧洲和美国)的公共卫生官员建议年轻女性接种HPV疫苗,以预防***和生殖器疣,并减少HPV感染引起的宫颈非典型增生带来的痛苦而昂贵的治疗。根据建议,年龄9-25岁且未暴露于HPV的妇女和女童都应接种HPV疫苗
[11]。尽管如此,由于各种原因许多妇女和女童还是没有接种HPV疫苗。在美国,只有约四分之一的女童接种了HPV疫苗,因为多数家庭还是担心疫苗的有效性或副作用
[12]。此外,第三世界国家不容易获得疫苗。比如肯尼亚,疫苗接种的成本超过一个家庭的平均年收入
[13]。另外,很多妇女已经暴露于HPV
[14],则对HPV的治疗提出要求。
靶向HPV16和HPV18的siRNA和新疗法的开发
RNA干扰最初发现于植物中,但很快被证明是一个覆盖低等和高等生物物种的通用过程。这是一个有效过程,会诱导双链RNA的形成,并导致特定靶标信使RNA的识别、结合和降解
[15]。近几年,RNAi不仅应用于各种生物研究,还应用于各种疗法的开发
[16]。 至今,至少开发了15个RNAi疗法,这些疗法处于不同的临床试验阶段或已经完成临床试验
[17],四种RNAi新药已经在美国和欧洲获批进入市场。
通过电脑软件进行in silico筛选得到一些靶向于HPV16 E7和HPV18 E7的候选siRNA序列后,这些序列化学合成后,用体外细胞***和体内HPV动物模型对候选小干扰核酸序列的生物学功能进行进一步的验证和筛选,通过体外细胞学实验证实外源同时导入相应的HPV16E7和HPV18E7siRNA后能明显抑制靶基因的mRNA表达水平,并在相应的棉尾兔动物模型中得到了很好的治疗效果。因此,使用化学合成的HPV16 E7和HPV18 E7修饰的siRNA-小分子偶联治疗HPV和HIV和/或HSV感染等开创了一种全新的治疗方法,是一种不同于传统小分子或单克隆抗体药物的新型药物,作用机理明确,靶点清晰,导入***独特有效。
用于体内siRNA传递的组氨酸-赖氨酸分枝状多肽(HKP)
尽管RNAi提供了一个非常有吸引力的技术来开发创新性疗法,但多个项目都没有成功。多数项目的失败都归结于siRNA的稳定性问题
[16]。裸siRNA必须经过修饰以避免降解,或用其他分子包裹(pack),以促进siRNA进入细胞或使siRNA功能化以降低靶基因表达
[18]。因此,导入方法的开发已经是siRNA疗法研究和开发的最重要的领域之一
[19]。
组氨酸-赖氨酸分枝状多肽(HKP,Histidine-Lysine Co-polymer)是一种带正电的分枝型聚合物(图2),其已经成功用于质粒DNA和siRNA的体内传导。我们已开展在各种组织类型中将HKP用于核酸体内导入,包括皮肤瘢痕、肝脏、肺脏、肿瘤、眼睛和大脑。
发明内容
本发明所要解决的技术问题是提供一种能够用于制备治疗HPV感染的靶向药物的药物组合物。
为解决以上技术问题,本发明采取如下技术方案:
一种核酸多肽纳米药物组合物,包括HPV16-E7和HPV18-E7的siRNA、这些siRNA与小分子药物的偶联物,以及适用于体内运送药物的药学可接受载体、以及载体与核酸组成的纳米药物。
其包括含靶向HPV16 E7和HPV18 E7的siRNA、以及适用于体内导入的载体,所述的载体为组氨酸-赖氨酸分枝状多肽(HKP)或其修饰物。
具体地,所述的HPV16 E7 siRNA包括HPV16 E7 siRNA-45#,所述的HPV16 E7 siRNA-45#的序列为5’-GCACCCUGGGCAUCCUGUGCCCCAU-3’。
具体地,所述的HPV16 E7-45#siRNA为双链,易于降解,需用无RNA酶的处理水进行溶解,并可以加入带正电荷的载体进行包裹,以提高稳定性。
所述组合物包含一种药学可接受载体,所述药学可接受载体可以选自包括但不限于如下的载体:生理盐水(saline)、糖溶液、多肽、聚合物、脂质、乳膏凝胶、胶束材料、金属纳米粒子、树枝状分子和HK聚合物。
所述的带正电的载体是组氨酸-赖氨酸分枝状多肽(HKP)。
这种共聚物描述于美国专利号7070807 B2、7163695 B2和7772201 B2的多项专利中,其全部内容通过引用并入本文。优选的,所述HKP载体是H3K4b、H3K(+H)4b、H2K4b或H3K(+N)4b,这些HKP具有赖氨酸主干,其四个分支包含多个重复的组氨酸、赖氨酸或天冬酰胺。
具体地,所述的HPV16 E7 siRNA包括HPV16-CRPV E7 siRNA-43#,所述的HPV16-CRPV E7 siRNA-43#的序列为5’-GGAAGACCUGCUGAUGGGCACCCU-3’。
更具体地,所述的HPV16-CRPV E7 siRNA-43#是根据CRPVE7(即棉毛兔***瘤病毒)和HPV16 E7的mRNA同源序列而设计出的siRNA序列。
具体地,所述的HPV18 E7 siRNA包括HPV18 E7 siRNA-44#,所述的HPV18 E7 siRNA-44#的序列为5'-GCUCAGCAGACGACCUUCGAGCAUU-3'。
具体地,所述的HPV18 E7 siRNA包括HPV18 E7 siRNA-46#,所述的HPV18 E7 siRNA-46#的序列为5'-GCUGUUUCUGAACACCCUGUCCUUU-3'。
具体地,所述的siRNA分子包括HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-44#, 所述的HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-44#混合成双靶点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-46#,所述的HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-46#混合成双靶点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV16 E7 siRNA-45#和HPV18 E7 siRNA-44#,所述的HPV16 E7 siRNA-45#和HPV18 E7 siRNA-44#混合成双靶点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV16 