WO2023125481A1 - 经过修饰的aav衣壳蛋白及其用途 - Google Patents
经过修饰的aav衣壳蛋白及其用途 Download PDFInfo
- Publication number
- WO2023125481A1 WO2023125481A1 PCT/CN2022/142185 CN2022142185W WO2023125481A1 WO 2023125481 A1 WO2023125481 A1 WO 2023125481A1 CN 2022142185 W CN2022142185 W CN 2022142185W WO 2023125481 A1 WO2023125481 A1 WO 2023125481A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- capsid protein
- seq
- aav
- amino acid
- aav8
- Prior art date
Links
- 108090000565 Capsid Proteins Proteins 0.000 title claims abstract description 137
- 102100023321 Ceruloplasmin Human genes 0.000 title claims abstract description 137
- 150000001413 amino acids Chemical class 0.000 claims abstract description 79
- 241000702421 Dependoparvovirus Species 0.000 claims abstract description 47
- 238000006467 substitution reaction Methods 0.000 claims abstract description 25
- 230000002207 retinal effect Effects 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 78
- 241001164825 Adeno-associated virus - 8 Species 0.000 claims description 73
- 229920001184 polypeptide Polymers 0.000 claims description 59
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 59
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 59
- 241000700605 Viruses Species 0.000 claims description 58
- 238000002347 injection Methods 0.000 claims description 49
- 239000007924 injection Substances 0.000 claims description 49
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 42
- 230000035772 mutation Effects 0.000 claims description 40
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 34
- 239000005557 antagonist Substances 0.000 claims description 33
- 239000008194 pharmaceutical composition Substances 0.000 claims description 33
- 201000010099 disease Diseases 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 30
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 23
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 23
- 102000004169 proteins and genes Human genes 0.000 claims description 23
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 claims description 21
- 208000004644 retinal vein occlusion Diseases 0.000 claims description 21
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 claims description 20
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 15
- 208000003456 Juvenile Arthritis Diseases 0.000 claims description 15
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 15
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 15
- 206010003246 arthritis Diseases 0.000 claims description 15
- 208000030533 eye disease Diseases 0.000 claims description 14
- 208000001344 Macular Edema Diseases 0.000 claims description 12
- 206010025415 Macular oedema Diseases 0.000 claims description 12
- 210000000234 capsid Anatomy 0.000 claims description 12
- 201000010230 macular retinal edema Diseases 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 230000002917 arthritic effect Effects 0.000 claims description 10
- 229960000403 etanercept Drugs 0.000 claims description 10
- 238000007918 intramuscular administration Methods 0.000 claims description 10
- 208000002780 macular degeneration Diseases 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 241001634120 Adeno-associated virus - 5 Species 0.000 claims description 9
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 9
- 229960002833 aflibercept Drugs 0.000 claims description 9
- 201000008482 osteoarthritis Diseases 0.000 claims description 9
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 9
- 241001655883 Adeno-associated virus - 1 Species 0.000 claims description 8
- 241000702423 Adeno-associated virus - 2 Species 0.000 claims description 8
- 241000202702 Adeno-associated virus - 3 Species 0.000 claims description 8
- 241000580270 Adeno-associated virus - 4 Species 0.000 claims description 8
- 241000972680 Adeno-associated virus - 6 Species 0.000 claims description 8
- 241001164823 Adeno-associated virus - 7 Species 0.000 claims description 8
- 241000649045 Adeno-associated virus 10 Species 0.000 claims description 8
- 206010012688 Diabetic retinal oedema Diseases 0.000 claims description 8
- 229960002964 adalimumab Drugs 0.000 claims description 8
- 208000002849 chondrocalcinosis Diseases 0.000 claims description 8
- 208000004575 Infectious Arthritis Diseases 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 7
- 208000014674 injury Diseases 0.000 claims description 7
- 230000037431 insertion Effects 0.000 claims description 7
- 238000003780 insertion Methods 0.000 claims description 7
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 7
- 201000001223 septic arthritis Diseases 0.000 claims description 7
- 230000008733 trauma Effects 0.000 claims description 7
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 206010002556 Ankylosing Spondylitis Diseases 0.000 claims description 6
- 206010053555 Arthritis bacterial Diseases 0.000 claims description 6
- 206010008690 Chondrocalcinosis pyrophosphate Diseases 0.000 claims description 6
- 208000005590 Choroidal Neovascularization Diseases 0.000 claims description 6
- 206010060823 Choroidal neovascularisation Diseases 0.000 claims description 6
- 201000005569 Gout Diseases 0.000 claims description 6
- 201000001263 Psoriatic Arthritis Diseases 0.000 claims description 6
- 208000036824 Psoriatic arthropathy Diseases 0.000 claims description 6
- 206010064930 age-related macular degeneration Diseases 0.000 claims description 6
- 208000019069 chronic childhood arthritis Diseases 0.000 claims description 6
- 238000001990 intravenous administration Methods 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000007920 subcutaneous administration Methods 0.000 claims description 6
- 241000649046 Adeno-associated virus 11 Species 0.000 claims description 5
- 241000649047 Adeno-associated virus 12 Species 0.000 claims description 5
- 108010008165 Etanercept Proteins 0.000 claims description 5
- 208000010412 Glaucoma Diseases 0.000 claims description 5
- 201000004681 Psoriasis Diseases 0.000 claims description 5
- 206010038933 Retinopathy of prematurity Diseases 0.000 claims description 5
- 206010046851 Uveitis Diseases 0.000 claims description 5
- 108010081667 aflibercept Proteins 0.000 claims description 5
- 208000011231 Crohn disease Diseases 0.000 claims description 4
- 206010065630 Iris neovascularisation Diseases 0.000 claims description 4
- 206010073286 Pathologic myopia Diseases 0.000 claims description 4
- 208000007135 Retinal Neovascularization Diseases 0.000 claims description 4
- 201000002661 Spondylitis Diseases 0.000 claims description 4
- 201000005667 central retinal vein occlusion Diseases 0.000 claims description 4
- 208000001309 degenerative myopia Diseases 0.000 claims description 4
- 230000004340 degenerative myopia Effects 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 201000011190 diabetic macular edema Diseases 0.000 claims description 4
- 201000007914 proliferative diabetic retinopathy Diseases 0.000 claims description 4
- 206010055665 Corneal neovascularisation Diseases 0.000 claims description 3
- 201000000159 corneal neovascularization Diseases 0.000 claims description 3
- 229960000598 infliximab Drugs 0.000 claims description 3
- 108700036276 KH902 fusion Proteins 0.000 claims description 2
- 229950005748 conbercept Drugs 0.000 claims description 2
- 229960003876 ranibizumab Drugs 0.000 claims description 2
- 102200158858 rs34435255 Human genes 0.000 claims 2
- 206010063381 Polypoidal choroidal vasculopathy Diseases 0.000 claims 1
- 239000008280 blood Substances 0.000 claims 1
- 210000004369 blood Anatomy 0.000 claims 1
- 229930182470 glycoside Natural products 0.000 claims 1
- 150000002338 glycosides Chemical class 0.000 claims 1
- 230000004242 retinal defects Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 57
- 230000014509 gene expression Effects 0.000 description 45
- 239000000047 product Substances 0.000 description 29
- 210000001519 tissue Anatomy 0.000 description 19
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 17
- 210000001508 eye Anatomy 0.000 description 16
- 239000000203 mixture Substances 0.000 description 14
- 230000008488 polyadenylation Effects 0.000 description 14
- 108091033319 polynucleotide Proteins 0.000 description 14
- 102000040430 polynucleotide Human genes 0.000 description 14
- 239000002157 polynucleotide Substances 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 210000001525 retina Anatomy 0.000 description 13
- 108020004705 Codon Proteins 0.000 description 12
- 239000013603 viral vector Substances 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 11
- 210000005252 bulbus oculi Anatomy 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 11
- 230000001105 regulatory effect Effects 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- 108091026890 Coding region Proteins 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- 208000022873 Ocular disease Diseases 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000033115 angiogenesis Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 210000000608 photoreceptor cell Anatomy 0.000 description 6
- 102220004761 rs33990858 Human genes 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000026683 transduction Effects 0.000 description 6
- 238000010361 transduction Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 230000003612 virological effect Effects 0.000 description 6
- 241000283690 Bos taurus Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 102100035846 Pigment epithelium-derived factor Human genes 0.000 description 5
- 210000003161 choroid Anatomy 0.000 description 5
- 239000000839 emulsion Substances 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 238000004806 packaging method and process Methods 0.000 description 5
- 108090000102 pigment epithelium-derived factor Proteins 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- BJHCYTJNPVGSBZ-YXSASFKJSA-N 1-[4-[6-amino-5-[(Z)-methoxyiminomethyl]pyrimidin-4-yl]oxy-2-chlorophenyl]-3-ethylurea Chemical compound CCNC(=O)Nc1ccc(Oc2ncnc(N)c2\C=N/OC)cc1Cl BJHCYTJNPVGSBZ-YXSASFKJSA-N 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical group C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 4
- 208000024304 Choroidal Effusions Diseases 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 241000283977 Oryctolagus Species 0.000 description 4
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 4
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 4
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 4
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 239000002552 dosage form Substances 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 210000001503 joint Anatomy 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 3
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 3
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 208000012659 Joint disease Diseases 0.000 description 3
- 241000282560 Macaca mulatta Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 108050001704 Opsin Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 description 3
- 208000025865 Ulcer Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000012761 co-transfection Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 210000002683 foot Anatomy 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001524 infective effect Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 206010023332 keratitis Diseases 0.000 description 3
- 206010025135 lupus erythematosus Diseases 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000001124 posttranscriptional effect Effects 0.000 description 3
- 208000032253 retinal ischemia Diseases 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 201000005671 spondyloarthropathy Diseases 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 3
- 231100000397 ulcer Toxicity 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 2
- -1 Bcl-Xl) Proteins 0.000 description 2
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 description 2
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 102000000503 Collagen Type II Human genes 0.000 description 2
- 108010041390 Collagen Type II Proteins 0.000 description 2
- 206010011017 Corneal graft rejection Diseases 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 2
- 208000007465 Giant cell arteritis Diseases 0.000 description 2
- 102000053171 Glial Fibrillary Acidic Human genes 0.000 description 2
- 102000034615 Glial cell line-derived neurotrophic factor Human genes 0.000 description 2
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 2
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 208000016604 Lyme disease Diseases 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 102100034268 Neural retina-specific leucine zipper protein Human genes 0.000 description 2
- 102000003683 Neurotrophin-4 Human genes 0.000 description 2
- 108090000099 Neurotrophin-4 Proteins 0.000 description 2
- 102100021584 Neurturin Human genes 0.000 description 2
- 102000010175 Opsin Human genes 0.000 description 2
- 206010034277 Pemphigoid Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 102100040756 Rhodopsin Human genes 0.000 description 2
- 108090000820 Rhodopsin Proteins 0.000 description 2
- 206010039710 Scleroderma Diseases 0.000 description 2
- 102000005406 Tissue Inhibitor of Metalloproteinase-3 Human genes 0.000 description 2
- 108010031429 Tissue Inhibitor of Metalloproteinase-3 Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 210000003423 ankle Anatomy 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003429 antifungal agent Substances 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 108010006025 bovine growth hormone Proteins 0.000 description 2
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 210000004240 ciliary body Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 201000001981 dermatomyositis Diseases 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 2
- 229960001743 golimumab Drugs 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 210000000554 iris Anatomy 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 208000018937 joint inflammation Diseases 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 229940097998 neurotrophin 4 Drugs 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 201000006651 patellofemoral pain syndrome Diseases 0.000 description 2
- 108091008695 photoreceptors Proteins 0.000 description 2
- 208000002574 reactive arthritis Diseases 0.000 description 2
- 101150066583 rep gene Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 210000003705 ribosome Anatomy 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 201000000306 sarcoidosis Diseases 0.000 description 2
- 210000003786 sclera Anatomy 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012772 sequence design Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 206010043207 temporal arteritis Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000002845 virion Anatomy 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- 239000013607 AAV vector Substances 0.000 description 1
- 102100028187 ATP-binding cassette sub-family C member 6 Human genes 0.000 description 1
- OIRDTQYFTABQOQ-KQYNXXCUSA-N Adenosine Natural products 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 1
- 206010001257 Adenoviral conjunctivitis Diseases 0.000 description 1
- 208000026326 Adult-onset Still disease Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-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
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000003120 Angiofibroma Diseases 0.000 description 1
- 102400000068 Angiostatin Human genes 0.000 description 1
- 108010079709 Angiostatins Proteins 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 208000031104 Arterial Occlusive disease Diseases 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 206010003267 Arthritis reactive Diseases 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 208000008035 Back Pain Diseases 0.000 description 1
- 208000027496 Behcet disease Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010006811 Bursitis Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 101150044789 Cap gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 235000014653 Carica parviflora Nutrition 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 208000002691 Choroiditis Diseases 0.000 description 1
- 206010008874 Chronic Fatigue Syndrome Diseases 0.000 description 1
- 241000243321 Cnidaria Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000023890 Complex Regional Pain Syndromes Diseases 0.000 description 1
- 208000013586 Complex regional pain syndrome type 1 Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 208000019878 Eales disease Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 208000007985 Erythema Infectiosum Diseases 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 206010016207 Familial Mediterranean fever Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 208000001640 Fibromyalgia Diseases 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000034286 G proteins Human genes 0.000 description 1
- 108091004242 G-Protein-Coupled Receptor Kinase 1 Proteins 0.000 description 1
- 102000004437 G-Protein-Coupled Receptor Kinase 1 Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 208000003098 Ganglion Cysts Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 description 1
- 102100039261 Guanine nucleotide-binding protein G(t) subunit alpha-1 Human genes 0.000 description 1
- 208000018565 Hemochromatosis Diseases 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 201000002563 Histoplasmosis Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 1
- 101000888178 Homo sapiens Guanine nucleotide-binding protein G(t) subunit alpha-1 Proteins 0.000 description 1
- 101001040800 Homo sapiens Integral membrane protein GPR180 Proteins 0.000 description 1
- 101001032837 Homo sapiens Metabotropic glutamate receptor 6 Proteins 0.000 description 1
- 101000729271 Homo sapiens Retinoid isomerohydrolase Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010051151 Hyperviscosity syndrome Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 108020005350 Initiator Codon Proteins 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100021244 Integral membrane protein GPR180 Human genes 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 206010061246 Intervertebral disc degeneration Diseases 0.000 description 1
- 101150105817 Irbp gene Proteins 0.000 description 1
- 208000012528 Juvenile dermatomyositis Diseases 0.000 description 1
- 229940127379 Kallikrein Inhibitors Drugs 0.000 description 1
- 208000011200 Kawasaki disease Diseases 0.000 description 1
- 208000001126 Keratosis Diseases 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 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
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010025412 Macular dystrophy congenital Diseases 0.000 description 1
- 206010025421 Macule Diseases 0.000 description 1
- 208000035719 Maculopathy Diseases 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 102100038300 Metabotropic glutamate receptor 6 Human genes 0.000 description 1
- 208000003250 Mixed connective tissue disease Diseases 0.000 description 1
- 206010062207 Mycobacterial infection Diseases 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- 210000005156 Müller Glia Anatomy 0.000 description 1
- 108050003956 Neural retina-specific leucine zipper proteins Proteins 0.000 description 1
- 201000004404 Neurofibroma Diseases 0.000 description 1
- 108010015406 Neurturin Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 239000012269 PD-1/PD-L1 inhibitor Substances 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 1
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 102000003827 Plasma Kallikrein Human genes 0.000 description 1
- 108090000113 Plasma Kallikrein Proteins 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 208000007048 Polymyalgia Rheumatica Diseases 0.000 description 1
- 208000003971 Posterior uveitis Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 201000004613 Pseudoxanthoma elasticum Diseases 0.000 description 1
- 206010037649 Pyogenic granuloma Diseases 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 101150116978 RPE65 gene Proteins 0.000 description 1
- 208000037418 Rare genetic eye disease Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000012322 Raynaud phenomenon Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 201000001947 Reflex Sympathetic Dystrophy Diseases 0.000 description 1
- 208000033464 Reiter syndrome Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 206010038848 Retinal detachment Diseases 0.000 description 1
- 101710097927 Retinal-binding protein Proteins 0.000 description 1
- 206010038910 Retinitis Diseases 0.000 description 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 1
- 102100031176 Retinoid isomerohydrolase Human genes 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 108090000799 Rhodopsin kinases Proteins 0.000 description 1
- 241000124015 Salix viminalis Species 0.000 description 1
- 206010039705 Scleritis Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- 208000027073 Stargardt disease Diseases 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N 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 1
- 241000282887 Suidae Species 0.000 description 1
- 208000005400 Synovial Cyst Diseases 0.000 description 1
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- 102000012753 TIE-2 Receptor Human genes 0.000 description 1
- 108010090091 TIE-2 Receptor Proteins 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 206010043189 Telangiectasia Diseases 0.000 description 1
- 208000000491 Tendinopathy Diseases 0.000 description 1
- 206010043255 Tendonitis Diseases 0.000 description 1
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 1
- 201000005485 Toxoplasmosis Diseases 0.000 description 1
- 206010044302 Tracheitis Diseases 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 108091008605 VEGF receptors Proteins 0.000 description 1
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 206010058990 Venous occlusion Diseases 0.000 description 1
- 208000014070 Vestibular schwannoma Diseases 0.000 description 1
- 208000010011 Vitamin A Deficiency Diseases 0.000 description 1
- 206010047663 Vitritis Diseases 0.000 description 1
- 241001492404 Woodchuck hepatitis virus Species 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000004064 acoustic neuroma Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 210000000411 amacrine cell Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 210000001742 aqueous humor Anatomy 0.000 description 1
- 208000021328 arterial occlusion Diseases 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003986 cell retinal photoreceptor Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 201000010415 childhood type dermatomyositis Diseases 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 201000007391 chondromalacia patellae Diseases 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 208000018180 degenerative disc disease Diseases 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 208000014906 developmental dysplasia of the hip Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 208000021373 epidemic keratoconjunctivitis Diseases 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 229940116862 faricimab Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical class O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000035474 group of disease Diseases 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 201000011066 hemangioma Diseases 0.000 description 1
- 208000013210 hematogenous Diseases 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 210000002287 horizontal cell Anatomy 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000013653 hyalitis Diseases 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 208000021600 intervertebral disc degenerative disease Diseases 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000010189 intracellular transport Effects 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 208000001725 mucocutaneous lymph node syndrome Diseases 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 208000029766 myalgic encephalomeyelitis/chronic fatigue syndrome Diseases 0.000 description 1
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 208000001491 myopia Diseases 0.000 description 1
- 230000004379 myopia Effects 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 201000003142 neovascular glaucoma Diseases 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000000414 obstructive effect Effects 0.000 description 1
- 230000000174 oncolytic effect Effects 0.000 description 1
- 210000001328 optic nerve Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940121653 pd-1/pd-l1 inhibitor Drugs 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000013326 plasmid cotransfection Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 208000005987 polymyositis Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 208000023558 pseudoxanthoma elasticum (inherited or acquired) Diseases 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 239000013608 rAAV vector Substances 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 102000024458 retinal binding proteins Human genes 0.000 description 1
- 230000004264 retinal detachment Effects 0.000 description 1
- 210000003994 retinal ganglion cell Anatomy 0.000 description 1
- 239000000790 retinal pigment Substances 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- 210000003583 retinal pigment epithelium Anatomy 0.000 description 1
- 108091010987 retinoic acid binding proteins Proteins 0.000 description 1
- 102000003702 retinoic acid receptors Human genes 0.000 description 1
- 108090000064 retinoic acid receptors Proteins 0.000 description 1
- 201000007714 retinoschisis Diseases 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 201000006476 shipyard eye Diseases 0.000 description 1
- 208000007056 sickle cell anemia Diseases 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 208000005198 spinal stenosis Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 208000009056 telangiectasis Diseases 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 201000004415 tendinitis Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002627 tracheal intubation Methods 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 201000007790 vitelliform macular dystrophy Diseases 0.000 description 1
- 210000004127 vitreous body Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
-
- 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
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/01—DNA viruses
- C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the invention relates to the field of biotechnology, in particular to a modified AAV capsid protein and its use.