E7 siRNA-45#和HPV18 E7 siRNA-46#,所述的HPV16 E7 siRNA-45#和HPV18 E7 siRNA-46#混合成双靶点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-44#、HPV18 E7 siRNA-46#,所述的HPV16-CRPV E7 siRNA-43#和HPV18 E7 siRNA-44#、HPV18 E7 siRNA-46#混合成三靶多效siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV16 E7 siRNA-45#和HPV18 E7 siRNA-44#、HPV18 E7 siRNA-46#,所述的HPV16 E7 siRNA-45#和HPV18 E7 siRNA-44#、HPV18 E7 siRNA-46#混合成三靶多点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV18 E7 siRNA-44#和HPV16-CRPV E7 siRNA-43#、HPV16 E7 siRNA-45#,所述的HPV18 E7 siRNA-44#和HPV16-CRPV E7 siRNA-43#、HPV16 E7 siRNA-45#混合成三靶多点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
具体地,所述的siRNA分子包括HPV18 E7 siRNA-46#和HPV16-CRPV E7 siRNA-43#、HPV16 E7 siRNA-45#,所述的HPV18 E7 siRNA-46#和HPV16-CRPV E7 siRNA-43#、HPV16 E7 siRNA-45#混合成三靶多点siRNA鸡尾酒可以用于增强其抗HPV、HIV和/或HSV的感染作用。
一种用于预防或治疗HPV感染的药物组合物,所述药物组合物的活性成分包括抑制HPV复制的siRNA分子和另一种分子,所述另一种分子包括抑制人体免疫调控相关基因的siRNA分子、抗HPV小分子化合物、***mRNA疫苗、或抗HPV单克隆抗体中的一种或多种。
上述的抑制人体免疫调控相关基因的siRNA分子,是抑制免疫检查点的siRNA分子,包括但不限于:抑制PD-1的siRNA分子,抑制PD-L1的siRNA分子,抑制LAG-3的siRNA分子,抑制TIM-3的siRNA分子,抑制VISTA的siRNA分子,抑制TIGIT的siRNA分子,和抑制CTLA-4/B7的siRNA分子。
上述的抗HPV小分子化合物,选自昔多福韦(Cidofovir)、布林昔多福韦(Brincidofovir)中的一种或多种,或者青蒿琥酯(artesunate)、二氢青蒿素(dihydroartemisinin)中的一种或多种。
上述的***mRNA疫苗,是一种采用HPV基因片段编码特定蛋白从而诱导人体形成对HPV感染具有保护效果的信使核糖核酸疫苗。
上述的抗HPV单克隆抗体,是一种治疗HPV感染引起的各种病症的治疗性抗体药物。
本发明所述的组合物中,所述的组氨酸-赖氨酸分枝状多肽(HKP)为带正电的分枝型组氨酸-赖氨酸聚合物,在各种组织类型中用于核酸传导。
具体地,所述的组氨酸-赖氨酸分枝状多肽的修饰物为增加一个组氨酸的分枝型组氨酸-赖氨酸聚合物(HKP+H),在各种组织类型中用于核酸传导,并诱导产生极低的免疫及炎症反应。
具体地,所述的组氨酸-赖氨酸分枝状多肽采用H3K4b,其由三个赖氨酸核心和四个分枝构成,该四个分枝均包括大量重复的组氨酸、赖氨酸,其具体结构参见图2。
具体地,所述的组氨酸-赖氨酸分枝状多肽的修饰物采用H3K(+H)4b,其具体结构为在H3K4b的分支上增加一个组氨酸,即H3K(+H)4b的结构为将图2中的侧链R采用R=KHHHKHHHKHHHHKHHHK进行替换。
一种核酸多肽纳米药物组合物,所述纳米药物包含一种药学上可接受的载体,所述载体将siRNA分子以特定的氮磷比(N:P)混合后,形成特定大小的纳米药物。
一种由HKP与siRNA药物或基于siRNA药物的组合物形成的纳米药物组合,所述HKP携带正电荷,而siRNA、siRNA与siRNA的组合物、siRNA与mRNA疫苗的组合物等携带负电荷,当HKP水溶液与siRNA或基于siRNA药物的组合物按特定质量比(如4:1)混合时,纳米颗粒会自组装形成。所述纳米颗粒的平均直径在50-300纳米的范围内,进一步优选的,所述纳米颗粒大小为80-150纳米。
具体地,所述的载体与所述的小核酸分子siRNA的N:P质量比例介于16:1到1:8之间。
优选地,所述的载体与所述的小核酸分子siRNA的N:P质量比例大于等于4:1。
具体地,单个siRNA分子结合由一个HPV基因编码的mRNA。其中HPV16或者HPV18的20-40核苷酸对以相同的“阅读框”***到棉尾兔Papilloma Virus的E7基因末尾形成融合蛋白,该20-40核苷酸对可以作为siRNA的攻击序列位点。
具体的,这种融合蛋白所形成的融合病毒可以感染棉尾兔皮肤并形成正常的感染斑痕。这种感染斑痕的变化将成为小干扰核酸治疗药效的指征。
本发明的HKP委托外包公司,按照本发明人拥有的专利技术合成。图3详述了HKP合成的具体步骤。
一种所述的小干扰核酸药物组合物在制备治疗HPV感染的靶向药物中的应用。
本发明第二方面提供一种用于预防或治疗HPV病毒感染的药物组合物,所述药物组合物的活性成分包括靶向HPV病毒的siRNA分子和抗HPV病毒的小分子化合物。
具体地,所述抑制HPV病毒的核苷酸类似物选自昔多福韦(Cidofovir)、布林昔多福韦(Brincidofovir)中的一种或多种。
具体地,所述青蒿素衍生物选自青蒿琥酯(artesunate)、二氢青蒿素(dihydroartemisinin)中的一种或多种。
具体地小核酸siRNA包括特殊2'-OMe,2'-F,2'-MOE,硫修饰的磷酸骨架,碱基的修饰,反义和的正义的5'端修饰等化学修饰的小核酸提高小核酸siRNA的稳定性,降低小核酸siRNA的脱靶效应和免疫反应。
具体地,修饰的小核酸siRNA包括特殊不对称结构的19+2双链,21+23双链等。