- Vectors used in gene therapy can be divided into viral vectors and non-viral vectors.
- viral vectors the most commonly used ones are adenoviral vectors, lentiviral vectors, adeno-associated viral vectors, and herpes simplex virus vectors.
- Adeno-associated virus belongs to the family Parvoviridae and the genus Dependovirus.
- the virion consists of a 25 nm icosahedral capsid comprising a 4.7 kb single-stranded DNA genome with two open reading frames: Rep and Cap.
- the nonstructural Rep gene encodes four regulatory proteins necessary for viral replication, while Cap encodes three structural proteins (VP1-3) that assemble into a 60-subunit capsid.
- the viral capsid mediates the ability of AAV vectors to overcome many biological barriers to viral transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and unencapsulation in the nucleus.
- AAV is widely used in many fields such as gene transduction, gene therapy, vaccination, and oncolytic therapy. It has many advantages such as low immunogenicity, long time for expressing foreign genes in vivo, and no host cell genome integration, and is widely used in experimental and clinical research.
- the present invention relates to the delivery of viral vectors, such as AAV viral vectors, containing a gene of interest to desired cells or tissues.
- viral vectors such as AAV viral vectors
- the present invention encompasses modified capsid proteins having one or more modifications (such as substitutions, insertions, or mutations) in amino acid sequence relative to the parent AAV capsid protein, when present
- a target tissue or target cell eg, retina, muscle, or joint cavity
- One aspect of the present invention provides a modified adeno-associated virus (AAV) capsid protein, the capsid protein comprising about 5-14 amino acid polypeptide substitutions relative to the parent AAV capsid protein, and wherein The AAV virus comprising the modified capsid protein has enhanced retinal cell infectivity compared to the AAV virus of the AAV capsid protein.
- AAV adeno-associated virus
- the polypeptide comprises selected from RGNRQ (SEQ ID NO: 1), QQNTARGNRQ (SEQ ID NO: 2), RGNRQAAQQNTA (SEQ ID NO: 3), RGNRQQNTA (SEQ ID NO: 4), RGNRQQQNTA (SEQ ID NO:5), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7), RGNQQPRPTSRQ (SEQ ID NO:8), RGNRQAAQQPTPTS (SEQ ID NO:9) or RGNRQQQPTTPTS (SEQ ID NO:9) :19) amino acid sequence; and the substitution is located at the 588th to 592nd amino acid of the parent AAV8 or the corresponding position of another serotype capsid protein.
- the capsid protein polypeptide comprises selected from QQNTARGNRQ (SEQ ID NO: 2), RGNRQQNTA (SEQ ID NO: 4), SGNTQ (SEQ ID NO: 6), RGNQQNTARQ (SEQ ID NO: 7) or the amino acid sequence of RGNRQQQPTPTS (SEQ ID NO: 19).
- the capsid protein further includes a mutation relative to amino acid positions 262-272 of the parental AAV8 capsid protein or the corresponding position of another serotype capsid protein, and the amino acid sequence after mutation is SQSGASNDNH (SEQ ID NO: 10).
- the capsid protein comprises a polypeptide substitution at amino acid 588-592 of the parent AAV8 or the corresponding position of another serotype capsid protein, and the polypeptide is RGNRQ (SEQ ID NO: 1); and relative to the mutation at positions 262-272 of the parental AAV8 capsid protein or the corresponding position of another serotype capsid protein, the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10).
- the AAV of the present invention can be derived from any serotype AAV, for example, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 , AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, or AAVrh10.
- the AAV of the present invention is selected from AAV8.
- the capsid protein further comprises amino acid mutations at positions D80 and/or V125 relative to the parental AAV8 capsid protein.
- said mutation is D80N and/or V125A.
- said mutation is D80Q and/or V125G.
- the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parental AAV8, and the polypeptide is RGNQQNTARQ (SEQ ID NO: 7); and relative to the parental AAV8 capsid protein D80 and Amino acid mutations at position V125, said mutations being D80N and V125A.
- the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8, and the polypeptide is RGNQQNTARQ (SEQ ID NO: 7); and relative to the parent AAV8 capsid Amino acid mutations at positions D80 and V125 of the protein, said mutations being D80Q and V125G.
- Another aspect of the present invention provides another modified adeno-associated virus (AAV) capsid protein, said capsid protein comprising an amino acid located after amino acid 589 of the parental AAV8 or the corresponding position of another serotype capsid protein A polypeptide is inserted, and wherein the AAV virus comprising the modified capsid protein has enhanced retinal cell infectivity compared to the AAV virus comprising the corresponding parental AAV capsid protein.
- AAV adeno-associated virus
- the polypeptide comprises an amino acid sequence selected from RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ (SEQ ID NO: 21 ) or RGDVSSPQQ (SEQ ID NO: 22).
- the AAV may be derived from any serotype of AAV, for example an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV - DJ9, AAVrh8, AAVrh8R or AAVrh10.
- the AAV serotype is AAV8.
- rAAV recombinant adeno-associated virus
- ii. comprising a heterologous nucleic acid encoding a gene product.
- the gene product is a VEGF antagonist or a TNF- ⁇ antagonist.
- the VEGF antagonist is selected from aflibercept, conbercept, ranibizumab, brucetizumab, preferably aflibercept.
- the TNF- ⁇ antagonist is selected from etanercept, infliximab, adalimumab, beselizumab or golimumab, preferably etanercept general.
- Another aspect of the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising:
- Another aspect of the present invention provides the recombinant adeno-associated virus according to the present invention for use as a medicine.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through vitreous, retinal, choroidal injection, intravenous, subcutaneous, intramuscular or joint cavity injection. In a preferred embodiment, the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through suprachoroidal injection. In another preferred embodiment, the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered by intramuscular or joint cavity injection.
- Another aspect of the present invention provides the recombinant adeno-associated virus or the pharmaceutical composition according to the present invention for use in therapy.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through suprachoroidal injection.
- Another aspect of the present invention provides the use of the recombinant adeno-associated virus in the preparation of medicines for preventing or treating eye diseases.
- Another aspect of the present invention provides a method for preventing or treating eye diseases, the method comprising administering an effective amount of the recombinant adeno-associated virus or the pharmaceutical composition according to the present invention to an individual in need.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through suprachoroidal injection.
- the ocular disease of the present invention is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascularization ocular disease, non-infectious uveitis or glaucoma.
- the eye disease of the present invention is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, Branch retinal vein occlusion, macular edema due to branch retinal vein occlusion, diabetic retinal edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoid choroidal vasculopathy, choroid secondary to degenerative myopia Neovascular or retinopathy of prematurity.
- Another aspect of the present invention provides the recombinant adeno-associated virus or the pharmaceutical composition according to the present invention for use in the treatment of eye diseases.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through suprachoroidal injection.
- the ocular disease of the present invention is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascularization ocular disease, non-infectious uveitis or glaucoma.
- the eye disease of the present invention is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, Branch retinal vein occlusion, macular edema due to branch retinal vein occlusion, diabetic retinal edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoid choroidal vasculopathy, choroid secondary to degenerative myopia Neovascular or retinopathy of prematurity.
- Another aspect of the present invention provides the use of the recombinant adeno-associated virus in the preparation of medicaments for preventing or treating arthritis or related diseases.
- Another aspect of the present invention provides a method for preventing or treating arthritic diseases or related conditions, the method comprising administering an effective amount of the recombinant adeno-associated virus or the pharmaceutical composition according to the present invention to an individual in need.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through intravenous, subcutaneous, intramuscular or joint cavity injection.
- the recombinant adeno-associated virus or the pharmaceutical composition described in the present invention is administered by intramuscular or joint cavity injection.
- the arthritic diseases or related diseases described in the present invention are selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease , Plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.
- Another aspect of the present invention provides the recombinant adeno-associated virus or pharmaceutical composition according to the present invention for use in the treatment of arthritis diseases or related conditions.
- the recombinant adeno-associated virus or the pharmaceutical composition of the present invention is administered through intravenous, subcutaneous, intramuscular or joint cavity injection.
- the recombinant adeno-associated virus or the pharmaceutical composition described in the present invention is administered by intramuscular or joint cavity injection.
- the arthritic diseases or related diseases described in the present invention are selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease , Plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.
- Figure 1A shows the amino acid sequence alignment of AAV capsid protein GH loop partial regions
- Figure 1B shows the amino acid sequence alignment of the AAV capsid protein GH loop partial region
- Figure 2 is a schematic diagram of the construction of the AAV8 capsid protein mutant plasmid
- Figure 3 shows the EGFP mRNA expression level of the mouse intravitreal injection of AAV8 mutant virus
- Figure 4 shows the EGFP fluorescence signal of retinal slices of mice injected with AAV8 mutant virus in the vitreous
- Figure 5 shows the retinal slice EGFP fluorescence signal of rat intravitreal injection of AAV8 mutant virus
- Figure 6 shows the EGFP fluorescence signal and DAPI signal of rabbit suprachoroidal injection of AAV8 mutant virus
- Fig. 7 is the EGFP fluorescence signal of ARPE19 cell culture AAV8 mutant virus
- Figure 8A shows the counting of EGFP-positive cells after AAV8 mutant virus infection of ARPE19 cells by flow cytometry
- Figure 8B shows flow cytometry counting of EGFP-positive cells after AAV8 mutant virus infected ARPE19 cells
- Figure 9 shows the relative content of genomic DNA of AAV8 mutant virus mCherry injected into the suprachoroidal space of rabbits
- Figure 10 shows the luciferase fluorescence intensity of the mouse intramuscularly injected AAV8 mutant virus
- Figure 11 shows the clinical score of mice with intraarticular/muscular injection of AAV8 mutant virus
- Figure 12 shows the paw thickness of mice injected with AAV8 mutant virus in the joint cavity/muscle.
- the present invention relates to AAV viral vectors comprising an AAV viral vector having increased infectivity to a target tissue or target cell (e.g. retina, muscle or joint cavity) compared to an unmodified parent AAV capsid protein, comprising the capsid protein rAAV expressing a target gene (such as VEGF antagonist, TNF- ⁇ antagonist) and its pharmaceutical composition and application.
- a target tissue or target cell e.g. retina, muscle or joint cavity
- a target gene such as VEGF antagonist, TNF- ⁇ antagonist
- the present invention provides a modified AAV capsid protein, wherein the modified AAV capsid protein comprises one or more amino acid modifications compared to a corresponding wild-type AAV or a parent AAV.
- the modifications include one or more amino acid substitutions, deletions or insertions relative to the parental capsid protein or AAV capsid protein.
- Such modifications confer increased infectivity to target tissues or cells (eg, retina, muscle, or joint cavity) compared to AAV viral vectors comprising the unmodified parent AAV capsid protein.
- the modified AAV capsid protein comprises a heterologous polypeptide substitution within a capsid protein serotype, such as VP1, belonging to: AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV 5 AAV type, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAVrh10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, Bovine AAV, AAV2.7m8, AAVShH10, AAV2.5T, AAV2.5T/7m8, AAV9/7m8, and AAV5/7m8.
- the capsid variant may be a chimeric capsid variant.
- the sequence of a chimeric capsid variant may comprise portions of two or more AAV capsid serotypes or variants thereof.
- the chimeric capsid comprises portions of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capsid protein serotypes.
- the modified AAV capsid protein comprises a heterologous polypeptide substitution of about 5-14 amino acids.
- the modified capsid protein of the invention comprises a heterologous polypeptide of about 5-14 amino acids at amino acid positions 588-592 of the parental AAV8 or the corresponding position of another serotype capsid protein replace.
- the amino acid sequences of AAV1-AAV9 are as described in patents such as US8962330B and US10041090B.
- the modified capsid protein of the present invention comprises a heterologous polypeptide substitution of about 5-14 amino acids at amino acids 588-592 of the parental AAV8.
- the 588th to 592nd amino acids of the parent/wild type AAV8 capsid protein are "QQNTA". Therefore, a specific embodiment of the present invention is that the modified capsid protein replaces the 588-592 amino acid "QQNTA" of the parental/wild-type AAV8 capsid protein with a heterologous polypeptide of 5-14 amino acids.
- the substitution sites corresponding to amino acids 588-592 of AAV8 are at the corresponding positions of other capsid proteins, for example as shown in FIG. 1A .
- amino acid 586-590 of AAV1 at amino acid 585-589 of AAV2, at amino acid 586-590 of AAV3, at amino acid 584-588 of AAV4, at amino acid 575-579 of AAV5, at amino acid 586 of AAV6 ⁇ 590 amino acids, at 587 ⁇ 591 amino acids of AAV7, at 586 ⁇ 590 amino acids of AAV9, at 588 ⁇ 592 amino acids of AAV10.
- the length is 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids or 11 amino acids.
- the polypeptide of the present invention comprises a polypeptide selected from RGNRQ (SEQ ID NO: 1), QQNTARGNRQ (SEQ ID NO: 2), RGNRQAAQQNTA (SEQ ID NO: 3), RGNRQQNTA (SEQ ID NO: 4 ), RGNRQQQNTA (SEQ ID NO:5), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7), RGNQQPRPTSRQ (SEQ ID NO:8), RGNRQAAQQPTTPTS (SEQ ID NO:9) or RGNRQQQPTPTS (SEQ ID NO:19) amino acid sequence.
- the polypeptide of the present invention comprises a polypeptide selected from QQNTARGNRQ (SEQ ID NO: 2), RGNRQQNTA (SEQ ID NO: 4), SGNTQ (SEQ ID NO: 6) or RGNQQNTARQ (SEQ ID NO: 7 ) or the amino acid sequence of RGNRQQQPTPTS (SEQ ID NO: 19).
- the modified AAV capsid protein further comprises a mutation at amino acid positions 262-272 relative to the parent AAV8 capsid protein or the corresponding position of another serotype capsid protein.
- the amino acids 262-272 of the parental/wild-type AAV8 capsid protein are "SNGTSGGATND”. Therefore, a specific embodiment of the present invention is that the modified capsid protein is mutated in the 262-272 amino acid "SNGTSGGATND" of the parent/wild type AAV8 capsid protein.
- the mutation sites corresponding to amino acids 262-272 of AAV8 are at the corresponding positions of other capsid proteins, such as amino acids 261-271 of AAV1, amino acids 261-271 of AAV2, and amino acids 261-271 of AAV3 at amino acid 255-265 of AAV4, at amino acid 251-261 of AAV5, at amino acid 261-271 of AAV6, at amino acid 262-272 of AAV7, at amino acid 261-271 of AAV9, at amino acid 261-271 of AAV10 262-272 amino acids.
- the mutation sites corresponding to amino acids 262-272 of AAV8 are at the corresponding positions of other capsid proteins, for example as shown in FIG. 1B .
- the mutated amino acid sequence of the present invention is SQSGASNDNH (SEQ ID NO: 10).
- the modified capsid protein of the present invention includes a polypeptide substitution at the 588-592 amino acid position of the parent AAV8 or the corresponding position of another serotype capsid protein, and the polypeptide is RGNRQ (SEQ ID NO: 1); With respect to the mutation at the 262nd to 272nd positions of the parental AAV8 capsid protein or the corresponding position of another serotype capsid protein, the amino acid sequence after the mutation is SQSGASNDNH (SEQ ID NO: 10) .
- the serotype of the parental capsid protein described in the present invention is AAV8.
- the modified AAV capsid protein of the present invention further comprises an amino acid mutation at position D80 and/or V125 relative to the parent AAV8 capsid protein.
- said mutation is D80N and/or V125A.
- said mutation is D80Q and/or V125G.
- the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8, and the polypeptide is RGNQQNTARQ (SEQ ID NO: 7); and relative to the parent AAV8 capsid protein D80 and amino acid mutations at position V125, which are D80N and V125A.
- the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8, and the polypeptide is RGNQQNTARQ (SEQ ID NO: 7); and relative to the parent AAV8 capsid protein Amino acid mutations at positions D80 and V125, said mutations being D80Q and V125G.
- the modified AAV capsid protein of the invention includes a polypeptide insertion after amino acid 589 relative to the parental AAV8 or the corresponding position of another serotype capsid protein.
- the insertion site is located after amino acid 589 of AAV8 or the corresponding position of other capsid proteins, for example, at amino acid 589 of AAV1, at amino acid 586 of AAV2, at amino acid 587 of AAV3, at amino acid 585 of AAV4, at amino acid 585 of AAV5 After amino acid 576 of AAV6, amino acid 587 of AAV7, amino acid 587 of AAV9, and amino acid 589 of AAV10.
- the inserted polypeptide comprises the amino acid sequence of RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ (SEQ ID NO: 21 ) or RGDVSSPQQ (SEQ ID NO: 22).
- the modified AAV capsid protein of the present invention comprises a polypeptide insertion after the 589th amino acid relative to the parent AAV8, and the inserted polypeptide comprises RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ ( SEQ ID NO:21) or the amino acid sequence of RGDVSSPQQ (SEQ ID NO:22).
- the modified capsid protein of the present invention also includes at least 85%, 90%, 95%, 98% of the amino acid sequence of the capsid protein undergoing amino acid substitution, mutation and/or insertion , 99% or 100% homologous sequences.
- an AAV comprising a modified capsid protein is at least 1-fold, 2-fold, 5-fold, 10-fold more infective to retinal cells compared to an AAV virus comprising the corresponding parental/wild-type capsid protein. times, 20 times, 50 times, 100 times or even higher.
- the retinal cells include RPE-19 cells (human retinal pigment epithelial cell line), retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Müller glia cells and retinal pigment epithelium.
- the AAV comprising the modified capsid protein has at least 1-fold, 2-fold, 5-fold increased infectivity to RPE cells , 10 times, 20 times, 50 times, 100 times or even higher.
- an AAV comprising a modified capsid protein is at least 1-fold more infective to retinal cells after suprachoroidal injection compared to an AAV virus comprising the corresponding parental/wild-type capsid protein , 2 times, 5 times, 10 times, 20 times, 50 times, 100 times or even higher.
- an AAV comprising a modified capsid protein is at least 1-fold, 2-fold more infective to retinal cells after intravitreal injection compared to an AAV virus comprising the corresponding parental/wild-type capsid protein. times, 5 times, 10 times, 20 times, 50 times, 100 times or even higher.
- rAAVs Recombinant adeno-associated viruses
- the gene delivery vectors of the present invention are recombinant adeno-associated viruses (rAAVs).
- rAAVs recombinant adeno-associated viruses
- the rAAV described herein is a single-chain AAV (ssAAV).
- ssAAV refers to rAAV having the coding sequence of the gene of interest and the complementary sequence on separate strands, and is packaged in a separate viral capsid.
- the recombinant adeno-associated virus (rAAV) of the present invention comprises: i) the modified capsid protein according to the present invention; ii) a heterologous nucleic acid comprising a gene product encoding.
- the rAAV described in the present invention comprises the aforementioned modified capsid protein of the present invention, so that the recombinant AAV (rAAV) described in the present invention has the same Enhanced cell or tissue (eg eye tissue or cells, muscle tissue or joint cavity, etc.) specific targeting ability or infectivity.
- Enhanced cell or tissue eg eye tissue or cells, muscle tissue or joint cavity, etc.
- the rAAV described herein comprises a heterologous nucleic acid encoding a gene product and a polynucleotide expression cassette of regulatory elements.
- regulatory element refers to a nucleic acid sequence that regulates the expression of a gene product to which it is operably linked.
- expression regulatory elements may include promoters, enhancers, internal ribosomal entry sites (IRES), transcription terminators, start codons preceding protein-encoding genes, intron splicing signals, and stop codons.
- the term may also include nucleic acid sequence design in which unwanted possible initiation codons are removed from the sequence in or out of frame. It may also include nucleic acid sequence design to remove unwanted possible splice sites. It may also include sequences directing the addition of polyadenylation or polyA, etc.
- the heterologous nucleic acid includes a nucleotide sequence encoding a gene product of a target gene, such as a therapeutic gene product.
- the gene product is interfering RNA.
- the gene product is an aptamer.
- the gene product is a polypeptide.
- the gene product is a site-specific nuclease provided for site-specific knockdown of gene function.
- the gene product is a polypeptide, protein, fusion protein, antibody, or the like.
- the gene product of the present invention is selected from VEGF antagonists, TNF- ⁇ antagonists, PD-1/PD-L1 inhibitors, Ang-2 inhibitors, plasma kallikrein inhibitors, endothelial inhibitors Oncostatin, Angiostatin, Pigment Epithelium-Derived Factor (PEDF), Soluble Tie-2 Receptor, Ang-2 Antagonist, CD Tissue Inhibitor of Metalloproteinase-3 (TIMP-3), Light Responsive Opsin (such as rhodopsin), anti-apoptotic polypeptides (such as Bcl-2, Bcl-Xl), glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2, neural rank protein (neurturin, NTN), Ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), neurotrophin-4 (NT4), brain-derived neurotrophic factor (BDNF), and functional variants and fragments thereof, contained in these proteins or polypeptides
- the gene product of the invention is selected from a VEGF antagonist.
- the VEGF antagonist is selected from any of the extracellular domains 1-7 of VEGF receptor 1 (VEGFR-1, or Flt) or receptor 2 (VEGFR-2, or Flk).
- a domain such as VEGFR-1 extracellular domain 2 (Flt d2) and/or VEGFR-2 extracellular domain 3 or 4.
- the VEGF antagonist comprises at least one item selected from the following group:
- a) a fusion protein comprising the extracellular domain 2 of VEGFR-1 and the extracellular domain 3 of VEGFR-2;
- the fusion protein has the amino acid sequence described in SEQ ID NO: 11 (Fltd2Flkd3);
- fusion protein comprising extracellular domain 2 of VEGFR-1, extracellular domain 3 and domain 4 of VEGFR-2;
- the fusion protein has SEQ ID NO: 12 (Fltd2 Flkd3, 4) said amino acid sequence;
- d) has the antibody heavy chain variable region sequence described in SEQ ID NO:15 and the antibody light chain variable region sequence described in SEQ ID NO:16.
- SEQ ID NO:12 is the sequence shown below:
- SEQ ID NO: 13 is the sequence shown below:
- SEQ ID NO: 14 is the sequence shown below:
- SEQ ID NO:15 is the sequence shown below:
- SEQ ID NO: 16 is the sequence shown below:
- the VEGF antagonist has the amino acid sequence as set forth in SEQ ID NO: 11:
- the nucleotide encoding the VEGF antagonist has a sequence as described in SEQ ID NO:23:
- the protein or polypeptide is selected from a TNF-alpha antagonist.
- the VEGF antagonist comprises etanercept, infliximab, adalimumab, becelitizumab, or golimumab.
- the TNF- ⁇ antagonist has the heavy chain variable region sequence described in SEQ ID NO: 17 and the light chain variable region sequence described in SEQ ID NO: 18.
- SEQ ID NO: 17 is the sequence shown below:
- SEQ ID NO: 18 is the sequence shown below:
- the TNF- ⁇ antagonist has the amino acid sequence described in SEQ ID NO:24:
- the nucleotide encoding TNF- ⁇ antagonist has the sequence as described in SEQ ID NO:25:
- the VEGF antagonist or TNF- ⁇ antagonist has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about Amino acid sequences that are 99% identical or 100% identical.
- the VEGF antagonist or TNF- ⁇ antagonist has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, Nucleotide sequences that are at least about 99% identical or 100% identical.
- the protein or polypeptide is selected to have one or more distinct antigen binding sites.
- the protein or polypeptide binds both VEGF and Ang-2.
- the protein or polypeptide is a bispecific antibody that simultaneously binds to VEGF and Ang-2, such as the Faricimab double antibody.
- codon optimization can be used to design the coding sequence for better expression of the gene of interest.
- the present invention can optimize the nucleotide codons encoding polypeptides, fusion proteins, antibodies or functional fragments thereof, so as to improve the protein expression level of target genes in target cells, tissues or species.
- the coding sequence is the portion of the mRNA sequence that encodes the amino acids used for transcription and translation. During transcription and translation, every three nucleotide codons in the 61 nucleotide codons are transcribed and translated into any one of the 20 amino acids. However, different cell types and different animal species have different frequencies of tRNAs encoding the same amino acid.
- the ribosomal transcription machinery may slow down, preventing efficient transcription. Therefore, those skilled in the art can improve the expression level of the target gene by "codon optimization".
- the nucleotide sequence encoding the product is modified with a synonymous codon sequence, for example, the codon that can be expressed at a higher frequency in the target cell is used to replace the codon that is expressed at a lower frequency, so as to increase the specificity of the target substance.
- the expression level in a cell, tissue or substance is, for example, increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%. Codon-optimized coding sequences can be designed by various methods.
- Such optimization can be performed using methods available online, published methods, or companies that provide codon optimization services.
- One method of codon optimization is described, for example, in International Patent Publication No. WO2015/012924, which is incorporated herein by reference.
- the entire length of the open reading frame (ORF) of the product was appropriately modified. However, in some embodiments, only one segment of the ORF may be altered.
- codon-optimized encoding nucleotides encoding polypeptides are generated.
- one or more expression control elements in the polynucleotide expression cassette of the present invention comprises a constitutive promoter region, the nucleotide sequence encoding the gene product is operably linked to the constitutive promoter, and the promoter is a promoter that is active in a wide range of cells, tissues or species, so The above-mentioned promoter promotes the expression of the target gene in specific cells or tissues in vivo or in vitro.
- examples of promoters include chicken ⁇ -actin promoter (CBA), cytomegalovirus promoter (CMV), CMV early enhancer/chicken ⁇ -actin (CBA) promoter/rabbit ⁇ -globin intron (CAG), elongation factor 1 ⁇ promoter (EF1 ⁇ ), human phosphoglycerate kinase promoter (PGK), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter promoter, mitochondrial heavy chain promoter, ubiquitin promoter, etc.
- CBA chicken ⁇ -actin promoter
- CMV cytomegalovirus promoter
- CBA CMV early enhancer/chicken ⁇ -actin
- CAG CAG
- EF1 ⁇ elongation factor 1 ⁇ promoter
- PGK human phosphoglycerate kinase promoter
- MNT promoter UB6 promoter
- CAG promoter CAG promoter
- RPE65 promoter opsin promote
- the regulatory element comprises an inducible promoter region to which the nucleotide sequence encoding the gene product is operably linked.
- the inducible promoter is a photoreceptor-specific promoter.
- Suitable photoreceptor-specific promoters include, for example, rhodopsin promoter, rhodopsin kinase promoter, beta phosphodiesterase gene promoter, retinitis pigmentosa gene promoter, interphotoreceptor retinoic acid binding protein (IRBP) gene Enhancer, IRBP gene promoter, opsin gene promoter, retinoschisis protein gene promoter, CRX homeodomain protein gene promoter, guanine nucleotide binding protein alpha transduction activity polypeptide 1 (GNAT1) gene promoter , neural retina-specific leucine zipper protein (NRL) gene promoter, human cone arrester protein (hCAR) promoter, and PR2.1, PR1.7, PR1.5 and PR1.1 promoters, R
- the expression control sequence in the polynucleotide expression cassette of the present invention may also contain a polyadenylation signal.
- the polyadenylation signal is also known as polyadenylation site, polyadenylation tail, Poly(A) site, Poly(A) signal or Poly(A) tail.
- a polyadenylation region refers to the covalent linkage of polyadenylic acid to a messenger RNA (mRNA) molecule. During protein biosynthesis, this is part of the way to produce mature mRNA ready for translation. In eukaryotes, polyadenylation is a mechanism by which mRNA molecules are interrupted at their 3' ends.
- the polyadenylation signal protects mRNA from exonuclease attack and is important for transcription termination, export of mRNA from the nucleus, and translation.
- the polyadenylation signal consists of multiple consecutive adenosine monophosphates, usually containing AAUAAA repeats.
- Some exemplary polyadenylation signals include simian vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or beta-globin.
- the polynucleotide expression cassette of the present invention may further comprise an inverted terminal repeat (ITR).
- ITR inverted terminal repeat
- a functional adenoviral inverted terminal repeat (ITR) refers to, for example, the ITR sequence used for integration, replication and packaging of AAV virions.
- the length of the ITR sequence is about 145 bp.
- substantially the entire sequence encoding ITR is used in the molecule, and those skilled in the art modify these ITR sequences according to conventional technical means in the field.
- An example of the use of ITR molecules in this application is a "cis" plasmid containing a gene of interest, where the gene sequence of interest and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences.
- AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types.
- the heterologous nucleotides encoding the gene of interest flank the AAV ITR (e.g., in the orientation 5'-ITR-gene of interest-ITR-3').
- the AAV ITR is selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITV, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITV, AAV10 ITR, AAV11 ITR, and AAV12 ITR.
- RNA export signals are cis-acting post-transcriptional regulatory elements that enhance RNA export from the nucleus.
- Exemplary RNA export sequences include, but are not limited to, sequences from the hepatitis B virus post-transcriptional regulatory element (HPRE) and woodchuck hepatitis virus post-transcriptional element (WPRE).
- polynucleotide expression cassette of the present invention may optionally contain other elements, including but not limited to restriction sites for facilitating cloning, for operably linking each element, or for regulating Regulatory elements of gene expression.
- restriction sites for facilitating cloning, for operably linking each element, or for regulating Regulatory elements of gene expression.
- examples are bacterial sequences of plasmid vectors, attp sites, attB sites of phage integrase vectors, promoter linkers, polyadenylation sequence linkers, and the like.
- the polynucleotide expression cassette described in the present invention comprises those skilled in the art according to the combination of two or more elements in the above-mentioned elements and the gene of interest, wherein the expression cassette of the polynucleotide
- the gene-of-interest coding sequence and associated regulatory elements in the cassette are flanked by 5' and 3' AAV ITR sequences.
- a polynucleotide expression cassette of the present invention may include, in 5' to 3' order, an N-terminal AAV ITR, an enhancer/promoter/intron, a Kozak sequence, a coding sequence for a gene product, WPRE or HPRE RNA output signal sequence, polyadenylation signal sequence, and C-terminal AAV ITR.
- the recombinant adeno-associated virus of the present invention comprises a polynucleotide expression cassette encoding a gene product, and the sequence from the 5' to the 3' end of the polynucleotide expression cassette includes:
- the gene product coding sequence is the nucleic acid sequence encoding the aforementioned gene product (such as the aforementioned VEGF antagonist or TNF- ⁇ antagonist).
- Recombinant viral vectors comprising the modified capsid proteins described herein can be produced using standard methods.
- the method comprises culturing a host cell containing an artificial genome comprising: a polynucleotide expression cassette in cis flanked by AAV ITRs, wherein the polynucleotide expression cassette in cis comprises a coding sequence for a gene product , the coding sequence is operably linked to an expression control element that will control the expression of the gene product in a human cell; a trans expression cassette lacking the AAV ITR, wherein the trans expression cassette encodes an AAV rep and a capsid protein, the The AAV rep and capsid proteins are operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cell in culture and supply the rep and cap proteins in trans; sufficient to permit passage of the AAV an adenoviral helper function for capsid protein replication and packaging of the artificial genome; and recovery of recombinant AAV packaging the artificial genome from the cell
- “Host cell” refers to any cell that contains or is capable of containing a substance of interest.
- the host cell is a mammalian cell.
- the host cells are photoreceptor cells, retinal pigment epithelial cells, keratinocytes, keratinocytes and/or tumor cells.
- Another aspect of the present invention is to provide a pharmaceutical composition
- a pharmaceutical composition comprising rAAV, said composition comprising the aforementioned recombinant adeno-associated virus of the present invention and a pharmaceutically acceptable diluent, carrier or excipient.
- a carrier or excipient includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, Preservatives or chemical stabilizers, etc.
- Exemplary carriers or excipients include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water.
- one suitable excipient or carrier includes saline, which can be formulated with various buffer solutions such as phosphate buffer.
- saline which can be formulated with various buffer solutions such as phosphate buffer.
- Prevention of the action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
- isotonic agents such as sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents which delay absorption, for example, aluminum monostearate and gelatin.
- buffer solutions may be suitably prepared as necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- Sterile injectable solutions are prepared by mixing the required amount of active rAAV with various other ingredients listed in the present invention in an appropriate solvent, followed by filter sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the dispersion medium and the required other ingredients.
- the rAAV compositions of the invention can also be formulated in neutral or saline form.
- Pharmaceutically acceptable salts include acid addition salts and salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid). Salts formed from the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.
- the solutions will be administered in a manner compatible with the dosage form and in a therapeutically effective amount.
- the formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.
- compositions of the present invention can be delivered to suitable host cells via delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles and the like.
- unit dosage form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of a drug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. active compound.
- the specification for the unit dosage forms of the invention are dictated by the unique properties of the active compound and the particular therapeutic effect to be achieved as well as the limitations inherent in the art of compounding such active compounds for use in treating individuals.
- a unit dose can be a certain amount of vector genome, or contain a certain amount of vector genome per milliliter, or contain a unit dose of a pharmaceutical composition measured using the multiplicity of infection (MOI), which means that the vector or viral genome and nucleic acid can be delivered Ratio or multiple of cells that arrive.
- MOI multiplicity of infection
- the pharmaceutical composition may be contained in a container, pack or dispenser, such as a syringe, together with instructions for administration.
- rAAV recombinant adeno-associated virus
- the present invention relates to a method for preventing or treating a disease or symptom, wherein the method comprises delivering a therapeutically effective amount of the rAAV virus or the pharmaceutical composition of the present invention to a subject.
- the rAAV described herein can be delivered to a subject according to any suitable method known in the art.
- rAAV suspended in a physiologically compatible carrier is preferably administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse , cattle, goats, pigs, guinea pigs, hamsters, chickens, turkeys, or non-human primates (eg, rhesus monkeys).
- a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse , cattle, goats, pigs, guinea pigs, hamsters, chickens, turkeys, or non-human primates (eg, rhesus monkeys).
- host animals do not include humans.
- the subject is a human.