图1A为HPV基因组。底部为E7基因的放大,三个准备***棉尾兔(Cotton tail Rabbit)基因组中的序列标记为黑色。
图1B为靶向HPV 16的野生型E7基因,以及靶向来自HPV 16和CRPV的杂合E7基因的siRNA。红色表示用于替代相应HPV 16片段的CRPV序列。CRPV E7 siRNA中的黄色区域表示密码子优化的结果。
图1C为嵌合人兔***瘤病毒(cH-RPV)的基因结构。将来自HPV 16 E7基因的3个抗原决定基因(epitope sequences)序列A、B和C***CRPV E7基因尾部的同一阅读框。
图2为组氨酸-赖氨酸分枝状多肽以及侧链增加一个组氨酸修饰的结构示意图,其中,R表示四个分支侧链的氨基酸顺序。
图3为HKP的合成步骤图。
图4为siRNA和HKP形成复合物的过程(左图)以及棉尾兔皮肤感染***瘤病毒模型(SIRAM)(右图)。
图5为HPV 16 E7 siRNA在SiHa细胞中的筛选结果(实时荧光定量方法检测靶基因的mRNA表达情况)。
图6为HPV-CRPV16 E7 siRNA在SiHa细胞中的筛选结果(实时荧光定量方法检测靶基因的mRNA表达情况)。
图7为HPV 18 E7 siRNA在Hela细胞中的筛选结果(实时荧光定量方法检测靶基因的mRNA表达情况)。
图8为Western方法检测siRNA对SiHa细胞中HPV16E7基因在蛋白表达水平的抑制效果。Western印迹(左)和定量数据(右)表明,siRNA能有效降低E7蛋白的表达, 敲低效果顺序为-45>-43>-44>-37,该结果符合实时荧光定量实验的结果。
图9siRNA(CRPV-43)在cH-RPV棉尾兔模型中的治疗效果,结果表明该siRNA对兔子的皮肤疣生长具有较好的抑制效果(L),数据见右(R)。
图10为不同siRNA治疗cH-RPV的数据总结,并高亮标记出有效的siRNA。
图11为体外实时荧光定量方法检测HPV16和HPV18 siRNA联合用药的效果1。即将HPV16-CRPV-43#siRNA和HPV18-44#siRNA或HPV18-46#siRNA转染进Siha细胞,两个siRNA比例为1:2、1:1、2:1,之后利用实时定量PCR的方法来检测相应的靶基因(HPV16E7)mRNA的表达情况,由此来确定两种siRNA的联合作用效果。
图12为体外实时荧光定量方法检测HPV16和HPV18 siRNA联合用药的效果2。即将HPV16-45#siRNA和HPV18-44#siRNA或HPV18-46#siRNA转染进Siha细胞,两个siRNA比例为1:2、1:1、2:1,之后利用实时定量PCR的方法来检测相应的靶基因(HPV16E7)mRNA的表达情况,由此来确定两种siRNA的联合作用效果。
图13为体外实时荧光定量方法检测HPV16和HPV18 siRNA联合用药的效果3。即将HPV18-44#siRNA和HPV16-CRPV-43#siRNA或HPV16-45#siRNA转染进Hela细胞,两个siRNA比例为1:2、1:1、2:1,之后利用实时定量PCR的方法来检测相应的靶基因(HPV18E7)mRNA的表达情况,由此来确定两种siRNA的联合作用效果。
图14为体外实时荧光定量方法检测HPV16和HPV18 siRNA联合用药的效果4。即将HPV18-46#siRNA和HPV16-CRPV-43#siRNA或HPV16-45#siRNA转染进Hela细胞,两个siRNA比例为1:2、1:1、2:1,之后利用实时定量PCR的方法来检测相应的靶基因(HPV18E7)mRNA的表达情况,由此来确定两种siRNA的联合作用效果。
图15为siRNA分子与核苷酸类似物的偶联方式,分子中均含有氨基、羟基和磷酸活性基团,对其进行分子改造制成适合固相合成使用的亚磷酰胺单体,可直接用于siRNA连接。
图16为siRNA分子与青蒿素衍生物的偶联方式,分子中含有羧酸或者羟基活性基团,可以通过加成反应将siRNA连接上去。
图17显示了一种在siRNA的一个末端偶联其他药物分子的通用方式。通过磷酸基团,可以将治疗HPV感染的特定小分子连接到抑制HPV复制的siRNA分子上。
图18为siRNA的修饰方式。A为磷酸骨架或碱基的修饰方式示意图,B显示在siRNA不同位点进行修饰,形成特殊不对称结构的19+2双链,21+23双链等。
下面结合具体的实施例对本发明做进一步详细的说明,但本发明不限于以下实施例。
实施例1.制备靶向HPV 16-E7、HPV 18-E7和cH-RPV-E7的有效siRNA双链
在初步研究中,我们已经证明25mer siRNA抑制特定基因的表达最有效。为了确保各siRNA敲低靶基因的疗效,在计算机设计(silico design)和随后的体外和体内试验中应该考虑siRNA的几个关键特征:
(1)具有最佳热力学性质,以结合靶序列;
(2)有足够的长度,以结合RISC;
(3)已经除去(或添加)免疫刺激基序;
(4)最小化“脱靶”的可能性;
(5)通过专利搜索,和目前专利没有冲突;
(6)多个序列在鸡尾酒中混合时无相互作用。
本发明中,我们设计了靶向保守基因序列的siRNA,所靶向的保守序列为尽可能多的HPV种类所共有,以增加siRNA的广泛适用性。此外,我们获得的初步结果已经证明,25mer siRNA比21mer siRNA更有效。我们用25mer siRNA来设计靶向早期基因E7的siRNA。具体的siRNA序列如下:
HPV18E7 siRNA的设计序列:
HPV18E7-31:GCAUGGACCUAAGGCAACAUUGCAA
HPV18E7-34:GGUUGACCUUCUAUGUCACGAGCAA
HPV18E7-36:GCAAUUAAGCGACUCAGAGGAAGAA
HPV18E7-38:CGAUGAAAUAGAUGGAGUUAAUCAU
HPV18E7-39:CGAGCCGAACCACAACGUCACACAA
HPV18E7-43:GCCAGAAUUGAGCUAGUAGUAGAAA
HPV18E7-44:GCUCAGCAGACGACCUUCGAGCAUU
HPV18E7-46:GCUGUUUCUGAACACCCUGUCCUUU
HPV16E7 siRNA的设计序列:
HPV16E7-34:GCAUGGAGAUACACCUACAUUGCAU
HPV16E7-35:GGAGAUACACCUACAUUGCAUGAAU
HPV16E7-36:GCAUGAAUAUAUGUUAGAUUUGCAA
HPV16E7-37:GGACAGAGCCCAUUACAAUAUUGUA
HPV16E7-38:GCCCAUUACAAUAUUGUAACCUUUU
HPV16E7-39:GCAAGUGUGACUCUACGCUUCGGUU
HPV16E7-40:GCGUACAAAGCACACACGUAGACAU
HPV16E7-41:CGUACAAAGCACACACGUAGACAUU
HPV16E7-42:GCACACACGUAGACAUUCGUACUUU
HPV16E7-43:GGAAGACCUGUUAAUGGGCACACUA
HPV16E7-44:CCUGUUAAUGGGCACACUAGGAAUU
HPV16E7-45:GCACACUAGGAAUUGUGUGCCCCAU
针对cH-RPV(嵌合人兔***瘤病毒)中E7基因的siRNA设计序列:
CRPE7-36:5’-GCAUGAAUAUAUGUUGGAUCUGCA-3’
CRPE7-37:5’-GGACAGAGCCCACUACAACAUCGU-3’
CRPE7-38:5’-GCCCACUACAACAUCGUGACCUUUU-3’
CRPE7-43:5’-GGAAGACCUGCUGAUGGGCACCCU-3’
CRPE7-44:5’-CCUGCUGAUGGGCACCCUGGGCAU-3’
CRPE7-45:5’-GCACCCUGGGCAUCCUGUGCCCCAU-3’
实施例2.在携带HPV基因的细胞系中筛选siRNA(图5,6,7)
SiHa是***细胞系,含HPV 16基因组,表达癌基因蛋白类E7。SiHa细胞系用来筛选在HPV 16和cH-RPV病毒株中靶向E7基因的siRNA的功能。用RPMI 1640培养基培养SiHa细胞,并含有10%FBS、100U/ml青霉素、100μg/ml链霉素,在37℃含10%CO2的培养箱中培养。按照厂商的说明,利用LipofectAmine 2000将siRNA转染至细胞。收集细胞,并用qRT-PCR评估E7的基因表达水平。此外,相同的细胞样品还用于ELISA和Westerrn分析。图5、6中的结果表示,HPV16E7siRNA-37#-40#-41#-42#-44#的效果比较好,cH-RPVE7siRNA-37#-43#-45#的效果比较好。
同样,HeLa***细胞的细胞基因组中融合了HPV 18基因组,用来筛选靶向HPV 18基因表达的siRNA。细胞培养在类似于如上所述的基质中。siRNA转染、qRT-PCR、ELISA和Western遵循同样的过程。图7中的结果表明,HPV18E7 siRNA-39#-44#-46#的效果比较好。
实施例3.Western方法确认能够有效敲低E7蛋白表达的siRNA序列
通过Western免疫印迹方法,我们进一步对cH-RPVE7基因的siRNA在抑制E7蛋白表达水平方面的效果。在图8中,Western印迹(左侧)和定量数据(右侧)表明,siRNA减少E7蛋白表达的效力如下:-45>-43>-44>-37,和qRT-PCR分析的结果一致。
实施例4.siRNA在皮肤感染兔动物模型(SIRAM)中的效果
试验采用的棉尾兔子品种为CRPV/NZW。为检测siRNA的治疗效果,前期我们在体外细胞筛选***中进行了验证,如图9所示在NZW兔皮肤中接种了6种不同的野生型和杂合病毒。每只动物戴上Elizabeth颈圈,以避免治疗部位受到其他动物的干扰。
在初步研究中使用6只动物。每只动物(L1-R1、L2-R2、L3-R3、L4-R4、L5-R5和L6-R6)分别感染了6种不同的病毒,如图9所示。感染后2周,***瘤的左侧用相应的试验siRNA、N.C.siRNA和Cidovofir(病毒感染小分子抑制剂)治疗,局部连续治疗5天。从第3周开始监测***瘤的生长情况,直至第5周末实验结束。同时进行拍照记录。右侧是左侧处理部位的非处理对照。如果siRNA有效,我们会在左侧观察到更小的或者根本观察不到***瘤。感染L5-R5部位的病毒比感染L2-R2、L3-R3和L4-R4的病毒更有活 力。感染野生型CRPV的L1-R1作为siRNA的特异性对照。因此,如果一个表位特异性的siRNA是有效的,它应该不会影响L1-R1部位,但是会对感染含有该表位病毒的部位产生影响,如L5-R5。
病毒如下所述:
L1-R1,wt CRPV DNA 5ug/site;
L2-R2,CRPV,含HPV 16E7/A 82-90融合病毒;
L3-R3,CRPV,含HPV 16E7/B 45-57融合病毒;
L4-R4,CRPV,含HPV 16E7/C 11-20融合病毒;
L5-R5,CRPV,含L2的HPV 16 E7/82-90融合病毒;
L6-R6,CRPV,含HPV 16E7的串联重复序列。
待皮肤上出现***瘤或感染病毒两周后,我们采用不同的siRNA来处理***瘤,以评价这些siRNA的疗效。以下为应用于动物的siRNA:
兔#3270,siRNA-CRPC-37
兔#3271,siRNA-CRPC-43
兔#3272,siRNA-CRPC-44
兔#3273,siRNA-CRPC-45
兔#3274,siRNA-NC;
兔#3275,Cidofovir,阳性对照
在该实验中,CRPV-43的治疗抑制了***瘤的生长(图9)。
siRNA抑制杂合人兔***瘤病毒(cH-RPV)生长的能力总结于图10。
实施例5.HPV16-18 siRNA体外联合作用细胞的相关实验
分别从HPV16和HPV18的siRNA中挑选出转染效果相对较好的两个siRNA,然后将HPV16-18 siRNA按照不同比例进行混合,并同时转染Siha细胞(特异性表达HPV16)和Hela细胞(特异性表达HPV18),之后利用实时定量PCR的方法来检测相应的靶基因(HPV16E7和HPV18E7)mRNA的表达情况,由此来确定两种siRNA的联合作用效果。
细胞准备:前一天准备Hela细胞和siha细胞,12孔细胞培养板,2×10
5个细胞/孔。
样品分组情况:
实验方法:
细胞常规4个小时转染方法(根据Lipofectamine 2000产品操作说明书做适当修改);