- a “therapeutically effective amount” refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic effect.
- a therapeutically effective amount of an rAAV virus or pharmaceutical composition can be determined based on factors such as the disease state, age, sex, and weight of the subject to be treated, and the ability of the rAAV virus or pharmaceutical composition to elicit a desired response in the subject. Variety. Dosage regimens may be adjusted to provide the optimum therapeutic response.
- a therapeutically effective amount is generally also an amount in which any toxic or detrimental effects of the rAAV virus or pharmaceutical composition are outweighed by the therapeutically beneficial effects.
- “Prophylactically effective amount” refers to an amount effective at the dose and time required to achieve the desired preventive effect, such as preventing or inhibiting various diseases.
- a prophylactic dose can be used in a subject before or at an early stage of the disease, and in some cases, the prophylactically effective dose can be greater or less than the therapeutically effective dose.
- the dosage to be administered depends largely on the condition and size of the subject being treated as well as the formulation of the treatment, frequency of treatment and route of administration.
- the regimen for continued treatment, including dosage, formulation, and frequency, may be guided by initial response and clinical judgment.
- the rAAV virus or pharmaceutical composition is administered to a subject once a day, once a week, once every two weeks, once a month, once every 2 months, once every 3 months, once every 6 months, Once a year or once every 2 years, once every 5 years, once in a lifetime.
- Exemplary routes of administration and delivery include intravenous (I.V.), intraarticular, intraperitoneal (I.P.), intraarterial, intramuscular, parenteral, subcutaneous, intrapleural, dermal, transdermal, parenteral, e.g., transdermal Mucosal, intracranial, intraspinal, oral (digestive), mucosal, respiratory, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoresis , In the eye, in the gland, in the organ, in the fallopian tube.
- I.V. intravenous
- I.P. intraperitoneal
- intraarterial intramuscular
- parenteral subcutaneous, intrapleural, dermal
- transdermal parenteral
- transdermal Mucosal intracranial, intraspinal
- mucosal respiratory, intranasal, intubation
- rAAV can be delivered to a mammalian subject by, for example, intraocular injection, subretinal injection, choroidal injection (e.g., suprachoroidal injection), or topical administration (e.g., eye drops) or by injection into the recipient eye.
- Internal tissues are used to affect the mammalian eye (eg intravitreal injection).
- "Ocular tissue” refers to any tissue derived from or contained in the eye. Non-limiting examples of ocular tissues include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous, macula, fovea, optic disc, lens, pupil, iris, aqueous fluid, cornea, conjunctival ciliary body, and optic nerve.
- rAAV or compositions described herein are administered by intraocular injection. In some embodiments, rAAV or compositions described herein are administered by intravitreal injection. In some embodiments, rAAV or compositions described herein are administered by subretinal injection. In some embodiments, rAAV or compositions described herein are administered by intrachoroidal (eg, suprachoroidal) injection. In some embodiments, rAAV or compositions described herein are administered by intravenous injection.
- delivery of rAAV to a mammalian subject can be by, for example, intramuscular injection into the mammalian subject.
- delivery of rAAV to a mammalian subject can be by, for example, intravenous injection into the mammalian subject.
- delivery of rAAV to a mammalian subject can be by, for example, intra-articular injection into the mammalian subject.
- Intra-articular injection is defined herein as injection or injection into a joint. Intra-articular injections are commonly used to administer drugs into joints affected by inflammation.
- the effect of administering a pharmaceutical composition comprising rAAV according to the present invention may be to prevent the progression of the disease, stop the progression of the disease, reverse the progression of the disease, and the like.
- the disease or disease is related to the target gene loaded by rAAV and the gene product expressed.
- the gene product is a VEGF antagonist
- the disease or condition to be prevented or treated may be a VEGF-related disease.
- VEGF-associated disease refers to a group of diseases associated with abnormal VEGF activity/signaling. Many studies have confirmed that if the excess of VEGF is abnormal, it can stimulate and induce pathological angiogenesis and cause eye diseases related to angiogenesis.
- Non-limiting exemplary angiogenesis-related eye diseases include angiogenesis-dependent cancers, angiogenesis-related eye diseases, solid tumors (e.g., lung cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, colon cancer, melanoma), hematogenous tumors (eg, leukemia, metastatic tumors), benign tumors (eg, hemangioma, acoustic neuroma, neurofibroma, tracheitis, and pyogenic granuloma), rheumatoid arthritis, psoriasis, erythema, Osier - Webber syndrome, myocardial angiogenesis, plaques, telangiectasia, hemophilic joints, or angiofibromas.
- solid tumors e.g., lung cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, colon cancer, melanoma
- hematogenous tumors eg, leukemia
- angiogenesis-related ocular diseases include, but are not limited to, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and retrolentic fibroplasia, epidemic keratoconjunctivitis, Vitamin A deficiency, excessive contact lens wear, atopic keratitis, bacterial keratitis, ulcers, ulcers, bacterial keratitis, ulcers, primary keratosis, rheumatoid arthritis, systemic lupus, multiple Arteritis, trauma, Wegeners sarcoidosis, scleritis, Steven Johnson disease, pemphigoid radial keratotomy, corneal graft rejection, sickle cell anemia, sarcoidosis, pseudoxanthoma elasticum, pemphigoid Gietz disease, venous occlusion, arterial occlusion, carotid
- the ocular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular eye disease, noninfectious uveitis, or glaucoma.
- the eye disease is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, retinal vein occlusion, Macular edema due to branch vein occlusion, diabetic retinal edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoid choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retina of prematurity lesion.
- macular degeneration can include acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration.
- the disease or condition to be prevented or treated may be an arthritic disease or a related condition.
- arthritic disease a condition in which inflammation of one or more joints is involved.
- Arthritis is understood herein to mean “joint pain” or “joint disease”.
- the arthritic disease is selected from adult-onset Still's disease, ankylosing spondylitis, arthritis, back pain, Behget's disease, blunt trauma, bursitis, calcium pyrophosphate deposition disease (CPPD), wrist Tube Syndrome, Chondromalacia Patellae, Chronic Fatigue Syndrome, Complex Regional Pain Syndrome, Cryobiline-Associated Periodic Syndrome (CAPS), Degenerative Disc Disease, Developmental Dysplasia of the Hip, Eller-Danlos, Familial Mediterranean fever, fibromyalgia, fifth disease, giant cell arteritis, gout, hemochromatosis, infectious arthritis, inflammatory arthritis, inflammatory bowel disease, joint replacement, juvenile arthritis, juvenile dermatomyositis ( JD), juvenile idiopathic arthritis (JIA), juvenile rheumatoid arthritis, juvenile scleroderma, Kawasaki disease, lupus, lupus in children and adolescents, Lyme disease, mixed connective tissue disease, myos
- the arthritic disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, plaque psoriasis , Psoriatic Arthritis, Ankylosing Spondylitis, Septic Arthritis, Arthritis, Juvenile Idiopathic Arthritis, Blunt Trauma, Joint Replacement, or Still's Disease.
- the arthritic disease is a joint disorder involving inflammation of one or more joints.
- the arthritic disease is selected from the group consisting of rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, Ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, and Still's disease.
- Site-directed mutagenesis and recombinant DNA techniques are used to generate variants of AAV8 that contain a polypeptide substitution of 5-14 amino acids at positions Q588-A592 (QQNTA) of the parental AAV8, or further include a polypeptide located at the parental AAV8 coat
- the mutation at the position corresponding to the N262-N272 amino acid of the shell protein, the amino acid sequence after the mutation is SQSGASNDNH (SEQ ID NO: 10); or further includes D80N, V125A, V125G mutations, as shown in Table 1.
- An exemplary No.12 capsid mutant plasmid is shown in Figure 2.
- capsid protein mutants inserts a polypeptide sequence after amino acid Q589 of the parent AAV8, as shown in Table 4.
- the viral packaging of the AAV8 mutant is generated by co-transfecting cells with three plasmids: the first plasmid (pAAV-CAG-EGFP) comprising the expression cassette (GFP or luciferase) flanked by the ITR of the gene of interest; a mutant plasmid encoding the Rep/Cap gene (second plasmid); and a third plasmid containing the adenovirus helper function gene.
- the cells were collected, lysed to release the virus, and the virus titer was determined by PCR, which was then used for later use.
- the purified AAV8 mutant virus was injected into the mouse vitreous at 7E+8vg/eye. 21 days after the vitreous injection, the eyeballs of the mice were taken, and the eyeballs were ground using a frozen homogenizer, RNA was extracted using a total RNA extraction kit, and cDNA was synthesized using a reverse transcription kit, and the specific EGFP primer probe group was determined by qPCR (wherein, The sequence of the upstream primer EGFP-F is: 5'-CACATGAAGCAGCACGACTT-3' (SEQ ID NO:26); the sequence of the downstream primer EGFP-R is: 5'-TCGTCCTTGAAGAAGATGGT-3' (SEQ ID NO:27); For: 5'-AGTCCGCCATGCCCGAAGGCT-3'-TAMRA, the sequence contained in the probe is shown in SEQ ID NO: 28), calculate the EGFP mRNA level of the normalized AAV8 mutant, that is, compare the effect of different AAV8 mutants on vitre
- the purified AAV8 mutant virus was injected into the vitreous of mice at 7E+8vg/eye; 21 days after the vitreous injection, the mouse eyeballs were taken, and the mouse eyeballs were fixed overnight with 4% paraformaldehyde.
- the mouse retina was stripped under a microscope, the mouse retina was spread, and the EGFP fluorescence signal was photographed confocally. The results are shown in Figure 4.
- the purified AAV8 mutant virus was injected into the rat vitreous at 1.92E+9vg/eye; 21 days after the vitreous injection, the rat eyeballs were taken; 4% paraformaldehyde was used to fix the rat eyeballs overnight; The rat retina was peeled off under a microscope; the rat retina was sliced, and the fluorescent signal of EGFP was captured by a fluorescent scanning microscope, and the results are shown in Figure 5.
- the purified AAV8 mutant virus was injected into the suprachoroidal space of New Zealand rabbits at 5E+10vg/eye; 14 days after the suprachoroidal injection, the eyeballs of New Zealand rabbits were taken; the eyeballs of New Zealand rabbits were fixed overnight with 4% paraformaldehyde; The eyeballs were dehydrated and tissue-embedded; frozen sections were then stained with nuclei (DAPI); the whole image was scanned using a confocal microscope to capture EGFP fluorescence signals and DAPI signals, and the results are shown in Figure 6 (in Figure 5, EGFP : autofluorescence, DAPI: nucleus, RGC: ganglion cell, INL: retinal inner nucleus; ONL: retinal outer nuclear layer; RPE: retinal epithelial cells), from the fluorescence map, the wild type has the effect on photoreceptor cell layer and RPE layer The transduction efficiency is not high, but the transduction efficiency of the mutant to photoreceptor cells and RPE layer is greatly improved
- the total amount of serum + medium is 1000 ⁇ L; after 16 hours of infection, the medium is changed every day, and the culture is continued until 72 hours, and the fluorescent signal of EGFP is confocally taken to observe the expression of fluorescent protein.
- Figure 7 shows the 48-hour infection of ARPE19 cells.
- AAV8-aflibercept virus and AAV No.12-aflibercept mutant virus were produced by co-transfection of the three plasmids described in Example 1, and the genome titer of each virus was measured by ddPCR; The genome titer of the virus in the body was diluted to 3.0E12vg/mL by buffer solution, and the number of cells was 3.0E5. Add 10 ⁇ L of virus to each well, change the medium after 16 hours of culture, and collect cells and cell supernatant after 72 hours; Freezing, repeated freezing and thawing 3 times, cell lysis, and ELISA method was used to measure the expression level of Trap protein. The results showed that the protein expression level of AAV8-aflibercept virus was 97ng/ml, and the protein expression level of AAV No.12-aflibercept virus was 1662ng/ml.
- AAV No.128-aflibercept virus was produced by co-transfection of the three plasmids described in Example 1, and the genome titer of each virus was measured by ddPCR; the genome titer of the virus was diluted to 1.0E13vg/ mL, injecting 100 ⁇ L into the suprachoroidal space at 4 mm above the monkey eye temporally, injecting 1 monkey and 2 eyes; 56 days later, the rhesus monkeys were executed, the eyeballs were taken, and various tissues of the monkey eyes (including aqueous humor, Choroid, conjunctiva, iris ciliary body, retina, sclera, vitreous body), after adding protease inhibitors, perform low-temperature tissue homogenate, take the supernatant, and measure the expression level of aflibercept protein (ng/ml) by ELISA, the results are as follows Table 6 shows.
- AAV8-luciferase and AAV No.128-luciferase viruses were produced by co-transfection of the three plasmids described in Example 1, and the genome titer of the virus was measured by ddPCR; the genome titer of the virus was diluted to 1.62E11vg/mL by buffer , injected into the gastrocnemius muscle of the right leg of the mouse, the volume is 50 ⁇ L, and 5 mice (Balb/c) were injected with each virus; 7 days, 14 days, 28 days, and 160 days after the virus injection, the mice were injected intraperitoneally A certain amount of fluorescein sodium salt substrate was used for in vivo imaging of mice, and fluorescence intensity analysis was performed on an in vivo imager. The results are shown in Table 7 and FIG. 10 .
- AAV8-No.128-etanercept and AAV No.128-adalimumab plasmids were respectively constructed; 2 ⁇ g of the plasmids were selected to transfect 293F cells with a single plasmid, and after 72 hours, the protein expression level of the plasmids was detected by ELISA method. The results showed that the expression level of AAV No.128-adalimumab plasmid was 1010 ng/mL, while the expression level of AAV No.128-etanercept plasmid was 2020 ng/mL.
- AAV No.128-etanercept virus was prepared according to the method described in Example 1 and the viral genome titer was determined, then diluted to 9.98E12vg/mL for later use.
- Positive control substance take adalimumab antigen solution 40mg/400 ⁇ l, dilute to 1.6 ⁇ g/ ⁇ l.
- Second modeling on the 21st day put 0.6ml bovine type II collagen (CII) (2mg/ml) and 0.6ml (4mg/ml) incomplete Freund's adjuvant into a film squeezer to prepare about 1ml milky white emulsion , each mouse was subcutaneously injected with 100 ⁇ l of the emulsion at the base of the tail.