逆转录(RT)-实时定量(Realtime)PCR技术。
基因敲除的实验可以通过检测经siRNA处理细胞内mRNA的变化来评估,用RT-PCR扩增从相应细胞中分离的RNA。选择合适的上下游引物是评价靶基因敲除和选择合适细胞系的初始步骤。用于RT-PCR分析的引物序列为:
HPV16 PCR引物序列如下:
HPV16-1:
16E6-1F(191-461):GGAATCCATATGCTGTATGT(PCR产物长度:270bp)
16E6-1B(191-461):CTACGTGTTCTTGATGATCT
HPV16-2:
16E6-2F(278-448):CAACATTAGAACAGCAATAC(PCR产物长度:170bp)
16E6-2B(278-448):ATGATCTGCAACAAGACATA
HPV16-E7-1:
16E7-1F(21-43):ATTGCATGAATATATGTTAGATT(PCR产物长度:250bp)
16E7-1B(248-270):CACAATTCCTAGTGTGCCCATTA
HPV18 PCR引物序列如下:
HPV18-1:
18E6-1F(65-84):ACACTTCACTGCAAGACATA(PCR产物长度:196bp)
18E6-1B:(241-260):CCATACACAGAGTCTGAATA
HPV18-2:
18E6-2F(107-126):AGACAGTATTGGAACTTACA(PCR产物长度:151bp)
18E6-2B(238-257):TACACAGAGTCTGAATAATG
HPV18-E7-1:
18E7-1F(38-54):TGCATTTAGAGCCCCAA(PCR产物长度:253bp)
18E7-1B(275-291):CACAAAGGACAGGGTGT
根据生产厂家的使用说明,使用RNeasy mini kit(加利福尼亚Qiagen)从细胞培养或肿瘤组织中提取总RNA。对于RT-PCR,根据生产厂家的说明,使用cDNA合成试剂盒(GE Healthcare,Chicago,IL)合成第一条cDNA链。PCR反应以较低的周期数开始,从25、30到35,以避免可能的扩增平台。用Geneamp 9700热循环仪和Taqman(ABI,CA)进行PCR分析。扩增产物经凝胶电泳分析。
HPV-16中E7基因在SiHa细胞系中表达PCR引物序列如下:
16E7-Forward:ATTGCATGAATATATGTTAGATT
16E7-Reverse:CACAATTCCTAGTGTGCCCATTA;
HPV-18中E7基因在HeLa细胞系中表达PCR引物序列如下:
18E7-1Forward:TGCATTTAGAGCCCCAA
18E7-1Reverse:CACAAAGGACAGGGTGT。
结果分析:
在图11-14的结果中,可以进行初步的判定:HPV16 siRNA(CRPE43#)和HPV18 siRNA(46#)的配对效果(siha细胞)相对较好。
实施例6.抗HPV的siRNA与小分子药物的偶联
图15显示了siRNA分子与核苷酸类似物等小分子药物的偶联方式及结构。昔多福韦、布林昔多福韦均为核苷酸类似物,分子中均含有氨基、羟基和磷酸活性基团。可以由一般核酸化学专业人员对其进行分子改造制成适合固相合成使用的亚磷酰胺单体。通过此种改造得到的亚磷酰胺单体可以直接用于siRNA的固相合成中并在siRNA的任意位置***一个或者多个昔多福韦或者布林昔多福韦。
图16显示了siRNA分子与青蒿素衍生物的偶联方式及结构。青蒿琥酯、二氢青蒿素均为青蒿素的衍生物,分子中含有羧酸或者羟基活性基团。可以通过加成反应、缩合反应等常见手段连接到siRNA的末端或者侧链上。此外,通过磷酸基团,可以有效将不同的分子连接到siRNA的一个末端(图17)。
实施例7.siRNA的修饰
包括特殊2'-OMe、2'-F、2'-MOE,硫修饰的磷酸骨架,碱基的修饰(图18A),反义和的正义的5'端修饰等化学修饰的小核酸提高小核酸siRNA的稳定性,降低小核酸siRNA的脱靶效应和免疫反应。
修饰的小核酸siRNA包括特殊不对称结构的19+2双链,21+23双链等(图18B)。
以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围内。
参考文献
1.吉斯曼L和豪森HZ,人***瘤病毒DNA:物理映射和遗传异质性。美国国家科学院学报,1976。73(4):1310-3。
2.班特尔-史卡尔L和祖尔豪森H,从生殖部位分离的一种有缺陷的人类细小病毒的DNA特征。病毒学,1984。134(1):52-63。
3.克劳福德L,人***状瘤病毒和***。自然,1984。310(5972):16。
4.吉斯曼L等,生殖器肿瘤中人***瘤病毒的存在。皮肤病学杂志,1984。83(1Suppl):26s-28s。
5.卡恩JA,预防宫颈上皮内瘤变的HPV疫苗。新英格兰医学杂志,2009。361(3):271-8。
6.来自FDA和CDC关于加德西及其安全性的信息。
7.特罗蒂埃H。和伯切尔AN,黏膜型人***瘤病毒感染及相关疾病的流行病学研究。公共卫生基因组学,2009。12(5-6):291-307。
8.海纳尔K等人。HPV16 E7癌基因在正常人上皮细胞中的表达引起上皮细胞向间充质细胞转化的分子改变。病毒学2009年8月15日[引用391 1];2009/06/26:[57-63]。可从以下网址获得:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19552933。
9.来自FDA和CDC关于加德西及其安全性的信息。
10.http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm172678.htm。
11.科霍特T,斯图尔特A。“新的报告审查了将强制年轻女性接种HPV疫苗的法律”。乔治华盛顿大学雅各布斯妇女健康研究所。
http://www.jiwh.org/content.cfm?sectionid=167
12.四分之一的美国少女接受了***疫苗”。
http://www.washingtonpost.com/wpdyn/content/article/2008/10/09/AR2008100901452.html?sub=new2。
13.希瑞适(Cervarix)在肯尼亚的营销。
14.“来自FDA和CDC关于加德西及其安全性的信息”。
15.夏普PA,RNA干扰—2001。基因研究进展,2001。15(5):485-90。
16.李L和沈Y。克服障碍来开发有效和安全的siRNA疗法。生物治疗的专家意见,2009。9(5):609-19。
17.http://www.clinicaltrials.gov/ct2/results?term=siRNA。
18.李BJ等人,siRNA在恒河猴SARS冠状病毒预防和治疗方案中的应用。《自然医学》,2005。11(9):944-51。
19.谢FY,伍德尔MC和路PY,利用体内siRNA传递进行药物发现和治疗开发。今日药物发现,2006。11(1-2):67-73。
Claims (32)
- 一种核酸多肽纳米药物组合物,其特征在于:包括HPV16-E7和HPV18-E7的siRNA、所述siRNA与小分子药物的偶联物,以及适用于体内运送药物的药学可接受载体,或者所述载体与所述siRNA或所述siRNA与小分子药物的偶联物组成的纳米药物,所示组合物通过局部用药或全身用药的方式,可用于由人***状瘤病毒感染导致的相关疾病的治疗和预防。
- 根据权利要求1所述的药物组合物,其特征在于:其核酸组成部分包括以HPV16-E7 mRNA和HPV18-E7 mRNA为靶向的至少一条siRNA、或者包括至少一条作为肿瘤特异抗原的mRNA,所述siRNA分子包括正义链和反义链,所述正义链的序列选自SEQ ID No.1~143中的任意一条,所述反义链选自SEQ ID No.144~286中与所述正义链互补的一条。
- 根据权利要求2所述的药物组合物,其特征在于:所述的siRNA为根据棉毛兔***瘤病毒和HPV16 E7的mRNA同源序列设计的序列。
- 根据权利要求1至3中任一项所述的药物组合物,其特征在于:所述的siRNA包括人HPV16-CRPV-E7 siRNA-43序列为正义链:5'-GGAAGACCUGCUGAUGGGCACCCU-3',反义链:5'-AGGGUGCCCAUCAGCAGGUCUUCC-3'。
- 根据权利要求1至3中任一项所述的药物组合物,其特征在于:所述的siRNA包括HPV16-E7 siRNA-45#的序列为正义链:5'-GCACCCUGGGCAUCCUGUGCCCCAU-3',反义链:5'-AUGGGGCACAGGAUGCCCAGGGUGC-3'。
- 根据权利要求2所述的药物组合物,其特征在于:所述的siRNA为根据棉毛兔***瘤病毒和HPV18 E7的mRNA设计的序列。
- 根据权利要求1或2或6所述的药物组合物,其特征在于:所述的siRNA包括HPV18-E7 siRNA-46#的序列为正义链:5'-GCUGUUUCUGAACACCCUGUCCUUU-3',反义链:5'-AAAGGACAGGGUGUUCAGAAACAGC-3'。
- 根据权利要求1或2或6所述的药物组合物,其特征在于:所述的siRNA包括所述的HPV18-E7 siRNA-44#的序列为正义链:5'-GCUCAGCAGACGACCUUCGAGCAUU-3',反义链:5'-AAUGCUCGAAGGUCGUCUGCUGAGC-3'。
- 根据权利要求1或2所述的药物组合物,其特征在于:所述的siRNA为双链,而且可以通过化学修饰来进一步优化靶向性和抑制效果。
- 根据权利要求1所述的药物组合物,其特征在于:所述的siRNA包括HPV16-CRPV-E7 siRNA-43#和HPV18-E7 siRNA-44#,所述的HPV16-CRPV-E7 siRNA-43#和HPV18-E7 siRNA-44#混合成双靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-CRPV-E7 siRNA-43#和HPV18-E7 siRNA-46#,所述的HPV16-CRPV-E7 siRNA-43#和HPV18-E7 siRNA-46#混合成双靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-E7 siRNA-45#和HPV18-E7 siRNA-44#,所述的HPV16-E7 siRNA-45#和HPV18-E7 siRNA-44#混合成双靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-E7 siRNA-45#和HPV18-E7 siRNA-46#,所述的HPV16-E7 siRNA-45#和HPV18-E7 siRNA-46#混合成双靶点siRNA抑制剂。
- 根据权利要求1所述的药物组合物,其特征在于:所述的siRNA包括HPV16-CRPV-E7 siRNA-43#、HPV16-E7 siRNA-45#和HPV18-E7 siRNA-44#,所述的HPV16-CRPV-E7 siRNA-43#、HPV16-E7 siRNA-45#和HPV18-E7 siRNA-44#混合成三靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-CRPV-E7 siRNA-43#、HPV16-E7 siRNA-45#和HPV18-E7 siRNA-46#,所述的HPV16-CRPV-E7 siRNA-43#、HPV16-E7 siRNA-45#和HPV18-E7 siRNA-46#混合成三靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-CRPV-E7 siRNA-43#、HPV18-E7 siRNA-44#和HPV18-E7 siRNA-46#,所述的HPV16-CRPV-E7 siRNA-43#、HPV18-E7 siRNA-44#和 HPV18-E7 siRNA-46#混合成三靶点siRNA抑制剂;和/或,所述的siRNA包括HPV16-E7 siRNA-45#、HPV18-E7 siRNA-44#和HPV18-E7 siRNA-46#,所述的HPV16-E7 siRNA-45#、HPV18-E7 siRNA-44#和HPV18-E7 siRNA-46#混合成三靶点siRNA抑制剂。