- CII bovine type II collagen
- 4mg/ml incomplete Freund's adjuvant
- the adalimumab group received 160 ⁇ g of adalimumab subcutaneously on the back (40 mg/400 ⁇ l of the stock solution was diluted to 1.6 ⁇ g/ ⁇ l, and 100 ⁇ l was injected subcutaneously with an insulin needle on the back), once every two weeks.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Ophthalmology & Optometry (AREA)
- Rheumatology (AREA)
- Wood Science & Technology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Physical Education & Sports Medicine (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Pain & Pain Management (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Vascular Medicine (AREA)
- Gastroenterology & Hepatology (AREA)
Abstract
本发明涉及经过修饰的腺相关病毒衣壳蛋白及其用途，所述衣壳蛋白相对于亲本AAV衣壳蛋白包含约5-14个氨基酸的取代，并且包含所述经过修饰的衣壳蛋白的AAV与包含未修饰的亲本AAV衣壳蛋白的AAV相比，对靶组织或靶细胞(例如视网膜细胞)具有增加的感染性。
Description
本发明涉及生物技术领域，具体涉及经过修饰的AAV衣壳蛋白及其用途。
用于基因治疗的载体可分为病毒载体与非病毒载体。病毒载体中，最为常用的有腺病毒载体、慢病毒载体、腺相关病毒载体、单纯疱疹病毒载体等。
腺相关病毒(AAV)属于细小病毒科(Parvoviridae)和伴随病毒(Dependovirus)属。病毒体由25nm二十面体衣壳组成，所述25nm二十面体衣壳包含具有以下两个开放阅读框的4.7kb单链DNA基因组：Rep和Cap。非结构性Rep基因编码病毒复制所必需的四种调节蛋白，而Cap编码装配成60亚基衣壳的三种结构蛋白(VP1-3)。该病毒衣壳介导AAV载体克服病毒转导的许多生物屏障，包括细胞表面受体结合、内吞作用、细胞内运输和细胞核中的解包封的能力。
AAV广泛用于基因转导、基因治疗、疫苗接种以及溶瘤治疗等多个领域，具有致病性低、感染组织广、宿主细胞范围广(可在增殖和非增殖细胞中感染和表达)、免疫源性低，在体内表达外源基因时间长、无宿主细胞基因组整合等诸多优点，在实验和临床研究中被广泛应用。美国FDA在2017年批准了首个AAV介导的基因治疗，其用于治疗罕见的遗传性眼病，患者通过单次视网膜下施用rAAV载体递送的治疗药物后，四年多时间里持续观察到治疗药物的表达。
但是，目前关于AAV的应用目前仍存在一些挑战，例如如何提高载体的组织及细胞靶向性及转导效率，以及如何提高特定载体的包装效率，以适应工业化生产等。
发明内容
本发明涉及将包含目的基因的病毒载体(如AAV病毒载体)递送至期望的细胞或组织。具体地，本发明包含经过修饰的衣壳蛋白，所述经过修饰的衣壳蛋白相对于亲本AAV衣壳蛋白具有在氨基酸序列上的一个或多个修饰(如取代、插入或突变)，当存在于AAV病毒载体中时，与包含未修饰的亲本AAV衣壳蛋白的AAV病毒载体相比，对靶组织或靶细胞(例如视网膜、肌肉或关节腔)具有增加的感染性。
本发明一方面提供了一种经过修饰的腺相关病毒(AAV)衣壳蛋白，所述衣壳蛋白相对于亲本AAV衣壳蛋白包含约5-14个氨基酸的多肽取代，并且其中与包含相应亲本AAV衣壳蛋白的AAV病毒相比，包含所述经过修饰的衣壳蛋白的AAV病毒具有增强的视网膜细胞感染性。
在一些具体的实施方案中，所述多肽包括选自RGNRQ(SEQ ID NO:1)，QQNTARGNRQ(SEQ ID NO:2)、RGNRQAAQQNTA(SEQ ID NO:3)、RGNRQQNTA(SEQ ID NO:4)、RGNRQQQNTA(SEQ ID NO:5)、SGNTQ(SEQ ID NO:6)、RGNQQNTARQ(SEQ ID NO:7)、RGNQQPRPTSRQ(SEQ ID NO:8)、RGNRQAAQQPTPTS(SEQ ID NO:9)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列；并且所述取代位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处。
在一些具体的实施方案中，所述衣壳蛋白多肽包括选自QQNTARGNRQ(SEQ ID NO:2)、RGNRQQNTA(SEQ ID NO:4)、SGNTQ(SEQ ID NO:6)、RGNQQNTARQ(SEQ ID NO:7)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列。
在一些具体的实施方案中，所述衣壳蛋白进一步包括相对于亲本AAV8衣壳蛋白第262～272位氨基酸或另一血清型衣壳蛋白的对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。在一个优选的实施方案中，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处的多肽取代，所述多肽为 RGNRQ(SEQ ID NO:1)；和相对于亲本AAV8衣壳蛋白第262～272位或另一血清型衣壳蛋白的对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。
在一些具体的实施方案中，本发明所述AAV可以源自任何血清型AAV，例如AAV血清型选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11，AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R或AAVrh10。在一个优选的实施方案中，本发明所述AAV选自AAV8。
在一些具体的实施方案中，所述衣壳蛋白进一步包括相对于亲本AAV8衣壳蛋白D80和/或V125位置处氨基酸的突变。在一个优选的实施方案中，所述突变为D80N和/或V125A。在另一个优选的实施方案中，所述突变为D80Q和/或V125G。在一个优选的具体实施方案中，衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80N和V125A。在另一个优选的具体实施方案中，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80Q和V125G。
本发明的另一方面提供了另一种经过修饰的腺相关病毒(AAV)衣壳蛋白，所述衣壳蛋白包括位于亲本AAV8第589位氨基酸或另一血清型衣壳蛋白的对应位置后的多肽插入，并且其中与包含相应亲本AAV衣壳蛋白的AAV病毒相比，包含所述经过修饰的衣壳蛋白的AAV病毒具有增强的视网膜细胞感染性。
在一些具体的实施方案中，所述多肽包含选自RGDLTTPQQ(SEQ ID NO:20)、RGDLNTPQQ(SEQ ID NO:21)或RGDVSSPQQ(SEQ ID NO:22)的氨基酸序列。所述AAV可以源自任何血清型AAV，例如AAV血清型选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11，AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R或AAVrh10。在一个优选的实施方案中，所述AAV血清型为AAV8。
本发明另一方面提供了一种重组腺相关病毒(rAAV)，包括：
i.本发明提供的所述经过修饰的衣壳蛋白；
ii.包含编码基因产物的异源核酸。
在一些具体的实施方案中，所述基因产物为VEGF拮抗剂或TNF-α拮抗剂。
在一些具体的实施方案中，所述VEGF拮抗剂选自阿柏西普、康柏西普、雷珠单抗、布洛赛珠单抗，优选阿柏西普。
在一些具体的实施方案中，所述TNF-α拮抗剂选自依那西普、英夫利昔单抗、阿达木单抗、培塞利珠单抗或戈利木单抗，优选依那西普。
本发明另一方面提供了一种药物组合物，其包括：
a)本发明提供的所述重组腺相关病毒；
b)药学上可接受的赋形剂。
本发明另一方面提供了用作药物的根据本发明所述的重组腺相关病毒。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过玻璃体、视网膜、脉络膜注射给药、静脉、皮下、肌肉或关节腔注射给药。在一个优选的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过脉络膜上腔注射给药。在另一个优选的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过肌肉或关节腔注射给药。
本发明另一方面提供了用于治疗的根据本发明所述的重组腺相关病毒或药物组合物。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过玻璃体、视网膜或脉络膜注射给药。在一个优选的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过脉络膜上腔注射给药。
本发明另一方面提供了所述的重组腺相关病毒在制备用于预防或治疗眼部疾病的药物中的用途。
本发明另一方面提供了一种预防或治疗眼部疾病的方法，所述方法包括向有需要的个体施用有效量的根据本发明所述的重组腺相关病毒或药物组合物。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过玻璃体、视网膜或脉络膜注射给药。在一个优选的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过脉络膜上腔注射给药。
在一些具体的实施方案中，本发明所述眼部疾病选自视网膜新生血管、脉络膜新生血管、虹膜新生血管、角膜新生血管性眼部疾病、非感染性葡萄膜炎或青光眼。在一些具体的实施方案中，本发明所述眼部疾病选自龄相关性黄斑变性、黄斑水肿、糖尿病性黄斑水肿、继发于视网膜静脉阻塞的黄斑水肿、视网膜静脉阻塞、视网膜中央静脉阻塞、视网膜分支静脉阻塞、视网膜分支静脉阻塞所致黄斑水肿、糖尿病性视网膜水肿、糖尿病性视网膜病变、增生性糖尿病视网膜病变、糖尿病性视网膜缺血、息肉状脉络膜血管病变、继发于退行性近视的脉络膜新生血管或早产儿视网膜病变。
本发明另一方面提供了用于眼部疾病治疗方法的根据本发明所述的重组腺相关病毒或药物组合物。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过玻璃体、视网膜或脉络膜注射给药。在一个优选的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过脉络膜上腔注射给药。
在一些具体的实施方案中，本发明所述眼部疾病选自视网膜新生血管、脉络膜新生血管、虹膜新生血管、角膜新生血管性眼部疾病、非感染性葡萄膜炎或青光眼。在一些具体的实施方案中，本发明所述眼部疾病选自龄相关性黄斑变性、黄斑水肿、糖尿病性黄斑水肿、继发于视网膜静脉阻塞的黄斑水肿、视网膜静脉阻塞、视网膜中央静脉阻塞、视网膜分支静脉阻塞、视网膜分支静脉阻塞所致黄斑水肿、糖尿病性视网膜水肿、糖尿病性视网膜病变、增生性糖尿病视网膜病变、糖尿病性视网膜缺血、息肉状脉络膜血管病变、继发于退行性近视的脉络膜新生血管或早产儿视网膜病变。
本发明另一方面提供了所述重组腺相关病毒在制备用于预防或治疗关节炎疾病或相关病症的药物中的用途。
本发明另一方面提供了一种预防或治疗关节炎疾病或相关病症的方法，所述方法包括向有需要的个体施用有效量的根据本发明所述的重组腺相关病毒或药物组合物。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过静脉、皮下、肌肉或关节腔注射给药。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过肌肉或关节腔注射给药。
在一些具体的实施方案中，本发明所述关节炎疾病或相关病症选自类风湿性关节炎，青少年类风湿性关节炎，骨关节炎，痛风，假性痛风，脊椎炎，克罗恩病、斑块型银屑病，银屑病关节炎，强直性脊柱炎，脓毒性关节炎，关节炎，青少年特发性关节炎，钝性创伤，关节置换或Still’s疾病。
本发明另一方面提供了用于关节炎疾病或相关病症治疗方法的根据本发明所述的重组腺相关病毒或药物组合物。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过静脉、皮下、肌肉或关节腔注射给药。
在一些具体的实施方案中，本发明所述的重组腺相关病毒或药物组合物通过肌肉或关节腔注射给药。
在一些具体的实施方案中，本发明所述关节炎疾病或相关病症选自类风湿性关节炎，青少年类风湿性关节炎，骨关节炎，痛风，假性痛风，脊椎炎，克罗恩病、斑块型银屑病，银屑病关节炎，强直性脊柱炎，脓毒性关节炎，关节炎，青少年特发性关节炎，钝性创伤，关节置换或Still’s疾病。
图1A显示了AAV衣壳蛋白GH环部分区域的氨基酸序列比对；
图1B显示了AAV衣壳蛋白GH环部分区域的氨基酸序列比对；
图2为AAV8衣壳蛋白突变体质粒的构建示意图；
图3显示了小鼠玻璃体内注射AAV8突变体病毒的EGFP mRNA表达水平；
图4显示了小鼠玻璃体内注射AAV8突变体病毒的视网膜铺片EGFP荧光信号；
图5显示了大鼠玻璃体内注射AAV8突变体病毒的视网膜铺片EGFP荧光信号；
图6显示了兔脉络膜上腔注射AAV8突变体病毒的EGFP荧光信号及DAPI信号；
图7为ARPE19细胞培养AAV8突变体病毒的EGFP荧光信号；
图8A显示了流式细胞术对AAV8突变体病毒感染ARPE19细胞后EGFP阳性细胞计数；
图8B显示了流式细胞术对AAV8突变体病毒感染ARPE19细胞后EGFP阳性细胞计数；
图9显示了兔脉络膜上腔注射AAV8突变体病毒mCherry基因组DNA相对含量；
图10显示了小鼠肌肉注射AAV8突变体病毒的luciferase荧光强度；
图11显示了小鼠关节腔/肌肉注射AAV8突变体病毒的临床评分；
图12显示了小鼠关节腔/肌肉注射AAV8突变体病毒的脚掌厚度。
本发明涉及与包含未修饰的亲本AAV衣壳蛋白的AAV病毒载体相比，对靶组织或靶细胞(例如视网膜、肌肉或关节腔)具有增加的感染性的衣壳蛋白、包含该衣壳蛋白并表达目的基因(例如VEGF拮抗剂、TNF-α拮抗剂)的rAAV及其药物组合物和应用。
衣壳蛋白
本发明提供了一种经过修饰的AAV衣壳蛋白，其中经过修饰的AAV衣壳蛋白与对应的野生型AAV或亲本AAV相比包括一个或多个氨基酸修饰。所述修饰包括相对于亲本衣壳蛋白或AAV衣壳蛋白的一个或多个氨基酸取代、缺失或插入。所述修饰与包含未修饰的亲本AAV衣壳蛋白的AAV病毒载体相比，对靶组织或靶细胞(例如视网膜、肌肉或关节腔)具有增加的感染性。
在特定实施方案中，经过修饰的AAV衣壳蛋白包括在属于以下的衣壳蛋白血清型例如VP1内的异源多肽取代：AAV 1型、AAV 2型、AAV 3型、AAV 4型、AAV 5型、AAV 6型、AAV 7型、AAV 8型、AAV 9型、AAV 10型、AAVrh10、禽类AAV、牛类AAV、犬类AAV、马类AAV、灵长类AAV、非灵长类AAV、牛类AAV、AAV2.7m8、AAVShH10、AAV2.5T、AAV2.5T/7m8、AAV9/7m8、和AAV5/7m8。在一些实施方案中，衣壳变体可以是嵌合衣壳变体。嵌合衣壳变体的序列可包含两个或多个AAV衣壳血清型或其变体的部分。在一些实施例中，嵌合衣壳包含2、3、4、5、6、7、8、9、10或更多不同的衣壳蛋白血清型的部分。
在特定实施方案中，经过修饰的AAV衣壳蛋白包括约5-14个氨基酸的异源多肽取代。在一些特定实施方案中，本发明经过修饰的衣壳蛋白包括在位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处的约5-14个氨基酸的异源多肽取代。AAV1～AAV9等的氨基酸序列如专利US8962330B、US10041090B等中所描述。在一个具体的实施方案中，本发明经过修饰的衣壳蛋白包括在位于亲本AAV8的第588～592位氨基酸处的约5-14个氨基酸的异源多肽取代。所述亲本/野生型AAV8衣壳蛋白的第588～592位氨基酸为“QQNTA”。因此本发明的一个具体实施方案为经过修饰的衣壳蛋白在亲本/野生型AAV8衣壳蛋白的第588～592位氨基酸“QQNTA”取代为5-14个氨基酸的异源多肽。与AAV8第588～592位氨基酸相对应的取代位点在其他衣壳蛋白的对应位置处，例如如图1A中所示。例如在AAV1的586～590氨基酸处，在AAV2的585～589氨基酸处，在AAV3的586～590氨基酸处，在AAV4的584～588氨基酸处，在AAV5的575～579氨基酸处，在AAV6的586～590氨基酸处，在AAV7的587～591氨基酸处，在AAV9的586～590氨基酸处，在AAV10的588～592氨基酸处。
对于上述的取代异源多肽，长度为5个氨基酸、6个氨基酸、7个氨基酸、8个氨基酸、9个氨基酸、10个氨基酸或11个氨基酸。在一些具体的实施方案中，本发明所述多肽包括选自 RGNRQ(SEQ ID NO:1)，QQNTARGNRQ(SEQ ID NO:2)、RGNRQAAQQNTA(SEQ ID NO:3)、RGNRQQNTA(SEQ ID NO:4)、RGNRQQQNTA(SEQ ID NO:5)、SGNTQ(SEQ ID NO:6)、RGNQQNTARQ(SEQ ID NO:7)、RGNQQPRPTSRQ(SEQ ID NO:8)、RGNRQAAQQPTPTS(SEQ ID NO:9)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列。在一些优选的实施方案中，本发明所述多肽包括选自QQNTARGNRQ(SEQ ID NO:2)、RGNRQQNTA(SEQ ID NO:4)、SGNTQ(SEQ ID NO:6)或RGNQQNTARQ(SEQ ID NO:7)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列。
在特定实施方案中，经过修饰的AAV衣壳蛋白进一步包括在相对于亲本AAV8衣壳蛋白第262～272位氨基酸或另一血清型衣壳蛋白的对应位置处的突变。所述亲本/野生型AAV8衣壳蛋白的第262～272位氨基酸为“SNGTSGGATND”。因此本发明的一个具体实施方案为，经过修饰的衣壳蛋白在亲本/野生型AAV8衣壳蛋白的第262～272位氨基酸“SNGTSGGATND”进行突变。与AAV8第262～272位氨基酸相对应的突变位点在其他衣壳蛋白的对应位置处，例如在AAV1的261～271氨基酸处，在AAV2的261～271氨基酸处，在AAV3的261～271氨基酸处，在AAV4的255～265氨基酸处，在AAV5的251～261氨基酸处，在AAV6的261～271氨基酸处，在AAV7的262～272氨基酸处，在AAV9的261～271氨基酸处，在AAV10的262～272氨基酸处。与AAV8第262～272位氨基酸相对应的突变位点在其他衣壳蛋白的对应位置处，例如如图1B中所示。在一些具体的实施方案中，本发明突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。
在一些具体的实施方案中，本发明所述经过修饰的衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处的多肽取代，所述多肽为RGNRQ(SEQ ID NO:1)；和相对于亲本AAV8衣壳蛋白第262～272位或另一血清型衣壳蛋白的对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。在一些优选的实施方案中，本发明所述的亲本衣壳蛋白血清型为AAV8。
在另一些实施方案中，本发明经过修饰的AAV衣壳蛋白进一步包括在相对于亲本AAV8衣壳蛋白第D80和/或V125位置处氨基酸的突变。在一些具体的实施方案中，所述突变为D80N和/或V125A。在一些具体的实施方案中，所述突变为D80Q和/或V125G。在一些优选的实施方案中，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80N和V125A。在另一些优选的实施方案中，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80Q和V125G。
在另一些实施方案中，本发明经过修饰的AAV衣壳蛋白包括在相对于亲本AAV8第589位氨基酸或另一血清型衣壳蛋白的对应位置后的多肽插入。插入位点位于AAV8第589位氨基酸或其他衣壳蛋白的对应位置之后，例如在AAV1的589氨基酸处，在AAV2的586氨基酸处，在AAV3的587氨基酸处，在AAV4的585氨基酸处，在AAV5的576氨基酸处，在AAV6的587氨基酸处，在AAV7的588氨基酸处，在AAV9的587氨基酸处，在AAV10的589氨基酸处之后。在一些具体的实施方案中，所述插入的多肽包含RGDLTTPQQ(SEQ ID NO:20)、RGDLNTPQQ(SEQ ID NO:21)或RGDVSSPQQ(SEQ ID NO:22)的氨基酸序列。在一个优选的实施方案中，本发明所述经过修饰的AAV衣壳蛋白包括在相对于亲本AAV8第589位氨基酸后的多肽插入，所述插入多肽包含RGDLTTPQQ(SEQ ID NO:20)、RGDLNTPQQ(SEQ ID NO:21)或RGDVSSPQQ(SEQ ID NO:22)的氨基酸序列。
在一些实施方案中，本发明所述经过修饰的衣壳蛋白还包括与上述经过氨基酸的取代、突变和/或插入的衣壳蛋白的氨基酸序列具有至少85％、90％、95％、98％、99％或100％同源性的序列。
在一些实施方案中，与包含相应于亲本/野生型衣壳蛋白的AAV病毒相比，包含经过修饰的衣壳蛋白的AAV对视网膜细胞的感染性增强至少1倍、2倍、5倍、10倍、20倍、50倍、100倍甚至更高。所述视网膜细胞包括RPE-19细胞(人视网膜色素上皮细胞系)、视网膜神经节 细胞、无长突细胞、水平细胞、双极细胞、感光细胞、视锥细胞、视杆细胞、Müller神经胶质细胞和视网膜色素上皮细胞等。
在一些具体的实施方案中，与包含相应于亲本/野生型衣壳蛋白的AAV病毒相比，包含经过修饰的衣壳蛋白的AAV对RPE细胞的感染性增强至少1倍、2倍、5倍、10倍、20倍、50倍、100倍甚至更高。
在一些具体的实施方案中，与包含相应于亲本/野生型衣壳蛋白的AAV病毒相比，包含经过修饰的衣壳蛋白的AAV经过脉络膜上腔注射后对视网膜细胞的感染性增强至少1倍、2倍、5倍、10倍、20倍、50倍、100倍甚至更高。
在一些具体的实施方案中，与包含相应于亲本/野生型衣壳蛋白的AAV病毒相比，包含经过修饰的衣壳蛋白的AAV经过玻璃体注射后对视网膜细胞的感染性增强至少1倍、2倍、5倍、10倍、20倍、50倍、100倍甚至更高。
重组腺相关病毒(rAAVs)
本发明的基因递送载体为重组腺相关病毒(rAAVs)。在一些实施方案中，本发明所述的rAAV是单链AAV(ssAAV)。其中ssAAV是指在分开的链上具有目的基因的编码序列和互补序列的rAAV，并且被包装在分开的病毒衣壳中。
在一些实施方案中，本发明所述重组腺相关病毒(rAAV)包括：i)根据本发明所述的经过修饰的衣壳蛋白；ii)包含编码基因产物的异源核酸。
在某些实施例中，本发明所述的rAAV包括本发明前述的经过修饰的衣壳蛋白，使得本发明所述的重组AAV(rAAV)相对于包含亲本或野生型的衣壳蛋白的rAAV具有增强的细胞或组织(例如眼组织或细胞、肌肉组织或关节腔等)特异性靶向能力或感染性。
在某些实施例中，本发明所述的rAAV包含编码基因产物的异源核酸以及调节元件的多核苷酸表达盒。术语“调节元件”是指调节与之可操作地相连的基因产物的表达的一段核酸序列。因此，表达调节元件可包括启动子、增强子、内部核糖体进入位点(IRES)、转录终止子、蛋白编码基因前的起始密码子、内含子剪接信号和终止密码子。该术语还可包括将框内外不想要的可能的起始密码子从所述序列中除去的核酸序列设计。其还可包括将不想要的可能的剪接位点除去的核酸序列设计。其还可包括指引添加多聚腺苷酸化或polyA的序列等。
本发明所述多核苷酸表达盒中，异源核酸包括对目的基因的基因产物例如治疗性基因产物进行编码的核苷酸序列。在一些实施例中，基因产物是干扰RNA。在一些实施例中，基因产物是适配体。在一些实施例中，基因产物是多肽。在一些实施例中，基因产物是提供用于对基因功能进行位点特异性敲低的位点特异性核酸酶。在一些优选的实施例中，基因产物是多肽、蛋白、融合蛋白、抗体等。
在一些实施方案中，本发明所述基因产物选自VEGF拮抗剂、TNF-α拮抗剂、PD-1/PD-L1抑制剂、Ang-2抑制剂、血浆激肽释放酶抑制剂、内皮抑素、肿瘤抑素、血管抑素、色素上皮衍生因子(PEDF)、可溶性Tie-2受体、Ang-2拮抗剂、CD组织金属蛋白酶抑制剂-3(TIMP-3)、光响应性视蛋白(例如视紫红质)、抗凋亡多肽(例如Bcl-2、Bcl-Xl)、神经胶质源性神经营养因子(GDNF)、成纤维细胞生长因子2、神经秩蛋白(neurturin,NTN)、睫状神经营养因子(CNTF)、神经生长因子(NGF)、神经营养蛋白-4(NT4)、脑源性神经营养因子(BDNF)以及其功能性变体和片段，包含与这些蛋白或多肽中的任何蛋白或多肽具有至少80％、至少85％、至少90％或至少95％的序列同一性的变体、以及包括这些蛋白、多肽或其变体中至少20％、至少30％、至少50％、至少60％、至少70％、至少80％或至少90％的片段。
在一个实施方案中，本发明所述基因产物选自VEGF拮抗剂。在一个具体的实施方案中，所述VEGF拮抗剂选自VEGF受体1(VEGFR-1，或Flt)或受体2(VEGFR-2，或Flk)的细胞外结构域1-7中的任一个结构域，例如VEGFR-1细胞外结构域2(Flt d2)和/或VEGFR-2细胞外结构域3或4。
在一个具体的实施方案中，所述VEGF拮抗剂包括选自下列组中的至少1项：
a)包含VEGFR-1的细胞外结构域2和VEGFR-2的细胞外结构域3的融合蛋白；示例性的，所述融合蛋白具有SEQ ID NO:11(Fltd2Flkd3)所述的氨基酸序列；
b)包含VEGFR-1的细胞外结构域2、VEGFR-2的细胞外结构域3和结构域4的融合蛋白；示例性的，所述融合蛋白具有SEQ ID NO:12(Fltd2 Flkd3，4)所述的氨基酸序列；
c)具有SEQ ID NO:13所述的抗体重链可变区序列和SEQ ID NO:14所述的抗体轻链可变区序列；
d)具有SEQ ID NO:15所述的抗体重链可变区序列和SEQ ID NO:16所述的抗体轻链可变区序列。
其中，SEQ ID NO:12为如下所示的序列：
SEQ ID NO:13为如下所示的序列：
SEQ ID NO:14为如下所示的序列：
SEQ ID NO:15为如下所示的序列：
SEQ ID NO:16为如下所示的序列：
在一个具体的实施方案中，所述VEGF拮抗剂具有如SEQ ID NO:11所述的氨基酸序列：
在一个具体的实施方案中，所述编码VEGF拮抗剂的核苷酸具有如SEQ ID NO:23所述的序列：
在一些实施方案中，所述蛋白或多肽选自TNF-α拮抗剂。在一些具体实施方案中，所述VEGF拮抗剂包括选自依那西普、英夫利昔单抗、阿达木单抗、培塞利珠单抗或戈利木单抗。
在一个具体的实施方案中，所述TNF-α拮抗剂具有SEQ ID NO:17所述的重链可变区序列和SEQ ID NO:18所述的轻链可变区序列。
SEQ ID NO:17为如下所示的序列：
SEQ ID NO:18为如下所示的序列：
在一个具体的实施方案中，所述TNF-α拮抗剂具有SEQ ID NO:24所述的氨基酸序列：
在一个具体的实施方案中，所述编码TNF-α拮抗剂的核苷酸具有如SEQ ID NO:25所述的序列：
在一些实施方案中，所述VEGF拮抗剂或TNF-α拮抗剂具有与上述氨基酸序列具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％同一性或100％同一性的氨基酸序列。
在一些实施方案中，所述VEGF拮抗剂或TNF-α拮抗剂具有与上述核苷酸序列具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％同一性或100％同一性的核苷酸序列。
在一些实施方案中，所述蛋白或多肽选自具有一个或多个不同的抗原结合位点。例如在一些实施方案中，所述蛋白或多肽同时结合VEGF和Ang-2。在一个具体的实施方案中，所述蛋白或多肽为同时结合VEGF和Ang-2的双特异性抗体，例如Faricimab双抗。
在本发明的一些实施例中，可以使用密码子优化将编码序列设计用于更好地表达目的基因。本发明可以对编码多肽、融合蛋白、抗体或其功能性片段的核苷酸密码子进行优化，以提高目的基因在靶细胞、组织或物种中的蛋白表达水平。编码序列是对用于转录和翻译的氨基酸进行编码的mRNA序列的一部分。在转录和翻译过程中，61个核苷酸密码子中的每三个核苷酸密码子被转录和翻译成20个氨基酸中的任一个氨基酸。然而，不同的细胞类型和不同的动物物种对相同氨基酸进行编码的tRNA频率不同。当基因序列含有不频繁表达的密码子时，核糖体转录机制可能会减慢，从而阻碍有效转录。因此本领域技术人员可以通过“密码子优化”来改善目的基因的表达水平。简言之，编码产物的核苷酸序列用同义密码子序列修饰，例如采用在靶细胞中能够更高频率表达的密码子来替换低频率表达的密码子，以此来提高目标物质在特定细胞、组织或物质中的表达水平，例如提高10％、20％、30％，40％，50％，60％，70％，80％，90％、100％或超过100％。密码子优化的编码序列可以通过各种不同的方法来设计。这种优化可以使用在线提供的方法、公开的方法或提供密码子优化服务的公司来进行。例如在国际专利公开号WO2015/012924中描述了一种密码子优化方法，其通过引用并入本文。产物的开放阅读框(ORF)的整个长度适当地被修改。然而，在一些实施方案中，只有ORF的一个片段可以被改变。通过使用这些方法之一，产生编码多肽的密码子优化的编码核苷酸。
在一些实施方案中，本发明所述多核苷酸表达盒中一种或多种表达调控元件。例如，调控元件包含组成型启动子区，编码基因产物的核苷酸序列与组成型启动子可操作地连接，所述启动子是广泛存在与细胞、组织或物种中具有活性的启动子，所述启动子促进体内或体外特定细胞或组织中目的基因的表达。在一些实施方式中，启动子的实例包含鸡β-肌动蛋白启动子(CBA)、巨细胞病毒启动子(CMV)、CMV早期增强子/鸡β-肌动蛋白(CBA)启动子/兔β-珠蛋白内含子(CAG)、延伸因子1α启动子(EF1α)、人类磷酸甘油酸激酶启动子(PGK)、 MNT启动子、UB6启动子、CAG启动子、RPE65启动子、视蛋白启动子、线粒体重链启动子、泛素启动子等。
在一些实施方案中，调控元件包含诱导性启动子区，编码基因产物的核苷酸序列与诱导性启动子可操作地连接。在一些实施方案中，所述诱导性启动子为光感受器特异性启动子。合适的光感受器特异性启动子包含例如视紫红质启动子、视紫红质激酶启动子、β磷酸二酯酶基因启动子、视网膜色素变性基因启动子、光感受器间维甲酸结合蛋白(IRBP)基因增强子、IRBP基因启动子、视蛋白基因启动子、视网膜劈裂蛋白基因启动子、CRX同源域蛋白基因启动子、鸟嘌呤核苷酸结合蛋白α转导活性多肽1(GNAT1)基因启动子、神经视网膜特异性亮氨酸拉链蛋白(NRL)基因启动子、人类视锥抑制蛋白(hCAR)启动子、以及PR2.1、PR1.7、PR1.5和PR1.1启动子、RPE特异性启动子(如RPE65基因启动子)、细胞视黄醛结合蛋白(CRALBP)基因启动子、色素上皮源性因子(PEDF aka serpin F1)基因启动子、卵黄样黄斑营养不良(VMD2)启动子、穆勒神经胶质细胞特异性启动子(如神经胶质原纤维酸性蛋白(GFAP)启动子)、双极特异性启动子(如GRM6启动子)等。
本发明所述多核苷酸表达盒中的表达调控序列还可以包含多腺苷酸化信号。所述多腺苷酸化信号也被称为多腺苷酸化位点、多腺苷酸尾、Poly(A)位点、Poly(A)信号或Poly(A)尾。多腺苷酸化区是指多聚腺苷酸与信使RNA(mRNA)分子的共价链结。在蛋白质生物合成的过程中，这是产生准备作翻译的成熟mRNA的方式的一部分。在真核生物中，多聚腺苷酸化是一种机制，令mRNA分子于它们的3'端中断。多腺苷酸化信号保护mRNA，免受核酸外切酶攻击，并且对转录终结、将mRNA从细胞核输出及进行翻译都十分重要。多腺苷酸化信号包含多个连续的腺苷一磷酸，通常含有AAUAAA重复序列。一些示例性的多腺苷酸化信号包含猴空泡病毒40(SV40)、人生长激素(HGH)、牛生长激素(BGH)或β-珠蛋白。
本发明所述的多核苷酸表达盒还可以包含反向末端重复序列(ITR)。功能性的腺病毒反向末端重复序列(ITR)是指如用于整合、复制和包装AAV病毒粒子的ITR序列。通常，ITR序列的长度约为145bp。优选地，分子中基本上使用了编码ITR的整个序列，以及本领域技术人员根据本领域的常规技术手段对这些ITR序列的修饰。在本申请中使用ITR分子的一个实例是包含目的基因的“顺式”质粒，其中目的基因序列和相关的调控元件侧接5'和3'AAV ITR序列。AAV ITR序列可以从任何已知的AAV获得，包括目前鉴定的哺乳动物AAV类型。在一些实施方案中，编码目的基因的异源核苷酸在AAV ITR的侧面(例如，在方向5’-ITR-目的基因-ITR-3’)。在一些实施例中，AAV ITR选自由AAV1 ITR，AAV2 ITR，AAV3 ITR，AAV4 ITV，AAV5 ITR、AAV6 ITR，AAV7 ITR，AAV8 ITR，AAV9 ITV，AAV10 ITR，AAV11 ITR和AAV12 ITR组成的组。
本发明所述的多核苷酸表达盒进一步包括位于编码序列下游和聚腺苷酸化位点上游的RNA输出信号。RNA输出信号是顺式作用的转录后调控元件，可增强RNA从细胞核中的输出。示例性的RNA输出序列包括但不限于来自乙型肝炎病毒转录后调控元件(HPRE)和土拨鼠肝炎病毒转录后元件(WPRE)序列。
本领域技术人员可以理解的是，本发明所述的多核苷酸表达盒可以任选包含其他元件，包括但不限于促进克隆的限制性位点，用于可操作性地连接各元件，或调节基因表达的调节元件。实例如质粒载体的细菌序列、噬菌体整合酶载体的attp位点、attB位点、启动子接头、多腺苷酸化序列接头等。
在某些实施方案中，本发明所述的多核苷酸表达盒包含本领域技术人员根据对上述所提到的元件中的两个或多个元件以及目的基因的组合产生，其中多核苷酸表达盒中目的基因编码序列和相关的调控元件的侧面是5'和3'AAV ITR序列。
例如，本发明的多核苷酸表达盒，按照5'到3'的顺序可以包括N端AAV ITR、增强子/启动子/内含子、Kozak序列，基因产物的编码序列，WPRE或HPRE RNA输出信号序列，多腺苷酸化信号序列、以及C端AAV ITR。
在一个具体的实施方案中，本发明所述的重组腺相关病毒包含编码基因产物的多核苷酸表达盒，所述多核苷酸表达盒从5'到3'端顺序包括：
(a)5'AAV ITR；
(b)CMV增强子；
(c)CBA启动子；
(d)鸡β-肌动蛋白内含子；
(e)基因产物编码序列；
(f)兔β-珠蛋白多聚腺苷酸话信号序列；
(g)3'AAV ITR。
所述基因产物编码序列为编码前述基因产物(例如前述的VEGF拮抗剂或TNF-α拮抗剂)的核酸序列。
可以使用标准方法生产包含本发明所述的经过修饰的衣壳蛋白的重组病毒载体。例如，所述方法包括培养含有人工基因组的宿主细胞，所述人工基因组包含：由AAV ITR侧接的顺式多核苷酸表达盒，其中所述顺式多核苷酸表达盒包含基因产物的编码序列，所述编码序列可操作地连接至将控制基因产物在人类细胞中的表达的表达控制元件；缺乏AAV ITR的反式表达盒，其中所述反式表达盒编码AAV rep和衣壳蛋白，所述AAV rep和衣壳蛋白可操作地连接至驱动所述AAV rep和衣壳蛋白在培养物中的所述宿主细胞中的表达并且反式供应rep和cap蛋白的表达控制元件；足以准许通过AAV衣壳蛋白复制和包装所述人工基因组的腺病毒辅助功能；以及从所述细胞培养物回收包裹所述人工基因组的重组AAV。
“宿主细胞”是指包含或能够包含目标物质的任何细胞。通常，宿主细胞是哺乳动物细胞。在一些实施方案中，宿主细胞是感光细胞，视网膜色素上皮细胞，角质形成细胞，角膜细胞和/或肿瘤细胞。
药物组合物
本发明的另一方面在于提供包含rAAV的药物组合物，所述组合物包含本发明前述的重组腺相关病毒和药学上可接受的稀释剂、载体或赋形剂。
本领域技术人员可以容易地选择合适的稀释剂、载体或赋形剂。如本文所用，载体或赋形剂包括任何的溶剂、分散介质、载体、涂料、稀释剂、抗菌剂和抗真菌剂、等渗剂、吸收延迟剂、缓冲剂、载体溶液、悬浮液、胶体、防腐剂或化学稳定剂等。
示例性的载体或赋形剂包括无菌生理盐水、乳糖、蔗糖、磷酸钙、明胶、葡聚糖、琼脂、果胶、花生油、芝麻油和水。例如，一种合适的赋形剂或载体包括盐水，其可与多种缓冲溶液(例如磷酸盐缓冲液)调配。可通过各种抗菌和抗真菌剂来防止微生物的作用，例如，对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等。在许多情况下，最好包括等渗剂，例如糖或氯化钠。通过在组合物中使用延迟吸收的试剂，例如单硬脂酸铝和明胶，可以使可注射组合物的吸收延长。
为了给予可注射水溶液，可以根据需要适当地配制缓冲溶液，并且首先用足够的盐水或葡萄糖使液体稀释剂等渗。这些特定的水溶液特别适合于静脉内，肌内，皮下和腹膜内给药。
无菌注射溶液的制备方法是将所需量的活性rAAV与本发明所列举的各种其他成分混合在适当的溶剂中，然后过滤灭菌。通常，通过将各种已灭菌活性成分并入无菌载体中制备分散体，该载体包含分散介质和所需的其他成分。
本发明的rAAV组合物也可以以中性或盐形式配制。药学上可接受的盐，包括酸加成盐及由无机酸(例如盐酸或磷酸)或有机酸(如乙酸、草酸、酒石酸、扁桃酸)形成。由自由羧基形成的盐也可来自无机碱，例如钠、钾、铵、钙或氢氧化铁，或者有机碱，例如异丙胺、三甲胺、组氨酸、普鲁卡因等。配制完成后，溶液将以与剂型相容的方式给药，并且以治疗有效量施用。所述制剂易于以各种剂型施用，例如可注射溶液、药物释放胶囊等。