- 一种用于预防或治疗HPV感染的药物组合物,其特征在于:所述药物组合物的活性成分包括抑制HPV复制的siRNA分子和另一种分子,所述另一种分子包括抑制人体免疫调控相关基因的siRNA分子、抗HPV小分子化合物、***mRNA疫苗、或抗HPV单克隆抗体中的一种或多种。
- 权利要求12所述的药物组合物,其特征在于:所述的抑制人体免疫调控相关基因的siRNA分子,是抑制免疫检查点的siRNA分子,选自抑制PD-1的siRNA分子、抑制PD-L1的siRNA分子、抑制LAG-3的siRNA分子、抑制TIM-3的siRNA分子、抑制VISTA的siRNA分子、抑制TIGIT的siRNA分子、或抑制CTLA-4/B7的siRNA分子中的一种或多种。
- 权利要求1所述的药物组合物,其特征在于:所述药学可接受载体为生理盐水、糖溶液、多肽、聚合物、脂质、乳膏凝胶、胶束材料、金属纳米粒子、树枝状分子或HK聚合物中的一种或多种。
- 根据权利要求14所述的药物组合物,其特征在于:所述药学可接受载体为多肽载体,所述多肽载体为适用于体内导入的载体材料,即带正电荷的组氨酸-赖氨酸分枝状多肽或其修饰物。
- 根据权利要求15所述的药物组合物,其特征在于:所述的组氨酸-赖氨酸分枝状多肽的修饰物为在每一个分支上增加一个组氨酸的分枝型组氨酸-赖氨酸聚合物。
- 根据权利要求15或16所述的药物组合物,其特征在于:所述的组氨酸-赖氨酸分枝状多肽采用H3K4b或者H3K(+H)4b。
- 根据权利要求1所述的药物组合物,其特征在于:所述的载体与所述的siRNA的N:P质量比例介于16:1到1:8之间。
- 上述权利要求中任意一条所述的组合物,其特征在于:包含至少2个siRNA分子和一个药用载体,siRNA分子结合至少2个编码部分人***瘤病毒多肽或蛋白质的mRNA分子。
- 上述权利要求中任意一条所述的组合物,其特征在于:siRNA鸡尾酒包含2种siRNA分子,比例为1:2,1:1或2:1。
- 根据权利要求1、14至18中任意一条所述的组合物,其特征在于:所述载体包括一种组氨酸-赖氨酸聚合物,该聚合物和一种、两种或多种siRNA分子形成核酸多肽纳米药物组合物,所述纳米药物直径为50-300nm。
- 治疗感染HPV的哺乳动物的方法,包括给予哺乳动物权利要求1至21中任一项所述组合物的药学有效量。
- 治疗感染HPV和HIV和/或HSV的哺乳动物的方法,包括给予哺乳动物权利要求1至21中任一项所述组合物的药学有效量。
- 治疗感染HPV和真菌感染的哺乳动物的方法,包括给予哺乳动物权利要求1至21中任一项所述组合物的药学有效量。
- 上述权利要求中任意一条所述的siRNA分子,单给siRNA结合由一个HPV基因编码的mRNA,其中HPV16或者HPV18的20-40核苷酸对以相同的“阅读框”***到棉尾兔Papilloma Virus的E7基因末尾形成融合蛋白,该20-40核苷酸对可以作为siRNA的攻击序列位点。
- 上述权利要求中任意一条所述的siRNA分子,所述的siRNA可以进行特定的化学修饰,包括特殊2'-OMe、2'-F、2'-MOE、硫修饰的磷酸骨架,碱基的修饰,反义和的正义的5'端修饰等化学修饰的小核酸提高siRNA的稳定性,降低siRNA的脱靶效应和免疫反应。
- 权利要求26所述的siRNA分子,所述修饰的siRNA包括特殊不对称结构的19+2双链,21+23双链等。
- 一种siRNA-小分子药物偶联物,其特征在于:所述siRNA-小分子药物偶联物由抑制HPV病毒的siRNA分子与抑制HPV病毒的小分子化合物通过共价键偶联形成。
- 根据权利要求28所述的siRNA-小分子药物偶联物,其特征在于:所述抑制HPV 病毒的小分子化合物为抑制HPV病毒的核苷酸类似物和/或青蒿素衍生物。
- 根据权利要求29所述的siRNA-小分子药物偶联物,其特征在于:抑制HPV病毒的核苷酸类似物选自昔多福韦、布林昔多福韦中的一种或多种。
- 根据权利要求29所述的siRNA-小分子药物偶联物,其特征在于:所述青蒿素衍生物选自青蒿琥酯、二氢青蒿素中的一种或多种。
- 如权利要求29所述的siRNA-小分子药物偶联物在预防或者治疗HPV引起的***前病变及皮肤损伤、***等疾病中的应用。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110592224.6 | 2021-05-28 | ||
CN202110592224.6A CN115400222A (zh) | 2021-05-28 | 2021-05-28 | 一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022247817A1 true WO2022247817A1 (zh) | 2022-12-01 |
Family
ID=84154947
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2022/094631 WO2022247817A1 (zh) | 2021-05-28 | 2022-05-24 | 一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN115400222A (zh) |
WO (1) | WO2022247817A1 (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120171290A1 (en) * | 2009-04-13 | 2012-07-05 | Coursaget Pierre L | Hpv particles and uses thereof |
US20130345284A1 (en) * | 2010-07-29 | 2013-12-26 | Alan Y. Lu | siRNA Compositions and Methods for Treatment of HPV and Other Infections |
-
2021
- 2021-05-28 CN CN202110592224.6A patent/CN115400222A/zh active Pending
-
2022