可以通过例如脂质体，纳米胶囊，微粒，微球，脂质颗粒，囊泡等递送载体将本发明的组合物递送至合适的宿主细胞中。
以单位剂型配制注射、口服或肠胃外组合物以易于施用是有利的。如本文所使用的，单位剂型是指适于作为单一剂量用于待治疗的受试者的物理离散单位；每个单位含有经计算与所需药物载剂关联产生期望的治疗效果的预定量的活性化合物。本发明的单位剂型的规格由 活性化合物的独特特性和待实现的特定治疗效果以及本领域中在混配这种活性化合物以用于治疗个体时固有的局限性决定。例如单位剂量的可以是一定量的载体基因组，或者每毫升中含有一定量的载体基因组，或者含有使用感染复数(MOI)测量药物组合物的单位剂量，MOI意指载体或病毒基因组与核酸可以递送到的细胞的比率或倍数。
药物组合物可以与施用说明书一起包含在容器、包装或分配器中，分配器例如注射器。
重组腺相关病毒(rAAV)的递送
本发明涉及一种用于预防或治疗疾病或症状的方法，其中所述方法包括将治疗有效量的本发明所述rAAV病毒或药物组合物递送至受试者。本发明所述的rAAV可以根据本领域已知的任何合适方法递送至受试者。例如，优选将悬浮于生理相容性载体中(例如，在组合物中)的rAAV施用于受试者，即宿主动物，例如人、小鼠、大鼠、猫、狗、绵羊、兔子、马、牛、山羊、猪、豚鼠、仓鼠、鸡、火鸡或非人类灵长类动物(例如，猕猴)。在一些实施例中，宿主动物不包括人类。在一些实施例中，受试者是人类。
“治疗有效量”是指以达到所需治疗效果所需的剂量和时间有效的量。治疗有效量的rAAV病毒或药物组合物可根据诸如待治疗受试者的疾病状态，年龄，性别和体重以及rAAV病毒或药物组合物在受试者中引发所需反应的能力之类的因素而变化。可以调节给药方案以提供最佳的治疗反应。治疗有效量通常也是其中rAAV病毒或药物组合物的任何毒性或有害作用被治疗有益作用所超过的量。“预防有效量”是指以达到所需预防效果所需的剂量和时间有效的量，例如预防或抑制各种病症。预防剂量可用于疾病发生前或早期的受试者，在某些情况下，预防有效量可大于或小于治疗有效量。给药剂量在很大程度上取决于被治疗对象的状况和大小以及治疗配方，治疗频率和给药途径。用于持续治疗的方案，包括剂量，制剂和频率，可以由初始反应和临床判断指导。
在一些实施方案中，rAAV病毒或药物组合物给予受试者每天一次、每周一次、每两周一次、每月一次、每2个月一次、每3个月一次、每6个月一次、每年一次或每2年一次、每5年一次、一生一次。
示例性的给药和递送途径包括静脉内(I.V.)，关节内，腹膜内(I.P.)，动脉内，肌肉内，胃肠外，皮下，胸膜内，真皮，透皮，胃肠外，例如透粘膜，颅内，脊柱内，口服(消化性)，粘膜，呼吸，鼻内，插管，肺内，肺内滴注，颊，舌下，血管内，鞘内，腔内，离子电渗疗法，眼内，腺内，器官内，输卵管内。
在一些实施方案中，rAAV递送至哺乳动物受试者可通过例如眼内注射、视网膜下注射、脉络膜注射(例如脉络膜上腔注射)或局部施用(例如滴眼液)或藉由注射至受眼部组织以此来影响哺乳动物的眼睛(例如玻璃体内注射)。“眼组织”是指源自或包含在眼睛中的任何组织。眼组织的非限制性实例包括神经元、视网膜(例如，感光细胞)、巩膜、脉络膜、视网膜、玻璃体、黄斑、中央凹、视盘、晶状体、瞳孔、虹膜、水液、角膜、结膜睫状体和视神经。视网膜位于眼睛的后部，由感光细胞组成。这些光感受器细胞(如视杆、视锥)通过辨别颜色和视野中的对比度赋予视力。在一些实施例中，通过眼内注射施用本发明中所述的rAAV或组合物。在一些实施例中，通过玻璃体内注射施用本发明中所述的rAAV或组合物。在一些实施例中，通过视网膜下注射施用本发明中所述的rAAV或组合物。在一些实施例中，通过脉络膜内(例如，脉络膜上腔)注射施用本发明中所述的rAAV或组合物。在一些实施例中，通过静脉注射施用本发明中所述的rAAV或组合物。
在一些实施方案中，rAAV递送至哺乳动物受试者可通过例如肌肉注射进入哺乳动物受试者。
在一些实施方案中，rAAV递送至哺乳动物受试者可通过例如静脉注射进入哺乳动物受试者。
在一些实施方案中，rAAV递送至哺乳动物受试者可通过例如关节内注射进入哺乳动物受试者。“关节内注射”这里定义为注射或注入到关节中。关节内注射通常用于将药物施用到受炎症影响的关节中。
施用包含本发明所述的rAAV或药物组合物的作用可以是预防病症发展、停止病症进展、逆转病症进展等。疾病或病症与rAAV所装载的目的基因及表达的基因产物有关。例如当基因产物为VEGF拮抗剂，预防或治疗的疾病或病症可以是VEGF相关的疾病。例如“VEGF有关的疾病”是指与异常VEGF活性/信号相关的一组疾病。许多研究已证实如果VEGF异常过量就可以刺激及诱导病理性血管新生，引起血管新生相关眼部疾病。非限制性示例性血管新生相关眼部疾病包括血管生成依赖性癌症，血管生成相关的眼部疾病，实体瘤(例如，肺癌，乳腺癌，肾癌，肝癌，胰腺癌，头颈癌，结肠癌，黑色素瘤)，血液源性肿瘤(例如白血病，转移性肿瘤)，良性肿瘤(例如血管瘤，听神经瘤，神经纤维瘤，气管炎和化脓性肉芽肿)，类风湿关节炎，牛皮癣，红肿，Osier-Webber综合征，心肌血管生成，斑块，毛细血管扩张，血友病关节或血管纤维瘤。
在一些实施例中，血管生成相关眼部疾病包括但不限于糖尿病视网膜病变、早产儿视网膜病变、黄斑变性、角膜移植物排斥反应、新生血管性青光眼和晶状体后纤维增生、流行性角膜结膜炎、维生素A缺乏症、接触镜过度磨损、特应性角膜炎，细菌性角膜炎，溃疡，溃疡，细菌性角膜炎，溃疡，原发性角化病、类风湿性关节炎、系统性狼疮、多动脉炎、创伤、韦格纳斯结节病、巩膜炎、史蒂芬约翰逊病、类天疱疮放射状角膜切开术、角膜移植排斥反应、镰状细胞贫血、结节病、弹性假黄瘤、佩吉茨病、静脉阻塞、动脉闭塞、颈动脉阻塞性疾病，慢性葡萄膜炎/玻璃炎、分枝杆菌感染、莱姆病、系统性红斑狼疮、早产儿视网膜病变、Eales病、白塞病、视网膜炎或脉络膜炎感染、假定的眼部组织胞浆菌病、Bests病、近视、视凹、Stargardt病、扁平部炎、慢性视网膜脱离，高粘血症综合征、弓形虫病、创伤或激光后并发症。
在一些实施例中，眼部疾病选自视网膜新生血管、脉络膜新生血管、虹膜新生血管、角膜新生血管性眼部疾病、非感染性葡萄膜炎或青光眼。
在一些实施例中，眼部疾病选自年龄相关性黄斑变性、黄斑水肿、糖尿病性黄斑水肿、继发于视网膜静脉阻塞的黄斑水肿、视网膜静脉阻塞、视网膜中央静脉阻塞、视网膜分支静脉阻塞、视网膜分支静脉阻塞所致黄斑水肿、糖尿病性视网膜水肿、糖尿病性视网膜病变、增生性糖尿病视网膜病变、糖尿病性视网膜缺血、息肉状脉络膜血管病变、继发于退行性近视的脉络膜新生血管或早产儿视网膜病变。黄斑变性的具体形式可以包含急性黄斑变性、非渗出性年龄相关黄斑变性和渗出性年龄相关黄斑变性。
例如当基因产物为TNF-α拮抗剂，预防或治疗的疾病或病症可以是关节炎疾病或相关病症。目前，估计有超过100种不同形式的关节炎。通常，关节紊乱被称为关节病，当涉及一个或多个关节的炎症时，这种紊乱被称之为关节炎。关节炎在本文中被理解为指“关节疼痛”或“关节疾病”。在一个优选实施方案中，关节炎疾病选自成人发作的Still病、强直性脊柱炎、关节炎、背痛、Behget病、钝性创伤、滑囊炎、焦磷酸钙沉积病(CPPD)、腕管综合征、髌骨软骨软化症、慢性疲劳综合征、复杂局部疼痛综合征、冷冻比林相关周期性综合征(CAPS)、退行性椎间盘疾病、发育性髋关节发育不良、埃勒-丹洛斯、家族性地中海热、纤维肌痛、第五疾病、巨细胞动脉炎、痛风、血色病、感染性关节炎、炎性关节炎，炎性肠病、关节置换、幼年关节炎、幼年皮肌炎(JD)、青少年特发性关节炎(JIA)、青少年类风湿性关节炎、青少年硬皮病、川崎病、狼疮、儿童和青少年狼疮、莱姆病、混合性结缔组织病、肌炎(包括多发性肌炎、皮肌炎)、骨关节炎(OA)、骨质疏松症、pagets、回旋风湿症、髌股痛综合征、儿童风湿性疾病、儿童SLE、风湿性多肌痛、假性痛风、银屑病关节炎、雷诺氏现象、反应性关节炎、反射性交感神经营养不良、赖特综合征、风湿热、风湿病、类风湿关节炎、硬皮病、脓毒性关节炎，干燥症、椎管狭窄、脊椎关节炎、斯蒂尔氏病、系统性青少年特发性关节炎和系统性红斑狼疮，儿童和青少年系统性红斑狼疮、系统性硬化、颞动脉炎、腱炎、血管炎和韦格纳肉芽肿。
在另一优选实施方案中，关节炎疾病选自类风湿性关节炎，青少年类风湿性关节炎，骨关节炎，痛风，假性痛风，脊椎炎，克罗恩病、斑块型银屑病，银屑病关节炎，强直性脊柱炎，脓毒性关节炎，关节炎，青少年特发性关节炎，钝性创伤，关节置换或Still’s疾病。
在更优选的实施方案中，关节炎疾病是涉及一个或多个关节炎症的关节紊乱。优选地，所述关节炎疾病选自类风湿关节炎(RA)、幼年类风湿性关节炎、骨关节炎(OA)、痛风、假痛风、脊椎关节炎(SpA)、银屑病关节炎、强直性脊柱炎、脓毒性关节炎，关节炎、幼年特发性关节炎和Still氏病。
下面将结合实施例对本发明作进一步阐述，但这些实施例不对本发明构成任何限制。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1突变体构建
使用定点诱变和重组DNA技术来生成AAV8的变体，所述变体包含位于亲本AAV8的第Q588～A592位氨基酸(QQNTA)5-14个氨基酸的一段多肽取代，或者进一步包括位于亲本AAV8衣壳蛋白第N262～N272位氨基酸对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)；或者进一步包括D80N、V125A、V125G位突变，如表1。示例性的No.12衣壳突变体质粒示意如图2。
表1
| 突变体 | Q588～A592氨基酸取代 |
| No.12 | RGNRQ(SEQ ID NO:1) |
| No.121 | QQNTARGNRQ(SEQ ID NO:2) |
| No.122 | RGNRQAAQQNTA(SEQ ID NO:3) |
| No.123 | RGNRQQNTA(SEQ ID NO:4) |
| No.124 | RGNRQQQNTA(SEQ ID NO:5) |
| No.125 | SGNTQ(SEQ ID NO:6) |
| No.128 | RGNQQNTARQ(SEQ ID NO:7) |
| No.129 | RGNQQPRPTSRQ(SEQ ID NO:8) |
| No.1213 | RGNRQAAQQPTPTS(SEQ ID NO:9) |
| No.1214 | RGNRQQQPTPTS(SEQ ID NO:19) |
表2
| 突变体 | Q588～A592氨基酸取代 | D80突变 | V125突变 |
| No.1281 | RGNQQNTARQ(SEQ ID NO:7) | D80N | V125A |
| No.1282 | RGNQQNTARQ(SEQ ID NO:7) | D80Q | V125G |
表3
| 突变体 | Q588～A592氨基酸取代 | N262～N272突变 |
| No.1312 | RGNRQ(SEQ ID NO:1) | SQSGASNDNH(SEQ ID NO:10) |
另一组衣壳蛋白突变体则是在亲本AAV8的第Q589位氨基酸后插入一段多肽序列，如表4。
表4
| 突变体 | 589位氨基酸后插入多肽序列 |
| No.15 | RGDLTTPQQ(SEQ ID NO:20) |
| No.151 | RGDLNTPQQ(SEQ ID NO:21) |
| No.152 | RGDVSSPQQ(SEQ ID NO:22) |
AAV8突变体的病毒包装通过三质粒共转染细胞生成：包括ITR侧接目的基因的表达盒(GFP或荧光素酶)的第一质粒(pAAV-CAG-EGFP)；本实施例1中构建的对Rep/Cap基因进行编码的突变体质粒(第二质粒)；以及含有腺病毒辅助功能基因的第三质粒。转染后收集细胞，裂解释放病毒，并通过PCR测定病毒滴度，后续备用。
实施例2小鼠玻璃体注射转染实验
2.1突变体的EGFP mRNA表达水平
将纯化后的AAV8突变体病毒，按7E+8vg/每眼进行小鼠玻璃体注射。玻璃体注射21d后，取小鼠眼球，使用冰冻匀浆机磨碎眼球，使用总RNA提取试剂盒提取RNA，并使用逆转录试剂盒合成cDNA，通过qPCR测定特异性EGFP引物探针组(其中，上游引物EGFP-F的序列为：5'-CACATGAAGCAGCACGACTT-3'(SEQ ID NO:26)；下游引物EGFP-R的序列为：5'-TCGTCCTTGAAGAAGATGGT-3'(SEQ ID NO:27)；探针为：5'-AGTCCGCCATGCCCGAAGGCT-3'-TAMRA，探针中所含序列如SEQ ID NO:28所示)，计算归一化AAV8突变体的EGFP mRNA水平，即比较不同AAV8突变体对玻璃体转染效果，结果如图3。
2.2视网膜铺片
将纯化后的AAV8突变体病毒，按7E+8vg/每眼进行小鼠玻璃体注射；玻璃体注射21d后，取小鼠眼球，4％多聚甲醛过夜固定小鼠眼球，使用手术显微镜剪在正置显微镜下剥离小鼠视网膜，对小鼠视网膜进行铺片，共聚焦拍摄EGFP荧光信号，结果如图4所示。
实施例3大鼠玻璃体注射转染实验
将纯化后的AAV8突变体病毒，按1.92E+9vg/每眼进行大鼠玻璃体注射；玻璃体注射21d后，取大鼠眼球；4％多聚甲醛过夜固定大鼠眼球；使用手术显微镜剪在正置显微镜下剥离大鼠视网膜；对大鼠视网膜进行铺片，荧光扫描显微镜拍摄EGFP荧光信号，结果如图5所示。
实施例4兔脉络膜上腔注射转染实验
将纯化后的AAV8突变体病毒，按5E+10vg/每眼进行新西兰兔的脉络膜上腔注射；脉络膜上腔注射14d后，取新西兰兔眼球；4％多聚甲醛过夜固定新西兰兔眼球；对固定的眼球进行脱水及组织包埋处理；进行冰冻切片，然后细胞核染色(DAPI)；使用共聚焦显微镜进行全图扫描，捕获EGFP荧光信号及DAPI信号，结果如图6所示(图5中，EGFP：自发荧光，DAPI：细胞核，RGC：神经节细胞，INL：视网膜内核层；ONL：视网膜外核层；RPE：视网膜上皮细胞)，从荧光图来看，野生型对感光细胞层及RPE层的转导效率不高，而突变体对感光细胞及RPE层的转导效率大幅度提高。
实施例5 ARPE19细胞感染实验
将ARPE19细胞种于12孔板中，培养至细胞融合度70-80％(一般细胞个数为1.0E+5个)；按所需比例加入病毒上清(4.5E+9Vg)，使得病毒上清量+培养基总量为1000μL；感染16h 后，每日更换培养基，继续培养至72小时，共聚焦拍摄EGFP荧光信号，观测荧光蛋白表达情况。ARPE19细胞48小时感染情况如图7所示。
同时，采用流式细胞术方法对EGFP阳性细胞数进行计数。以2E+5细胞数于6孔板细胞皿培养，加入1E+10vg已包装的突变体病毒，37℃培养48h；使用胰蛋白酶消化细胞，收集细胞，并用300μL无菌PBS重悬细胞；使用罗氏流式细胞术选用FITC(488nm)通道测量，设定测量总细胞数为10万，计数EGFP阳性细胞数。结果如图8A、8B所示。
实施例6兔脉络膜上腔注射转染实验
按照实施例1中所述三质粒共转染方法制备包括不同突变体病毒AAV-mCherry(mCherry是一种珊瑚中分离出来的蛋白，此处用作一种红色荧光染料示踪剂)，并通过ddPCR测量每种病毒的基因组滴度；将每种突变体的病毒的基因组滴度通过缓冲液稀释至1.0E12vg/mL，在兔眼颞上方4mm处进行脉络膜上腔注射，每眼100μL，每种病毒注射2只兔，4只眼；5天后，处死新西兰兔，取眼球，分离视网膜/脉络膜组织后，进行低温组织匀浆，取上清，通过病毒基因组DNA提取试剂盒提取病毒基因组DNA，通过qPCR测定每种病毒转导视网膜/脉络膜病毒基因组DNA相对含量，结果如表5及图9所示。
表5
实施例7感染APRE细胞蛋白表达实验
按照实施例1中所述三质粒共转染生产AAV8-阿柏西普病毒和AAV No.12-阿柏西普突变体病毒，并通过ddPCR测量每种病毒的基因组滴度；将每种突变体的病毒的基因组滴度通过缓冲液稀释至3.0E12vg/mL，细胞数为3.0E5，每孔加入10μL病毒，培养16h后进行换液，72h后进行收集细胞及细胞上清液；通过液氮冷冻，反复冻融3次，进行细胞裂解，选用ELISA方法测定Trap蛋白表达水平。结果表明AAV8-阿柏西普病毒的蛋白表达量为97ng/ml，AAV No.12-阿柏西普病毒的蛋白表达量为1662ng/ml。
实施例8恒河猴脉络膜上腔注射蛋白表达实验
按照实施例1中所述三质粒共转染生产AAV No.128-阿柏西普病毒，并通过ddPCR测量每种病毒的基因组滴度；将病毒的基因组滴度通过缓冲液稀释至1.0E13vg/mL，在猴眼颞上方4mm处进行脉络膜上腔注射，每眼100μL，注射1只猴，2只眼；56天后，处死恒河猴，取眼球，分离猴眼各个组织(包括，房水，脉络膜，结膜，虹膜睫状体，视网膜，巩膜，玻璃体)，加入蛋白酶抑制剂后，进行低温组织匀浆，取上清，通过ELISA测定阿柏西普蛋白表达水平(ng/ml)，结果如表6所示。
表6
BLQ*：未检测到。
实施例9小鼠肌肉注射转染实验
按照实施例1中所述三质粒共转染生产AAV8-luciferase和AAV No.128-luciferase病毒，并通过ddPCR测量病毒的基因组滴度；将病毒的基因组滴度通过缓冲液稀释至1.62E11vg/mL，在小鼠右腿腓肠肌进行注射，体积为50μL，每种病毒注射5只鼠(Balb/c)；在病毒注射后的7天、14天、28天、160天，分别于小鼠腹腔注射一定量的荧光素钠盐底物，进行小鼠活体成像，于活体成像仪进行荧光强度分析，结果如表7及图10所示。
表7
实施例10转染293F细胞蛋白表达实验
分别构建AAV8-No.128-依那西普、AAV No.128-阿达木单抗质粒；选取2μg质粒，进行单质粒转染293F细胞，待72h后，通过ELISA方法检测质粒的蛋白表达水平。结果表明，AAV No.128-阿达木单抗质粒的表达水平为1010ng/mL，而AAV No.128-依那西普质粒的表达水平为2020ng/mL。
实施例11小鼠关节腔/肌肉注射有效性实验
1)样品溶液制备
供试品制备：AAV No.128-依那西普病毒按照实施例1中所述方法进行制备并测定病毒基因组滴度，然后稀释至9.98E12vg/mL，备用。
阳性对照品：取阿达木单抗原液40mg/400μl，稀释至1.6μg/μl。
2)造模
第0天初次造模：选用6周龄的DBA/1雄性小鼠，先将0.6ml牛II型胶原(CII)溶液(2mg/ml)与0.6ml(4mg/ml)完全弗氏佐剂装入挤膜器中，制备得到1ml左右乳白色乳剂。小鼠每只尾根部皮下注射100μl乳剂，观察尾根部肿胀且乳剂未漏出，小鼠未死亡，即说明给药基本成功。
第21天二次造模：将0.6ml牛II型胶原(CII)(2mg/ml)与0.6ml(4mg/ml)不完全弗氏佐剂装入挤膜器中，制备得到1ml左右乳白色乳剂，小鼠每只尾根部皮下注射100μl乳剂。
3)给药
第22天给药，给药第0天，关节腔内注射AAV No.128-依那西普病毒样品溶液10μl/ankle(双腿均注射)。
第22天给药，给药第0天，临近关节腔肌肉注射AAV No.128-依那西普病毒样品溶液100μl/ankle(双腿均注射)。
第22天，阿达木单抗组背部皮下给阿达木单抗160μg(原液40mg/400μl稀释至1.6μg/μl，背部皮下使用胰岛素针注射100μl)，每两周给药一次。
4)结果观察
从第23天起，给药第2天，使用电子游标卡尺监控测量脚掌厚度，同时对关节炎症情况进行临床评分，每两天一测。评分标准如下表8：
表8
5)结果
脚掌厚度测量及对关节炎症情况进行临床评分的结果如图11及图12所示。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (34)
- 一种经过修饰的腺相关病毒(AAV)衣壳蛋白，其特征在于，所述衣壳蛋白相对于亲本AAV衣壳蛋白包含约5-14个氨基酸的多肽取代，并且其中与包含相应亲本AAV衣壳蛋白的AAV相比，包含所述经过修饰的衣壳蛋白的AAV具有增强的视网膜细胞感染性。
- 根据权利要求1所述的衣壳蛋白，其特征在于，所述多肽包括选自RGNRQ(SEQ ID NO:1)，QQNTARGNRQ(SEQ ID NO:2)、RGNRQAAQQNTA(SEQ ID NO:3)、RGNRQQNTA(SEQ ID NO:4)、RGNRQQQNTA(SEQ ID NO:5)、SGNTQ(SEQ ID NO:6)、RGNQQNTARQ(SEQ ID NO:7)、RGNQQPRPTSRQ(SEQ ID NO:8)、RGNRQAAQQPTPTS(SEQ ID NO:9)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列；并且所述取代位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处。
- 根据权利要求2所述的衣壳蛋白，其特征在于，所述多肽包括选自RGNRQ(SEQ ID NO:1)、QQNTARGNRQ(SEQ ID NO:2)、RGNRQAAQQNTA(SEQ ID NO:3)、RGNRQQNTA(SEQ ID NO:4)、SGNTQ(SEQ ID NO:6)、RGNQQNTARQ(SEQ ID NO:7)或RGNRQQQPTPTS(SEQ ID NO:19)的氨基酸序列。
- 根据权利要求2或3所述的衣壳蛋白，其特征在于，所述衣壳蛋白进一步包括相对于亲本AAV8衣壳蛋白第262～272位氨基酸或另一血清型衣壳蛋白的对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。
- 根据权利要求1-4任一项所述的衣壳蛋白，其特征在于，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸或另一血清型衣壳蛋白的对应位置处的多肽取代，所述多肽为RGNRQ(SEQ ID NO:1)；和相对于亲本AAV8衣壳蛋白第262～272位或另一血清型衣壳蛋白的对应位置处的突变，突变后的氨基酸序列为SQSGASNDNH(SEQ ID NO:10)。
- 根据权利要求1-5任一项所述的衣壳蛋白，其特征在于，所述AAV血清型选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11，AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R和AAVrh10。
- 根据权利要求6所述的衣壳蛋白，其特征在于，所述AAV血清型为AAV8。
- 根据权利要求1-7任一项所述的衣壳蛋白，其特征在于，所述衣壳蛋白进一步包括相对于亲本AAV8衣壳蛋白D80和/或V125位置处氨基酸的突变。
- 根据权利要求8所述的衣壳蛋白，其特征在于，所述突变为D80N和/或V125A。
- 根据权利要求8所述的衣壳蛋白，其特征在于，所述突变为D80Q和/或V125G。
- 根据权利要求8或9所述的衣壳蛋白，其特征在于，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80N和V125A。
- 根据权利要求8或10所述的衣壳蛋白，其特征在于，所述衣壳蛋白包括位于亲本AAV8的第588～592位氨基酸的多肽取代，所述多肽为RGNQQNTARQ(SEQ ID NO:7)；和相对于亲本AAV8衣壳蛋白D80和V125位置处的氨基酸突变，所述突变为D80Q和V125G。
- 一种经过修饰的腺相关病毒(AAV)衣壳蛋白，其特征在于，所述衣壳蛋白包括位于亲本AAV8第589位氨基酸或另一血清型衣壳蛋白的对应位置后的多肽插入，并且其中与包含相应亲本AAV衣壳蛋白的AAV病毒相比，包含所述经过修饰的衣壳蛋白的AAV病毒具有增强的视网膜细胞感染性。
- 根据权利要求13所述的衣壳蛋白，其特征在于，所述多肽包含选自RGDLTTPQQ(SEQ ID NO:20)、RGDLNTPQQ(SEQ ID NO:21)和RGDVSSPQQ(SEQ ID NO:22)的氨基酸序列。
- 根据权利要求13或14所述的衣壳蛋白，其特征在于，所述AAV血清型选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11，AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R和AAVrh10。
- 根据权利要求15所述的衣壳蛋白，其特征在于，所述AAV血清型为AAV8。
- 一种重组腺相关病毒(rAAV)，其包括：i.前述权利要求1-16中任一项所述的经过修饰的衣壳蛋白；ii.包含编码基因产物的异源核酸。
- 根据权利要求17所述的重组腺相关病毒，其特征在于，所述基因产物为VEGF拮抗剂或TNF-α拮抗剂。
- 根据权利要求18所述的重组腺相关病毒，其特征在于，所述VEGF拮抗剂选自阿柏西普、康柏西普、雷珠单抗、布洛赛珠单抗。
- 根据权利要求19所述的重组腺相关病毒，其特征在于，所述VEGF拮抗剂选自阿柏西普。
- 根据权利要求18所述的重组腺相关病毒，其特征在于，所述TNF-α拮抗剂选自：依那西普、英夫利昔单抗、阿达木单抗、培塞利珠单抗、戈利木单抗。
- 根据权利要求21所述的重组腺相关病毒，其特征在于，所述TNF-α拮抗剂选自依那西普。
- 一种药物组合物，其包括：a)权利要求17-22任一项所述的重组腺相关病毒；b)药学上可接受的赋形剂。
- 权利要求17-22任一项所述的重组腺相关病毒在制备用于预防或治疗眼部疾病的药物中的用途。
- 一种预防或治疗眼部疾病的方法，所述方法包括向有需要的个体施用有效量的权利要求17-22任一项所述的重组腺相关病毒或权利要求23所述的药物组合物。
- 根据权利要求25所述的方法，其特征在于，通过玻璃体、视网膜或脉络膜注射给药。
- 根据权利要求26所述的方法，其特征在于，通过脉络膜上腔注射给药。
- 根据权利要求24所述的用途或权利要求25-27任一项所述的方法，其特征在于，所述眼部疾病选自视网膜新生血管、脉络膜新生血管、虹膜新生血管、角膜新生血管性眼部疾病、非感染性葡萄膜炎或青光眼。
- 根据权利要求24所述的用途或权利要求25-27任一项所述的方法，其特征在于，所述眼部疾病选自年龄相关性黄斑变性、黄斑水肿、糖尿病性黄斑水肿、继发于视网膜静脉阻塞的黄斑水肿、视网膜静脉阻塞、视网膜中央静脉阻塞、视网膜分支静脉阻塞、视网膜分支静脉阻塞所致黄斑水肿、糖尿病性视网膜水肿、糖尿病性视网膜病变、增生性糖尿病视网膜病变、糖尿病性视网膜缺血、息肉状脉络膜血管病变、继发于退行性近视的脉络膜新生血管或早产儿视网膜病变。
- 权利要求17-22任一项所述的重组腺相关病毒在制备用于预防或治疗关节炎疾病或相关病症的药物中的用途。
- 一种治疗关节炎疾病或相关病症的方法，所述方法包括向有需要的个体施用有效量的权利要求17-22任一项所述的重组腺相关病毒或权利要求23所述的药物组合物。
- 根据权利要求31所述的方法，其特征在于，通过静脉、皮下、肌肉或关节注射给药。
- 根据权利要求32所述的方法，其特征在于，通过肌肉或关节内注射给药。
- 根据权利要求30所述的用途或权利要求31-33任一项所述的方法，所述关节炎疾病或相关病症选自类风湿性关节炎，青少年类风湿性关节炎，骨关节炎，痛风，假性痛风，脊椎炎，克罗恩病、斑块型银屑病，银屑病关节炎，强直性脊柱炎，脓毒性关节炎，关节炎，青少年特发性关节炎，钝性创伤，关节置换或Still’s疾病。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111628667.2 | 2021-12-28 | ||
| CN202111628667 | 2021-12-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023125481A1 true WO2023125481A1 (zh) | 2023-07-06 |
Family
ID=86997846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/142185 WO2023125481A1 (zh) | 2021-12-28 | 2022-12-27 | 经过修饰的aav衣壳蛋白及其用途 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN116444626A (zh) |
| WO (1) | WO2023125481A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2912525A1 (en) * | 2013-05-16 | 2014-11-20 | Applied Genetic Technologies Corporation | Promoters, expression cassettes, vectors, kits, and methods for the treatment of achromatopsia and other diseases |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106062200A (zh) * | 2014-02-17 | 2016-10-26 | 伦敦国王学院 | 腺相关病毒载体 |
| US20170049910A1 (en) * | 2013-02-08 | 2017-02-23 | The Trustees Of The University Of Pennsylvania | Enhanced aav-mediated gene transfer for retinal therapies |
| CN107405507A (zh) * | 2015-03-02 | 2017-11-28 | 阿德夫拉姆生物技术股份有限公司 | 用于将多核苷酸玻璃体内递送到视网膜视锥的组合物和方法 |
| WO2018075798A1 (en) * | 2016-10-19 | 2018-04-26 | Adverum Biotechnologies, Inc. | Modified aav capsids and uses thereof |
| CN110461368A (zh) * | 2017-06-30 | 2019-11-15 | 加利福尼亚大学董事会 | 具有变异衣壳的腺相关病毒病毒体及其使用方法 |
| CN111770999A (zh) * | 2017-11-27 | 2020-10-13 | 4D分子治疗有限公司 | 腺相关病毒变体衣壳和用于抑制血管生成的应用 |
| WO2021009684A1 (en) * | 2019-07-15 | 2021-01-21 | Meiragtx Uk Ii Limited | Modified aav capsid proteins for treatment of arthritic disease |
| CN112352050A (zh) * | 2018-02-27 | 2021-02-09 | 宾夕法尼亚州大学信托人 | 新颖腺相关病毒（aav）载体、具有降低的衣壳脱酰胺化的aav载体及其用途 |
| WO2021168509A1 (en) * | 2020-02-25 | 2021-09-02 | Children's Medical Research Institute | Adeno-associated virus capsid polypeptides and vectors |
| CN113518824A (zh) * | 2019-10-16 | 2021-10-19 | 上海药明康德新药开发有限公司 | 新的aav变体 |
-
2022
- 2022-12-27 WO PCT/CN2022/142185 patent/WO2023125481A1/zh unknown
- 2022-12-27 CN CN202211691417.8A patent/CN116444626A/zh active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170049910A1 (en) * | 2013-02-08 | 2017-02-23 | The Trustees Of The University Of Pennsylvania | Enhanced aav-mediated gene transfer for retinal therapies |
| CN106062200A (zh) * | 2014-02-17 | 2016-10-26 | 伦敦国王学院 | 腺相关病毒载体 |
| CN107405507A (zh) * | 2015-03-02 | 2017-11-28 | 阿德夫拉姆生物技术股份有限公司 | 用于将多核苷酸玻璃体内递送到视网膜视锥的组合物和方法 |
| WO2018075798A1 (en) * | 2016-10-19 | 2018-04-26 | Adverum Biotechnologies, Inc. | Modified aav capsids and uses thereof |
| CN110461368A (zh) * | 2017-06-30 | 2019-11-15 | 加利福尼亚大学董事会 | 具有变异衣壳的腺相关病毒病毒体及其使用方法 |
| CN111770999A (zh) * | 2017-11-27 | 2020-10-13 | 4D分子治疗有限公司 | 腺相关病毒变体衣壳和用于抑制血管生成的应用 |
| CN112352050A (zh) * | 2018-02-27 | 2021-02-09 | 宾夕法尼亚州大学信托人 | 新颖腺相关病毒（aav）载体、具有降低的衣壳脱酰胺化的aav载体及其用途 |
| WO2021009684A1 (en) * | 2019-07-15 | 2021-01-21 | Meiragtx Uk Ii Limited | Modified aav capsid proteins for treatment of arthritic disease |
| CN113518824A (zh) * | 2019-10-16 | 2021-10-19 | 上海药明康德新药开发有限公司 | 新的aav变体 |
| WO2021168509A1 (en) * | 2020-02-25 | 2021-09-02 | Children's Medical Research Institute | Adeno-associated virus capsid polypeptides and vectors |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116444626A (zh) | 2023-07-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7416443B2 (ja) | 高形質導入効率ｒＡＡＶベクター、組成物、および使用方法 | |
| US20210269487A1 (en) | Recombinant aav1, aav5, and aav6 capsid mutants and uses thereof | |
| US20230381341A1 (en) | Adeno-associated viruses for ocular delivery of gene therapy | |
| JP6072772B2 (ja) | 変異体キャプシドを有するアデノ関連ウイルスビリオンおよびその使用方法 | |
| JP2024015194A (ja) | アデノ随伴ウイルス変異キャプシドおよび血管新生の阻害のための使用 | |
| JP2020510424A (ja) | 改変ａａｖカプシドおよびその使用 | |
| CN113121652A (zh) | 视网膜和肌肉高亲和性腺相关病毒衣壳蛋白及相关应用 | |
| US20170348387A1 (en) | Aav-mediated gene therapy for nphp5 lca-ciliopathy | |
| CN113121655B (zh) | 眼部和肌肉特异靶向型腺相关病毒载体及其应用 | |
| WO2023125481A1 (zh) | 经过修饰的aav衣壳蛋白及其用途 | |
| US20230321280A1 (en) | Compositions and methods for the treatment of ocular diseases | |
| CN111876432A (zh) | 一组肝靶向新型腺相关病毒的获得及其应用 | |
| CA3088079A1 (en) | Compositions and methods for treating retinal disorders | |
| WO2022222869A1 (zh) | 重组腺相关病毒及其应用 | |
| CA3186830A1 (en) | Composition and method for treating eye diseases | |
| CN113121653A (zh) | 肌肉和视网膜特异性的新型腺相关病毒衣壳蛋白 | |
| US20190330308A1 (en) | Modified soluble vegf receptor-1 genes and vectors for gene therapy | |
| EP4368203A1 (en) | Construction and use of anti-vegf antibody in-vivo expression system | |
| JP2022547158A (ja) | Ｔｆｅｂの活性化により網膜色素上皮細胞のリソソーム機能を回復する方法 | |
| JP7495756B2 (ja) | 医薬の製造におけるＣＹＰ４Ｖ２およびＲｄＣＶＦの使用 | |
| WO2024067776A1 (zh) | 核酸构建体及其用途 | |
| WO2023207792A1 (zh) | 新型aav衣壳改造株及其用途 | |
| WO2021114662A1 (zh) | CYP4V2和RdCVF在制备药物中的用途 | |
| CN116568815A (zh) | 用于基因疗法的眼部递送的腺相关病毒 | |
| JP2022523050A (ja) | 合理的設計により増強された新規なａａｖウイルスによる網膜における高効率の形質導入および側方への広がり |
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: 22914735 Country of ref document: EP Kind code of ref document: A1 |