- 2022-05-24 WO PCT/CN2022/094631 patent/WO2022247817A1/zh unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120171290A1 (en) * | 2009-04-13 | 2012-07-05 | Coursaget Pierre L | Hpv particles and uses thereof |
US20130345284A1 (en) * | 2010-07-29 | 2013-12-26 | Alan Y. Lu | siRNA Compositions and Methods for Treatment of HPV and Other Infections |
Also Published As
Publication number | Publication date |
---|---|
CN115400222A (zh) | 2022-11-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Pal et al. | Human papillomavirus E6 and E7: the cervical cancer hallmarks and targets for therapy | |
Hu et al. | TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy | |
CN107349226B (zh) | 甲病毒在制备抗肿瘤药物方面的应用 | |
US9506063B2 (en) | SiRNA compositions and methods for treatment of HPV and other infections | |
US20120136040A1 (en) | Hybrid interfering rna | |
JP2021515589A (ja) | エンドソーム脱出能を有するペプチド核酸複合体及びその用途 | |
JPWO2008139938A1 (ja) | ヒトパピローマウイルス16型遺伝子を標的とする二本鎖核酸分子及びそれを含む医薬 | |
CN109371021B (zh) | 一种使用CRISPR/Cas9治疗HPV阳性的宫颈上皮内瘤变的方法 | |
WO2008154482A2 (en) | Sirna compositions and methods of use in treatment of ocular diseases | |
US9801953B2 (en) | Nanoparticles carrying nucleic acid cassettes for expressing RNA | |
JP2004535813A (ja) | 遺伝子発現のサイレンシング | |
WO2018160690A1 (en) | Rna pharmaceutical formulations for prophylactic and therapeutic treatment of zika virus infection | |
JP6727381B2 (ja) | Hpv感染に係わる癌の治療用組成物 | |
Squiquera et al. | Ranpirnase eradicates human papillomavirus in cultured cells and heals anogenital warts in a Phase I study | |
JP2004505892A (ja) | ヒトパピローマウイルス(hpv)−感染細胞の治療 | |
Singhania et al. | Suppl 2: RNA Interference for the Treatment of Papillomavirus Disease | |
Ren et al. | An effective and biocompatible polyethylenimine based vaginal suppository for gene delivery | |
Graham et al. | Human tumor growth is inhibited by a vaccinia virus carrying the E2 gene of bovine papillomavirus | |
WO2019096796A1 (en) | Non-human papillomaviruses for gene delivery in vitro and in vivo | |
Zhao et al. | Identification of a 17-nucleotide splicing enhancer in HPV-16 L1 that counteracts the effect of multiple hnRNP A1-binding splicing silencers | |
WO2022247817A1 (zh) | 一种用于人***状瘤病毒感染治疗和预防的核酸多肽纳米药物组合物 | |
WO2022033469A1 (zh) | 重组溶瘤病毒及其构建方法和用途 | |
Simões et al. | Papillomavirus: Viral vectors in the gene therapy and new therapeutic targets | |
Márquez-Gutiérrez et al. | Effect of combined antisense oligodeoxynucleotides directed against the human papillomavirus type 16 on cervical carcinoma cells | |
JP2022543610A (ja) | 遺伝子改変エンテロウイルスベクター |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22810542 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |