WO2022247917A1 - 衣壳变异的重组腺相关病毒及其应用 - Google Patents

衣壳变异的重组腺相关病毒及其应用 Download PDF

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WO2022247917A1
WO2022247917A1 PCT/CN2022/095422 CN2022095422W WO2022247917A1 WO 2022247917 A1 WO2022247917 A1 WO 2022247917A1 CN 2022095422 W CN2022095422 W CN 2022095422W WO 2022247917 A1 WO2022247917 A1 WO 2022247917A1
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seq
aav
cells
capsid protein
retinal
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PCT/CN2022/095422
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French (fr)
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张文涛
廖成
宁威
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上海瑞宏迪医药有限公司
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Priority to AU2022281825A priority Critical patent/AU2022281825A1/en
Priority to KR1020237043802A priority patent/KR20240014477A/ko
Priority to CN202280028081.9A priority patent/CN117157309A/zh
Priority to BR112023024375A priority patent/BR112023024375A2/pt
Priority to EP22810641.5A priority patent/EP4349852A1/en
Priority to CA3219898A priority patent/CA3219898A1/en
Publication of WO2022247917A1 publication Critical patent/WO2022247917A1/zh

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Definitions

  • the present disclosure relates to the technical field of recombinant adeno-associated virus (rAAV), in particular to a rAAV virion with capsid variation and its application for delivering gene products to target cells (eg, retinal cells).
  • rAAV recombinant adeno-associated virus
  • Adeno-associated virus belongs to the genus Dependovirus of the family Parvoviridae, and is a small (25 nm), non-enveloped, single-stranded DNA virus with nucleic acid encapsulated by an icosahedral capsid (cap).
  • AAV contains two open reading frames rep and cap, rep is used to encode four proteins (Rep78, Rep68, Rep52, and Rep40) necessary for genome replication, and cap is used to encode three structural proteins required for viral capsid assembly ( VP1-3).
  • AAV shows great potential for the treatment of genetic diseases and genetic diseases, and can be used for gene complement therapy (also known as gene enhancement therapy). It restores the loss or disorder of gene function caused by mutation by complementing the missing gene function, thereby restoring the biological function of the target cell to a normal physiological state.
  • gene complement therapy also known as gene enhancement therapy.
  • the safety and long-term expression of the AAV vectors currently used in the clinic are genetically engineered to be latent in the absence of a helper virus, and have been extensively tested in rodent models, non-human primates, and in multiple human trials.
  • Test MacLaren et al. Lancet. 2014 Mar 29; 383(9923): 1129-37.; Maguire et al. N Engl J Med. 2008 May 22; 358(21): 2240-8; Simonelli et al. Mol Ther .2010 Mar;18(3):643-50; Nathwani et al.N Engl J Med.2014 Nov 20;371(21):1994-2004).
  • AAV capsid proteins naturally occur in different compositions and structures, and different capsids have different tissue phagocytosis. Multiple homologous primate and non-human primate AAV serotypes have been identified and different engineered AAV variants (also called AAV serotypes) have been developed. Although AAV vectors have shown a certain degree of diffuse infection in different organisms and tissues, local injection in the lesion area is used in most clinical trials, especially in the application of ocular gene therapy. The main current ocular clinical implementations are subretinal injection (ie, the cavity formed after injection of fluid between the RPE and the photoreceptor) and intravitreal injection.
  • Subretinal injection allows AAV to fully contact RPE and photoreceptor cells, and the effect of local infection is better, but the relative risk of injection operation is relatively high, and it is easy to cause retinal detachment.
  • AAV preparations will first be uniformly distributed in the vitreous humor and then diffusely infect the retina layer by layer. Due to the dense structure of the retinal layer and the composition of complex cell populations, the natural serotypes AAV8 and AAV2, which have a strong ability to infiltrate the eye, are less effective when injected into the vitreous cavity, while some engineered capsids are effective in the implementation of the vitreous cavity. When injected, it shows strong infection ability (such as AAVDJ, AAV2.7M8, etc.).
  • rAAV recombinant adeno-associated virus
  • the present disclosure provides variant AAV capsid proteins and gene products they can carry, rAAV virions containing the capsid proteins, pharmaceutical compositions, infection of cells (e.g., retinal cells) by the rAAV virions, treatment and prevention Methods and pharmaceutical uses for disease (eg, ocular disease).
  • AAV adeno-associated virus
  • AAV adeno-associated virus
  • LAETTRP SEQ ID NO: 11
  • polypeptide consisting of SEQ ID NO: 11
  • LGDTTRP SEQ ID NO: 12
  • a polypeptide consisting of SEQ ID NO: 12 SEQ ID NO: 12
  • LGETTRN SEQ ID NO: 13
  • polypeptide consisting of SEQ ID NO: 13;
  • KADTTKN (SEQ ID NO: 14) or a polypeptide consisting of SEQ ID NO: 14;
  • KDDTTRN SEQ ID NO: 15
  • polypeptide consisting of SEQ ID NO: 15;
  • LADTTKN (SEQ ID NO: 16) or a polypeptide consisting of SEQ ID NO: 16.
  • AAV adeno-associated virus
  • SEQ ID NO: 35 adeno-associated virus
  • X1 is selected from L or K
  • X2 is selected from G
  • D or A is selected from D or E
  • X3 is selected from D or E
  • X4 is selected from R or K
  • X5 is selected from P or N.
  • the aforementioned 1)-6) or the polypeptide represented by SEQ ID NO: 35 has 1-4 spacer amino acids (Y 1 -Y 4 ) at its amino terminus and/or carboxyl terminus.
  • the spacer amino acids include, but are not limited to, A, L, G, S and T.
  • AAV adeno-associated virus
  • X 1 is selected from L or K
  • X 2 is selected from G, D or A
  • X 3 is selected from D or E
  • X 4 is selected from R or K
  • X 5 is selected from P or N
  • Y 1 , Y 2 , Y 3 , Y 4 can be independently present or absent
  • Y 1 , Y 2 , Y 3 , Y 4 can be independently selected from A, L, G , S and T.
  • Y 1 is L
  • Y 2 is A
  • Y 3 is A
  • Y 4 is absent.
  • AAV adeno-associated virus
  • LALAETTRPA SEQ ID NO: 17
  • polypeptide consisting of SEQ ID NO: 17
  • LALGDTTRPA SEQ ID NO: 18
  • polypeptide consisting of SEQ ID NO: 18;
  • LAKDDTTRNA SEQ ID NO: 21
  • polypeptide consisting of SEQ ID NO: 21
  • a polypeptide comprising LALADTTKNA (SEQ ID NO: 22) or consisting of SEQ ID NO: 22.
  • the above-mentioned AAV capsid protein is an AAV2 capsid protein, or an AAV9 capsid protein.
  • the aforementioned 1) to 6), 1-1) to 6-1), the polypeptide of SEQ ID NO: 35 or 36 is located in the GH loop or IV loop (ring domain IV) of the parent AAV capsid protein
  • the solvent accessible part of the GH loop or IV loop of the AAV capsid protein see van Vliet et al. (2006) Mol.Ther.14:809; Padron et al. (2005) J.Virol.79:5047; and Shen et al. (2007) Mol. Ther. 15:1955).
  • Parental AAV capsid protein refers to the capsid protein of the same AAV serotype without the inserted polypeptide (including wild-type AAV serotype or its variant capsid protein, such as the AAV2 capsid protein shown in SEQ ID NO: 1 of the present disclosure).
  • Capsid protein or the AAV9 capsid protein shown in SEQ ID NO: 37, the AAV2 capsid protein may or may not have the V708I mutation).
  • the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (including AAVrh10).
  • the aforementioned inserted polypeptide is located within amino acid residues 411 to 650, or 432 to 640, or 570 to 671, or 570 to 614 of the parent AAV capsid protein, Or 570 to 610, or 580 to 600, or 570 to 575, or 575 to 580, or 580 to 585, or 585 to 590, or 590 to 600 , or 600 to 614.
  • amino acid residues 570 to 611 of the parental AAV2 capsid protein within amino acid residues 571 to 612 of the parental AAV1 capsid protein, and within amino acid residues 560 to 601 of the parental AAV5 capsid protein , within amino acid residues 571 to 612 of the parental AAV6 capsid protein, within amino acid residues 572 to 613 of the parental AAV7 capsid protein, within amino acid residues 573 to 614 of the parental AAV8 capsid protein, within the parental AAV9 capsid protein Within amino acid residues 571 to 612 of the capsid protein, or within amino acid residues 573 to 614 of the parental AAV10 (including AAVrh10) capsid protein.
  • the aforementioned inserted polypeptide is located between amino acid residues 587 and 588 of the parent AAV2 capsid protein, or between amino acid residues 588 and 589 of the parent AAV9 capsid protein, or other parent AAV serum The corresponding position of the type capsid protein. In some embodiments, the inserted polypeptide is located between amino acid residues 587 and 588 of the parent AAV2 capsid protein, or between amino acid residues 588 and 589 of the parent AAV9 capsid protein, or other parent AAV serotypes The corresponding position of the capsid protein.
  • serotypes are eg selected from AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV10 (including AAVrh10).
  • the sequences corresponding to amino acids 570-611 of the capsid protein VP1 of AAV2 in different AAV serotypes are well known in the art (see e.g. Figure 6 of WO2012145601A, and AAV1 of GenBank Accession No. NP_049542; AAV5 of AAD13756; AAV6 of AAB95459 ; AAV7 of YP_077178; AAV8 of YP_077180; AAV9 of AAS99264 and AAV10 of AAT46337).
  • the inserted polypeptide is located between amino acid residues 590 and 591 of the parental AAV1 capsid protein, between amino acid residues 575 and 576 of the parental AAV5 capsid protein, and between amino acid residues 575 and 576 of the parental AAV6 capsid protein. Between residues 590 and 591, between amino acid residues 589 and 590 of the parental AAV7 capsid protein, between amino acid residues 590 and 591 of the parental AAV8 capsid protein, parental AAV10 (including AAVrh10) Between amino acid residues 588 and 589 of the capsid protein.
  • Amino acid residue counts in the present disclosure are natural counts from the N-terminus relative to the encoded amino acid sequence of VP1 of the AAV capsid protein.
  • the inserted polypeptide is located between amino acid residues 587 and 588 of the AAV2 capsid protein
  • the polypeptide is located between 587 and 588 of the amino acid sequence encoded by VP1 of the AAV2 capsid protein, which is between
  • the encoded amino acid sequence of VP2 corresponds to positions 450 to 451
  • the encoded amino acid sequence of VP3 corresponds to positions 385 to 386.
  • the aforementioned inserted polypeptide is located between amino acid residues 450 and 460 of the parent AAV capsid protein, such as amino acid residue 453 of the parent AAV2, amino acid residue 454 of the parent AAV1, and amino acid residue 454 of the parent AAV6.
  • amino acid residue 456 of parental AAV7, amino acid residue 456 of parental AAV8, amino acid residue 454 of parental AAV9, or amino acid residue 456 of parental AAV10 (including AAVrh10) are incorporated herein in full.
  • the present disclosure provides a variant AAV capsid protein comprising a polypeptide of the foregoing 1) to 6), 1-1) to 6-1), SEQ ID NO: 35 or 36.
  • the aforementioned variant AAV capsid proteins of the present disclosure further comprise point mutations (including substitutions, deletions and/or additions) of one or more amino acid residues.
  • the point mutation of the amino acid residue is located at position 1, 15, 34, 57, 66, 81, 101, 109, 144, 164, 176, 188, 196, 226, 236, 240, 250, 312 , 363, 368, 449, 456, 463, 472, 484, 524, 535, 551, 593, 698, 708, 719, 721 and 735 bits or any combination thereof.
  • the point mutation (substitution) of the amino acid residue is selected from 1L, 15P, 34A, 57D, 66K, 81Q, 101R, 109T, 144K or M, 164K, 176P, 188I, 196Y, 226E, 236V, One or any combination of 240T, 250S, 312K, 363L, 368H, 449D, 456K, 463Y, 472N, 484C, 524T, 535S, 551S, 593E, 698V, 708I, 719M, 721L and 735Q, such as 312K, 449D, 472N , 551S, 698V, 735Q, 273F, 444F, 500F, 730F, 708I, such as 708I.
  • the point mutation (substitution) of the amino acid residue is selected from M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, Y273F, Y40FF, Y7 or any of them Combinations, such as one or more of N312K, N449D, D472N, N551S, I698V, L735Q, Y273F, Y444F, Y500F, Y
  • the point mutation (substitution) of the amino acid residue is 708I and/or 449D, or V708I and/or N449D. In some specific embodiments, the mutation (substitution) of the amino acid residue is 273F, 444F, 500F and/or 730F, or Y273F, Y444F, Y500F and/or Y730F.
  • the above-mentioned point mutations are relative to the corresponding positions of the corresponding parental AAV capsid protein, for example, relative to the corresponding positions of the parental AAV2 capsid protein.
  • the AAV capsids of the present disclosure are chimeric capsids.
  • the capsid includes a portion of an AAV capsid of a first AAV serotype including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and a portion of a second serotype of an AAV capsid.
  • AAV9, AAV10 (including AAVrh10).
  • the AAV capsid may be AAV2G9, which comprises sequences from AAV2 and AAV9, and the sequence of AAV2G9 in US20160017005 is incorporated herein in its entirety.
  • the variant AAV capsid proteins of the disclosure are isolated, and/or purified.
  • the present disclosure provides variant AAV2 capsid proteins, relative to the corresponding parental AAV2 capsid protein (such as shown in SEQ ID NO: 1), in the capsid protein GH loop or IV loop (loop domain IV) Include polypeptide, described polypeptide is selected from aforementioned 1) to 6), 1-1) to 6-1), SEQ ID NO:35 or 36.
  • the polypeptide comprises or is SEQ ID NO: 12 or 18.
  • the polypeptide is located between amino acid residues 587 and 588 of VP1 of the parent AAV2 capsid protein.
  • the present disclosure provides variant AAV9 capsid proteins, relative to the corresponding parental AAV9 capsid protein (eg, set forth in SEQ ID NO: 37), in the GH loop or IV loop (ring domain IV) of the capsid protein
  • polypeptide described polypeptide is selected from aforementioned 1) to 6), 1-1) to 6-1), SEQ ID NO:35 or 36.
  • the polypeptide comprises or is SEQ ID NO: 12 or 18.
  • the polypeptide is located between amino acid residues 588 and 589 of VP1 of the parent AAV9 capsid protein.
  • the present disclosure provides a variant AAV capsid protein having an amino acid sequence at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • variant AAV capsid proteins provided by the present disclosure have the following properties:
  • rAAV adeno-associated virus
  • rAAV adeno-associated virus
  • the heterologous polynucleotide comprises a polynucleotide that expresses or encodes a gene product.
  • the gene product is heterologous to AAV.
  • the gene product is heterologous or endogenous to the target cell.
  • the gene product is one or more (eg, 2, 3, 4).
  • the heterologous polynucleotide includes regulatory sequences that regulate the expression or encoding of a gene product.
  • the gene product is therapeutic or prophylactic for a disease, condition.
  • the gene product is selected from interfering RNA (RNAi), aptamers, polypeptides.
  • RNAi interfering RNA
  • aptamers aptamers
  • polypeptides polypeptides
  • the gene product is RNAi, such as RNAi that reduces, decreases the level of an apoptotic factor or an angiogenic factor in a cell.
  • RNAi can be shRNA or siRNA that reduces levels of pro-apoptotic or pro-apoptotic gene products in cells, including, for example, the Bax, Bid, Bak, and Bad gene products (see US 7,846,730, which is incorporated in its entirety ).
  • RNAi can be directed against angiogenic products, such as VEGF (for example Cand5, see US2011/0143400, US2008/0188437, which are incorporated in their entirety), VEGFR1 (for example Sirna-027, see Kaiser et al.
  • VEGFR2 eg, Kou et al. (2005) Biochem. 44:15064, and incorporated in its entirety.
  • the gene product is an aptamer, such as a specific aptamer for VEGF (for example, 5'-cgcaaucagugaaugcuuauacauccg-3', see Ng et al. (2006) Nat.Rev.Drug Discovery 5:123, and Lee et al. (2005) Proc.Natl.Acad.Sci.USA 102:18902, and incorporated in its entirety), specific aptamers against PDGF (eg E10030, see Ni and Hui (2009) Ophthalmologica 223:401, and Akiyama et al. (2006) J. Cell Physiol. 207:407, incorporated in its entirety).
  • a specific aptamer for VEGF for example, 5'-cgcaaucagugaaugcuuauacauccg-3', see Ng et al. (2006) Nat.Rev.Drug Discovery 5:123, and Lee et al. (2005) Proc.Natl.Acad.Sci.USA
  • the gene product is a polypeptide.
  • the gene product is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances retinal cell function.
  • the polypeptide can enhance the function of retinal cells, for example, enhance the function of rod or cone photoreceptor cells, retinal ganglion cells, Muller cell bipolar cells, amacrine cells, horizontal cells or retinal pigment epithelial cells function.
  • the polypeptide comprises or is selected from: neuroprotective polypeptides (eg, GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (eg, soluble vascular endothelial growth factor (VEGF) receptor, anti- VEGF antibody or antigen-binding fragment thereof, endostatin (endostatin), tumor statin (tumstatin), angiostatin (angiostatin), soluble Flt polypeptide and fusion protein thereof with Fc region (see Lai et al. (2005) Mol.Ther .12:659, Pechan et al.
  • neuroprotective polypeptides eg, GDNF, CNTF, NT4, NGF, and NTN
  • anti-angiogenic polypeptides eg, soluble vascular endothelial growth factor (VEGF) receptor, anti- VEGF antibody or antigen-binding fragment thereof, endostatin (endostatin), tumor statin (tumstatin),
  • PEDF pigment epithelium-derived factor
  • TMP-3 tissue inhibitor of metalloproteinase-3
  • light response sex opsins such as rhodopsin
  • anti-apoptotic polypeptides such as Bcl-2, Bcl-Xl
  • the polypeptides include, but are not limited to: epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis protein.
  • the polypeptides include, but are not limited to: retinoschisin, retinitis pigmentosa GTPase regulator (RGPR) interacting protein-1 (GenBank Accession NO.Q96KN7, Q9EPQ2, Q9GLM3), peripheral Protein-2 (Prph2) (GenBank Accession NO.NP_000313), retinal pigment epithelium-specific protein (RPE65) (GenBank Accession NO.AAC39660).
  • RGPR retinoschisin
  • RGPR retinitis pigmentosa GTPase regulator
  • the polypeptides include, but are not limited to, polypeptides that induce choroideremia when defective or missing, such as CHM (choroidermia (Rab escort protein 1)) (Donnelly et al. (1994) Hum.Mol.
  • CHM choroidermia (Rab escort protein 1)
  • the gene product provides a site-specific endonuclease for site-specific knockdown of gene function, e.g., wherein the endonuclease knocks out an allele associated with a retinal disease .
  • a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.
  • the site-specific endonucleases are, for example, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), wherein such site-specific endonucleases are non-naturally occurring and Modified to target specific genes.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • the gene product of the present disclosure is an anti-angiogenic agent, including an anti-angiogenic polypeptide, for example, an anti-VEGF antibody or an antigen-binding fragment thereof; another example is a VEGF antagonist (such as a VEGF-A, B, C antagonist ) or PDGF antagonists.
  • an anti-angiogenic polypeptide for example, an anti-VEGF antibody or an antigen-binding fragment thereof
  • another example is a VEGF antagonist (such as a VEGF-A, B, C antagonist ) or PDGF antagonists.
  • VEGF antagonists include, but are not limited to, ranibizumab, bevacizumab, aflibercept, KH902 VEGF receptor-Fc fusion protein, 2C3 antibody, ORA102, Pegaptanib, bevasiranib, SIRNA-027, decursin, decursinol, picropodophyllin, bisabolone (guggulsterone), PLG101, eicosanoid LXA4, PTK787, pazopanib, axitinib, CDDO-Me, CDDO-Imm, shikonin, ⁇ -hydroxyiso Beta-hydroxyisovalerylshikonin, or ganglioside GM3, DC101 antibody, Mab25 antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody, 5F12 antibody, VA01 antibody, BL2 antibody, VEGF-related protein, sFLT01, sFLT02, Peptide B3, TG100801, so
  • Ranibizumab See US7,060,269 (Fig. 1 thereof) for the sequence information of Bevacizumab See US6,054,297 (Fig. 1 thereof) for the sequence information of Aflibercept See Do et al. (Br J Ophthalmol. 2009, 93: 144-9) for the sequence information of , which is hereby incorporated by reference in its entirety.
  • the VEGF antagonist comprises or is the naturally occurring protein sFlt-1, or a functional fragment thereof (e.g., sFlt-1 domain 2, see sFlt-1 sequence information in US5,861,484, sFlt-1 in US2013/0323302 sFlt-1 domain 2 sequence information, incorporated in its entirety).
  • sFlt-1 domain 2 see sFlt-1 sequence information in US5,861,484, sFlt-1 in US2013/0323302 sFlt-1 domain 2 sequence information, incorporated in its entirety.
  • the VEGF antagonist is a VEGF-binding fusion protein
  • sequence information of the VEGF-binding fusion protein in US7,635,474 is incorporated herein in its entirety.
  • the amino acid sequence of aflibercept is as shown in SEQ ID NO: 38, and a polynucleotide sequence encoding SEQ ID NO: 38 is provided, for example, a codon-optimized polynucleotide sequence , as shown in any of SEQ ID NO: 39-41.
  • polynucleotides comprise polynucleotides (regulatory sequences) of any one of the following (a)-(h) or any combination:
  • polyA polyadenylation signal
  • the AAV ITR need not have a wild-type nucleotide sequence and can be altered by insertion, deletion or substitution of nucleotides, or the AAV ITR can be derived from any of several AAV serotypes, such as AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.
  • the 5'ITR and 3'ITR are the 5'ITR and 3'ITR of AAV2.
  • the nucleotide sequence encoding the gene product is operably linked to a tissue-specific or cell type-specific regulatory element, such as a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), and For example a regulatory element that confers selective expression of an operably linked gene within a photoreceptor cell.
  • a tissue-specific or cell type-specific regulatory element such as a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), and For example a regulatory element that confers selective expression of an operably linked gene within a photoreceptor cell.
  • any combination of (a)-(h) can satisfy the gene product (for example, anti-angiogenic agent (or anti-angiogenic polypeptide), and for example, anti-VEGF antibody or its antigen-binding fragment, Arbor Functions expressed in ocular tissues (e.g., aqueous humor, retinal tissues) of a subject, for example.
  • the gene product for example, anti-angiogenic agent (or anti-angiogenic polypeptide), and for example, anti-VEGF antibody or its antigen-binding fragment, Arbor Functions expressed in ocular tissues (e.g., aqueous humor, retinal tissues) of a subject, for example.
  • the polynucleotide of any one of (a)-(h) or any combination thereof is operably linked to the aforementioned polynucleotide encoding a gene product.
  • the 5'ITR and/or 3'ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8; eg, derived from AAV2, AAV9.
  • the 5'UTR and/or 3'UTR are derived from hemopexin (HPX), ⁇ globin (hemoglobin subunit beta, HBB), HSPB1, CCL13, Xenopus globulin (Xenopus globin).
  • HPX hemopexin
  • ⁇ globin hemoglobin subunit beta, HBB
  • HSPB1 hemoglobin subunit beta, HBB
  • CCL13 Xenopus globulin
  • Xenopus globulin Xenopus globulin
  • the promoter can be a constitutive promoter or an inducible promoter.
  • the promoter is selected from the group consisting of cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, UB6 promoter, chicken ⁇ -actin promoter, CAG promoter, RPE65 promoter, CBh promoter, EFS promoter, EF1 (eg, EF-1 ⁇ ) promoter, PGK promoter, SV40 promoter, Ubi promoter, opsin promoter, or any combination thereof.
  • the opsin promoters include, but are not limited to, rhodopsin promoters, rhodopsin kinase promoters (Young et al. (2003) Ophthalmol. (2007) J. Gene Med. 9: 1015), retinitis pigmentosa gene promoter (Nicoud et al. .55:225).
  • the promoter is a CMV promoter whose sequence is shown in SEQ ID NO: 29.
  • the enhancer is selected from Ubi, CMV, RSV, IRBP gene enhancer (Nicoud et al. (2007) J. Gene Med. 9: 1015) or any combination thereof.
  • the enhancer is a CMV enhancer, the sequence of which is shown in SEQ ID NO:28.
  • the intron is selected from MVM, SV40, ⁇ Globin, EF1 (eg EF-1 ⁇ ), hybrid intron, or any combination thereof.
  • the polyA is selected from PA75polyA, SV40polyA, hGH polyA, BGH polyA, rbGlob polyA or any combination thereof.
  • polyA is SV40polyA, and its sequence is shown in SEQ ID NO:34.
  • the post-transcriptional regulatory element is selected from WPRE, HPRE or a combination thereof.
  • the post-transcriptional regulatory element is WPRE, the sequence of which is shown in SEQ ID NO: 33.
  • polynucleotides comprise polynucleotides (regulatory sequences) of any one of the following (a)-(g) or any combination:
  • sequence of polynucleotides from the 5' end and the 3' end is (optional regulatory sequences are in brackets):
  • the rAAV virions are infectious; in other embodiments, the rAAV virions are not.
  • the aforementioned rAAV virions of the present disclosure exhibit at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold enhanced infectivity to cells compared to an AAV virion comprising the corresponding parental AAV capsid protein. , at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold or more infectivity of cells selected from:
  • the aforementioned rAAV virions of the present disclosure exhibit at least a 2-fold, at least 3-fold, at least 4-fold enhancement in the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the inner limiting membrane (ILM). , at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or 50-fold more capable of passing through the ILM.
  • ILM inner limiting membrane
  • the aforementioned rAAV virions of the present disclosure selectively infect retinal cells, for example, the aforementioned rAAV virions of the present disclosure are 2 times, 3 times, 4 times, 5 times stronger than non-retinal cells (such as non-eye tissue cells) times, 10 times, 15 times, 20 times, 25 times, 50 times or more specifically infected retinal cells.
  • non-retinal cells such as non-eye tissue cells
  • the aforementioned rAAV virions of the present disclosure are more sensitive to gene products (e.g., aflibercept) than rAAV virions comprising an AAV2.7m8 capsid protein.
  • the expression level of the whole eye tissue is 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times or more than 50 times.
  • the aforementioned rAAV virions of the present disclosure are more sensitive to gene products (e.g., aflibercept) than rAAV virions comprising an AAV2.7m8 capsid protein.
  • the expression level of aqueous humor is 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times or more than 50 times.
  • the aforementioned rAAV virions of the present disclosure are more sensitive to gene products (e.g., aflibercept) than rAAV virions comprising an AAV2.7m8 capsid protein.
  • the expression in the vitreous is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold stronger.
  • the aforementioned rAAV virions of the present disclosure are more sensitive to gene products (e.g., aflibercept) than rAAV virions comprising an AAV2.7m8 capsid protein.
  • the expression level in retinal tissue is 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times or more than 50 times stronger.
  • the rAAV virions are administered via intravitreal injection.
  • the present disclosure provides a polynucleotide encoding aflibercept, and its encoded amino acid sequence is shown in SEQ ID NO: 38.
  • the present disclosure provides a codon-optimized polynucleotide encoding aflibercept, as set forth in any of SEQ ID NO: 39-41 or at least 80%, 85%, 90% identical thereto %, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the present disclosure provides polynucleotides encoding any of the gene products of the foregoing disclosure.
  • the present disclosure provides polynucleotides encoding any of the aforementioned AAV capsid proteins of the present disclosure.
  • the aforementioned polynucleotide may be RNA, DNA or cDNA.
  • the aforementioned polynucleotides are isolated polynucleotides.
  • a polynucleotide of the present disclosure may also be in the form of, may be present in, and/or may be part of a vector, such as a plasmid, cosmid, YAC, or viral vector.
  • a vector may, for example, be an expression vector, ie a vector which provides for expression of a polynucleotide in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.).
  • polynucleotides of the present disclosure may be prepared or obtained by known means (eg, by automated DNA synthesis and/or recombinant DNA techniques), and/or may be isolated from suitable natural sources.
  • the present disclosure provides a vector comprising:
  • an isolated polynucleotide encoding any of the aforementioned variant AAV capsid proteins of the present disclosure (as shown in any of SEQ ID NOs: 3-9 and 23-27 or at least 90% or 95% identical thereto and/or the aforementioned heterologous polynucleotides encoding gene products (such as the polynucleotide encoding Aflibercept (aflibercept) shown in SEQ ID NO: 38, any of SEQ ID NOs: 39-41 A polynucleotide indicated or having at least 90% or 95% sequence identity thereto).
  • the polynucleotide encoding the variant AAV capsid protein of (b) and the heterologous polynucleotide encoding the gene product are in different vectors.
  • the present disclosure provides host cells comprising any of the aforementioned polynucleotides or (expression) vectors of the present disclosure.
  • the host cell may be an isolated cell, such as a cell from in vitro cell culture. Such cells are used to produce any of the aforementioned rAAV capsid proteins, gene products or rAAV virions of the present disclosure, also known as production cells.
  • the producer cells are bacterial cells, fungal cells or mammalian cells.
  • Exemplary producer cells include, but are not limited to, HeLa, CHO, 293 (including 293T), Vero, NIH 3T3, Huh-7, BHK, PC12, COS (including COS-7), RAT1, HepG2 cells, and the like.
  • Exemplary mammalian cells include, but are not limited to, 293 (293T), COS, HeLa, Vero, 3T3, C3H10T1/2, CHO cells.
  • As the production cells amphibian cells, insect cells, plant cells and any other cells used to express proteins and virus particles in the art can also be used.
  • Exemplary insect cells include, but are not limited to, Spodoptera frugiperda, Drosophila cell lines, or mosquito cell lines, such as Aedes albopictus derived cell lines, including but not limited to Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, Ha2302, Hz2E5, HighFive (Invitrogen, CA, USA), AO38 and BM-N cells.
  • Spodoptera frugiperda Drosophila cell lines
  • mosquito cell lines such as Aedes albopictus derived cell lines, including but not limited to Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, Ha2302, Hz2E5, HighFive (Invitrogen, CA, USA), AO38 and BM-N cells.
  • rAAV virions Methods of producing rAAV virions are routine in the art. This disclosure introduces the production and preparation methods of rAAV virus particles in WO200028004, WO200123001, WO2004112727, WO2005005610, WO2005072364, WO2013123503, WO2015191508 and US20130195801.
  • the rAAV virions may have properties that enhance delivery efficiency, enable efficient packaging, and successfully infect target cells (eg, mammalian or human cells) with high frequency and minimal toxicity.
  • the present disclosure provides methods for producing and preparing rAAV virions, comprising packaging any polynucleotide of the present disclosure into an AAV capsid.
  • a method for producing and preparing rAAV virions comprising: the polynucleotide encoding a gene product of the foregoing disclosure or its (expression) vector, the polynucleotide encoding any AAV capsid protein of the foregoing disclosure or The (expression) vector, and helper function plasmid (for example, pHelper), are introduced into production cells (for example, 293 cells), packaged and purified to obtain rAAV virus particles.
  • helper function plasmid for example, pHelper
  • a production method for producing and preparing rAAV virions comprising:
  • the vectors expressing Rep and Cap genes contain polynucleotides encoding any AAV capsid protein of the foregoing disclosure;
  • an rAAV virion production system for producing any of the aforementioned rAAV virions of the present disclosure, comprising:
  • any heterologous polynucleotide of the present disclosure comprising a gene product encoding gene product or its (expression) vector (eg, pGOI plasmid);
  • sufficient AAV rep functions and auxiliary functions are provided by packaging cells or the three plasmids pHelper, pR2C9, and pGOI, and the packaging cells may contain the three plasmids pHelper, pR2C9, and pGOI.
  • the Rep gene encodes a non-structural protein that regulates functions, such as replication of the AAV genome, and can be selected from Rep78, Rep68, Rep52, and Rep40.
  • Rep78 and Rep68 are usually transcribed from the p5 promoter, while Rep52 and Rep40 are usually transcribed from the p19 promoter.
  • the Cap gene encodes the structural proteins VP1, VP2 and/or VP3 that assemble to form the capsid capsid. Cap genes are usually transcribed from the p40 promoter.
  • rAAV virions produced by the AAV production method or production system described above are provided.
  • the active ingredient is for example selected from: any rAAV virion of the aforementioned disclosure, encoding SEQ ID NO: 39-41 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% % identity sequence to the polynucleotide of the gene product.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of polynucleotides (such as VEGF inhibitors, and polynucleotides encoding aflibercept) or rAAV virus particles.
  • the unit dose of the pharmaceutical composition contains 0.1 to 10 ⁇ 10 13 copies of gene products (such as VEGF inhibitors, such as aflibercept).
  • the concentration of rAAV virions in the pharmaceutical composition is 1 ⁇ 10 8 per milliliter or more, usually no more than 1 ⁇ 10 15 per milliliter.
  • cells can be transfected with any of the aforementioned rAAV virions of the present disclosure, and the cells can then be transferred or transplanted into a subject.
  • the pharmaceutical composition contains any rAAV virion of the present disclosure, and the rAAV virion is encapsulated with a polynucleotide, and the polynucleotide is shown in any one of SEQ ID NO: 39-41 or is related thereto. Sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity.
  • the present disclosure provides methods or uses of any AAV capsid protein, rAAV virion, pharmaceutical composition, or polynucleotide encoding aflibercept of the foregoing disclosure for treating, alleviating or improving diseases or symptoms.
  • a method of delivering a gene product to a subject in need comprising administering to the subject an effective amount of any of the aforementioned AAV capsid proteins, rAAV virions, pharmaceutical compositions, encoding AAV A polynucleotide of aflibercept.
  • a method of delivering a gene product to a target cell comprising combining said target cell with any of the aforementioned AAV capsid proteins of the present disclosure, rAAV virions, pharmaceutical compositions, aflibercept-encoding polynucleotide contacts.
  • the target cells are selected from hepatocytes, pancreatic cells, skeletal muscle cells, cardiomyocytes, fibroblasts, retinal cells, synovial joint cells, lung cells, T cells, neurons, glial cells, stem cells , endothelial cells or cancer cells.
  • the target cells are in vitro; in other embodiments, the target cells are in vivo.
  • a method for specifically infecting retinal cells comprising intraocularly administering a prophylactically or therapeutically effective amount of any of the aforementioned AAV capsid proteins of the present disclosure, rAAV virions, pharmaceutical compositions, encoding aflibercept (aflibercept) ), for example by intravitreal injection or subretinal injection.
  • aflibercept aflibercept
  • it provides the pharmaceutical application of any AAV capsid protein, rAAV virion, pharmaceutical composition, and polynucleotide encoding aflibercept in the present disclosure to prepare a medicament for specifically infecting retinal cells.
  • any AAV capsid protein, rAAV virion, pharmaceutical composition, or polynucleotide encoding aflibercept of the foregoing disclosure is provided for treating retinal cells selected from the group consisting of: Disease or condition: photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells.
  • the retinal cells are photoreceptor cells, such as rods or cones.
  • any AAV capsid protein, rAAV virion, pharmaceutical composition, or polynucleotide encoding aflibercept of the foregoing disclosure is provided for treating retinal cells selected from the group consisting of: Diseases or Conditions: Acute macular neuroretinopathy; Behcet's disease; Choroidal neovascularization; Diabetic uveitis; Histoplasmosis; Macular degenerations such as acute macular degeneration, non-exudative age-related macular age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma, which affects a point or location at the back of the eye; Central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease
  • any of the AAV capsid proteins, rAAV virions, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure are provided for the treatment of ocular diseases.
  • Internal diseases include, but are not limited to: age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion , branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, or diabetic retinal edema.
  • AMD age-related macular degeneration
  • wet AMD dry AMD
  • retinal neovascularization choroidal neovascularization
  • diabetic retinopathy proliferative diabetic retinopathy
  • retinal vein occlusion central retinal vein occlusion
  • any AAV capsid protein, rAAV virion, pharmaceutical composition, or polynucleotide encoding aflibercept of the present disclosure for treating retinal-related diseases are provided.
  • the present disclosure provides any of the aforementioned AAV capsid proteins, rAAV virions, pharmaceutical compositions, and polynucleotides encoding aflibercept for the treatment of the aforementioned eye diseases (such as retina-related diseases) , which is administered by intraocular injection.
  • intraocular injection includes by intravitreal injection, by subretinal injection, by suprachoroidal injection, or by any other convenient mode or route of administration that will result in delivery of rAAV virions to the eye.
  • Other convenient modes or routes of administration include, but are not limited to, intravenous, intraarterial, periocular, intracameral, subconjunctival and subballoon injections and topical and intranasal administration.
  • Fig. 1 is a schematic diagram of the AAV2 capsid protein sequence, the starting sites of VP1, VP2, and VP3, and the loop domains I-V.
  • Fig. 2 is a schematic diagram of a variant AAV capsid protein of the present disclosure with a peptide segment inserted between positions 587 and 588, with a mutation at position 708.
  • Fig. 3 is a graph showing the fluorescent signal detection results of rAAV vector viruses AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV2 seq5, AAV2 seq6 and AAV2, AAV2.7m8 of the present disclosure infected mouse retina.
  • Figure 4A and Figure 4B are the detection results of retinal fluorescence signals of mice infected with rAAV vector viruses AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV9 seq2 and AAV2, AAV2.7m8 of the present disclosure
  • Figure 4A is the ophthalmoscopy of the first week Test results
  • Fig. 4B is the results of ophthalmoscope test in the fourth week.
  • Figure 5A is a schematic diagram of the vector structure containing a nucleic acid molecule expressing VEGF Trap (aflibercept);
  • Figure 5B is a schematic diagram of the AAV packaging plasmid.
  • Figure 6 shows the detection results of rAAV vector virus AAV2 seq2 and AAV2.7m8 expressing the target gene (aflibercept) in mouse eyeballs.
  • Figure 7 shows the detection results of rAAV vector virus AAV2 seq2 and AAV 2.7m8 expressing the target gene protein in rabbit eyeballs, which is the detection of the target gene (aflibercept) protein content in aqueous humor 14 days after virus injection.
  • Figure 8 shows the detection results of the rAAV vector virus AAV2 seq2 and AAV 2.7m8 expressing the target gene protein in the rabbit eyeball, which is the detection of the molar concentration of the target gene protein in the aqueous humor 28 days after the virus injection.
  • AAV is an acronym for Adeno-Associated Virus, and may be used to refer to the virus itself or its derivatives. Unless otherwise indicated, this covers all AAV subtypes as well as naturally occurring and recombinant forms.
  • AAV includes, but is not limited to, AAV Type 1 (AAV1), AAV Type 2 (AAV2), AAV Type 3 (AAV3), AAV Type 4 (AAV4), AAV Type 5 (AAV5), AAV Type 6 (AAV6), AAV AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAVrh10 type (AAVrh10), and different species of avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV.
  • AAV is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid containing inverted terminal repeats (ITRs) flanked by inverted terminal repeats (ITRs) that serve as viral origins of replication and packaging signals.
  • Three open reading frames ORFs).
  • the Rep ORF encodes four nonstructural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly.
  • the Cap ORF encodes three structural proteins (VP1-3) that assemble to form the 60-mer viral capsid.
  • the ORF present as an alternative reading frame within the cap gene generates assembly activating protein (AAP), a viral protein that localizes the AAV capsid protein in the nucleus and plays a role in capsid assembly.
  • AAP assembly activating protein
  • NC_002077.1 AAV1
  • AF063497.1 AAV1
  • NC_001401.2 AAV2
  • AF043303.1 AAV2
  • J01901.1 AAV2
  • U48704.1 AAV3
  • NC_001729.1 AAV3
  • NC_001829.1 AAV4
  • U89790.1 AAV4
  • NC_006152.1 AAV5
  • AF085716.1 AAV5
  • AF028704.1 AAV6
  • NC_006260.1 AAV7
  • AF513851.1 AAV7
  • AF513852.1 AAV8
  • NC_006261.1 AAV8
  • AY530579.1 AAV9
  • AAV virion or “AAV virion” refers to a virion composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.
  • rAAV refers to recombinant adeno-associated virus
  • recombinant applied to polynucleotides means that the polynucleotides are the product of various combinations of cloning, restriction, or ligation steps, and result in polynucleotides different from those found in nature. Other processes for acidic constructs.
  • a recombinant virus is a viral particle that includes a recombinant polynucleotide.
  • an "AAV virion” includes heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as gene products to be delivered to target cells (e.g., transgenes, RNAi, etc.), it is often referred to as "recombinant AAV (rAAV) virion” or “rAAV virion” or "rAAV vector virus”.
  • rAAV recombinant AAV
  • rAAV virion or "rAAV virion” or "rAAV vector virus”.
  • the heterologous polynucleotide is flanked by at least one, and usually two, AAV inverted terminal repeats (ITRs).
  • rAAV vector encompasses rAAV virions comprising rAAV polynucleotides; and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotide encoding rAAV (ss-rAAV); encoding rAAV double-stranded polynucleotide (ds-rAAV), such as a plasmid encoding rAAV; etc.).
  • ss-rAAV single-stranded polynucleotide encoding rAAV
  • ds-rAAV double-stranded polynucleotide
  • AAV variant refers to a viral particle consisting of: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein is relative to the corresponding parent
  • the AAV capsid protein comprises at least one amino acid difference (e.g., an amino acid substitution, insertion, or deletion), wherein the AAV capsid protein differs from or does not correspond to the amino acid sequence of a naturally occurring AAV capsid protein; and optionally, (b) includes an amino acid sequence encoding A heterologous polynucleotide of a heterologous gene product, wherein the variant AAV capsid protein confers increased binding to a heparan or heparan sulfate proteoglycan compared to binding to an AAV virion comprising the corresponding parental AAV capsid protein .
  • Packaging refers to the series of intracellular events that lead to assembly and encapsidation of AAV particles.
  • the "rep” and “cap” genes refer to the polynucleotide sequences encoding the replication and capsidation proteins of the adeno-associated virus.
  • AAV rep and cap are referred to herein as AAV "packaging genes”.
  • Helper virus refers to a virus that allows AAV (eg, wild-type AAV) to be replicated and packaged by mammalian cells.
  • Various helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia.
  • Adenoviruses cover many different subgroups, but the most commonly used is adenovirus type 5 in subgroup C; viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV) and cytomegalovirus (CMV ) and pseudorabies virus (PRV).
  • HSV herpes simplex virus
  • EBV Epstein-Barr virus
  • CMV cytomegalovirus
  • PRV pseudorabies virus
  • Many adenoviruses, herpesviruses, etc. of human, non-human mammalian and avian origin are known and available from institutions such as the ATCC.
  • Helper virus function or “helper function” refers to the functions encoded in the helper virus genome that allow AAV replication and packaging (in combination with other requirements for replication and packaging described herein).
  • helper virus function can be provided in a number of ways, including by providing a helper virus or providing, for example, a polynucleotide sequence encoding the necessary function to the producer cell in transit.
  • infectious virus or virus particle is one that comprises a suitably assembled viral capsid and is capable of delivering polynucleotide components into cells for which the virus species has tropism, not necessarily implying any replication competence of the virus.
  • Assays for enumerating infectious viral particles are known in the art. Viral infectivity can be expressed as the ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See eg Grainger et al. (2005) Mol. Ther. 11:S337 (describing the TCID50 infectious titer assay); Zolotukhin et al. (1999) Gene Ther. 6:973 .
  • Tropism refers to the preferential targeting of a virus (eg, AAV) to cells of a particular host species or to particular cell types within a host species.
  • a virus eg, AAV
  • a virus that can infect heart, lung, liver, and muscle cells has a broader (ie, increased) tropism relative to a virus that can infect only lung and muscle cells.
  • Tropism can also encompass the dependence of the virus on specific types of cell surface molecules of the host. For example, some viruses may only infect cells with surface glycosaminoglycans, while others may only infect cells with sialic acid (this dependence allows the use of various cell lines lacking specific classes of molecules as potential host cells for viral infection carry out testing).
  • viral tropism describes the relative preferences of viruses.
  • the first virus may be able to infect all cell types but be more successful in infecting these cells with surface glycosaminoglycans.
  • the second virus also prefers the same characteristics (for example, the second virus is also more successful in infecting these cells with surface glycosaminoglycans)
  • the second virus can be considered to have similar (or consistent) tropism even though the absolute transduction efficiencies were not similar.
  • a second virus may be more effective than the first at infecting a given cell type of each tested, but if the relative preferences are similar (or consistent), the second virus may still be considered to have Similar (or identical) tropism of viruses.
  • the tropism of a virion comprising a variant AAV capsid protein of the disclosure is not altered relative to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a variant AAV capsid protein of the disclosure is extended (ie, broadened) relative to a naturally occurring virion. In some embodiments, virions comprising variant AAV capsid proteins of the disclosure have reduced tropism relative to naturally occurring virions.
  • Polynucleotide means a polymeric form of nucleotides of any length, or analogs thereof, including deoxyribonucleotides or ribonucleotides.
  • a polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure can be performed before or after the assembly of the polymer.
  • Polynucleotides refer interchangeably to double-stranded molecules and single-stranded molecules, and unless otherwise indicated, polynucleotides of the present disclosure encompass the double-stranded form as well as the two complementary single-stranded forms that make up the double-stranded form.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both compared sequences is occupied by the same nucleotide or subunit of an amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules at that position are Homologous.
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned.
  • Sequence similarity can be determined in a number of different ways. To determine sequence identity, methods and computer programs can be used to align sequences, including BLAST, available on the Internet at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG) package of Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment techniques are described in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), Doolittle ed., Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that tolerate gaps in sequences.
  • GCG Genetics Computing Group
  • Gene refers to a polynucleotide containing at least one open reading frame capable of encoding, upon transcription and sometimes translation, a specific gene product.
  • Gene or coding sequence refers to an in vitro or in vivo nucleotide sequence that encodes a gene product.
  • a gene consists or consists essentially of a coding sequence, ie, a sequence that encodes a gene product.
  • the gene includes additional non-coding sequences.
  • a gene may or may not contain regions preceding and following the coding region (eg, 5'UTR, 3'UTR, and intervening sequences (introns) between individual coding segments (exons)).
  • a “gene product” is a molecule, such as a polypeptide, aptamer, interfering RNA, mRNA, etc., resulting from the expression of a specific gene.
  • a “gene product” is a polypeptide, peptide, protein, or interfering RNA, including short interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA).
  • the gene product is a therapeutic gene product, such as a therapeutic polypeptide.
  • the therapeutic gene product confers a beneficial effect on the cell, tissue or mammal in which it is located, and the beneficial effect includes improving the sign or symptom of the condition or disease, preventing or inhibiting the condition or disease, or conferring a desired characteristic.
  • “Gene product” and “product of gene expression of interest” are used interchangeably when the gene encodes a polypeptide.
  • RNA interfering agent or “RNAi agent” includes any agent (or a polynucleotide encoding such an agent) that can be used to alter the expression of a gene (as defined above).
  • RNAi agents known to those of ordinary skill in the art include, but are not limited to, (i) siRNA agents ("small interfering” or “short interfering RNA” (or siRNA)); (ii) antisense RNA; (iii) CRISPR agents (iv) zinc finger nuclease reagents; and (v) transcription activator-like effector nuclease (TALEN) reagents.
  • siRNA agents are RNA duplexes targeting nucleotides of a gene of interest ("target gene").
  • RNA duplex refers to the structure formed by the complementary pairing between two regions of an RNA molecule, forming a region of double-stranded RNA (dsRNA).
  • dsRNA double-stranded RNA
  • An siRNA targets" a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to that of the target gene.
  • the duplex length of the siRNA is less than 30 nucleotides.
  • the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length.
  • the duplex is 19-25 nucleotides in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure.
  • siRNA reagents containing hairpins may also be referred to as "shRNA (short hairpin RNA) reagents".
  • the hairpin structure may also contain a loop portion located between the two sequences forming the duplex.
  • the loops may vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length.
  • the hairpin structure may also contain a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length.
  • the level of expression products (e.g., mRNA, polypeptide, etc.) of a target gene is reduced by siRNA reagents (e.g., siRNA, shRNA, etc.)
  • siRNA reagents e.g., siRNA, shRNA, etc.
  • a segment of nucleotide length eg, 20-21 nucleotide sequences
  • the short interfering RNA is about 19-25 nt in length.
  • siRNA and/or shRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.
  • the nucleic acid sequence may also include a polyadenylation signal.
  • the polyadenylation signal is a synthetic minimal polyadenylation signal.
  • antisense RNA is RNA complementary to gene expression product.
  • an antisense RNA that targets a specific mRNA is an RNA-based agent that is complementary to the mRNA (or can be a modified RNA), wherein hybridization of the antisense RNA to the mRNA alters the expression of the mRNA (e.g., by altering the stability of the RNA). sex, altering the translation of RNA, etc.).
  • Nucleic acids encoding antisense RNA are also included in "antisense RNA”.
  • VEGF refers to vascular endothelial growth factor that induces angiogenesis or the angiogenic process, including various isoforms of VEGF (also known as vascular permeability factor (VPF) produced by, for example, alternative splicing of the VEGF-A/VPF gene and VEGF-A) (see Figure 2(A) and (B) of US Patent Application Publication No. 20120100136), comprising VEGF121, VEGF165 and VEGF189.
  • VPF vascular permeability factor
  • VEGF includes VEGF-related angiogenic factors, such as PIGF (placental growth factor), VEGF-B, VEGF-C, VEGF-D, and VEGF-E, which pass through the cognate VEFG receptor (ie VEGFR) function to induce angiogenesis or the process of angiogenesis.
  • PIGF placental growth factor
  • VEGF-B vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • VEGF-D vascular endothelial growth factor
  • VEGF-E VEGF-related angiogenic factors
  • sFlt-1 or “sFlt-1 protein” herein refers to a sequence that is at least 90% or more homologous to a naturally occurring human sFLT-1 sequence such that the sFlt-1 protein or polypeptide binds to VEGF and/or a VEGF receptor Polypeptide sequences or functional fragments thereof.
  • Polypeptide refers to polymers of amino acids of any length, and also include polymers of amino acids that are modified, such as disulfide bond formation, glycosylation, lipidation, phosphorylation, or with labeling components conjugate.
  • a “regulatory element” or “regulatory sequence” is a nucleotide sequence that participates in molecular interactions that contribute to the regulation of the function of a polynucleotide, including replication, repetition, transcription, splicing, translation of the polynucleotide or degradation. Regulation can affect the frequency, speed or specificity of a process and can be enhancing or inhibitory in nature.
  • Known control elements include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a region of DNA that is capable, under certain conditions, of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) of the promoter.
  • an "expression vector” is a vector that includes a region encoding a gene product of interest and is used to effect expression of the gene product in an intended target cell, the vector comprising a polynucleotide encoding the gene product of interest.
  • Expression vectors also include control elements operably linked to the coding region to facilitate expression of the gene product in the target.
  • control elements such as promoters, enhancers, UTRs, miRNA targeting sequences, etc., and the gene or genes operably linked thereto for expression is sometimes referred to as an "expression cassette.”
  • Many expression cassettes are known and available in the art, or can be readily constructed from components available in the art.
  • “Operatively linked” or “operably linked” refers to the juxtaposition of genetic elements, wherein the elements are in a relationship permitting their operation in an intended manner.
  • a promoter is operably linked to an encoding polynucleotide sequence if it facilitates the initiation of transcription of the encoding polynucleotide sequence, there may be intervening nucleic acid residues between the promoter and the encoding polynucleotide sequence, so long as this functional relationship.
  • administering refers to delivering a vector for recombinant gene or protein expression to a cell, a cell and/or an organ of a subject or a subject. Such administration or introduction can occur in vivo, in vitro or ex vivo.
  • Vectors for expression of gene products can be introduced into cells by transfection, which generally means insertion of heterologous DNA into In a cell; infection, which usually refers to the introduction by an infectious agent, ie, a virus; or transduction, which usually means the stable infection of a cell with a cell or the transfer of genetic material from one microorganism to another by a viral agent such as a bacteriophage .
  • Transformation is generally used to refer to bacteria comprising heterologous DNA or cells expressing oncogenes and which have switched to a continuous mode of growth, such as tumor cells.
  • the vector used to "transform” cells may be a plasmid, virus or other vehicle.
  • a cell is often referred to as “transduced,” “infected,” “transfected,” or “transformed,” depending on the means used to administer, introduce, or insert heterologous DNA (ie, a vector) into the cell.
  • Transduction”, “transfection” and “transformation” are used interchangeably herein, regardless of the method of introduction of the heterologous DNA.
  • “Host cell” refers to a cell that has been transduced, infected, transfected or transformed with a vector and encompasses the originally transduced, infected, transfected or transformed cell and its progeny.
  • Vectors can be plasmids, virus particles, phage, and the like. Culture conditions such as temperature, pH, etc. will be apparent to those skilled in the art.
  • Treatment is generally used to mean obtaining a desired pharmacological and/or physiological effect.
  • the effect may be preventive in terms of completely or partially preventing the disease or its symptoms, such as reducing the likelihood of the disease or its symptoms occurring in the subject, and/or in partially or completely curing the disease and/or adverse effects caused by the disease. Aspects of the response can be therapeutic.
  • Treatment covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not been diagnosed as having the disease; (b) inhibiting or halting the development of the disease or (c) amelioration of the disease (or the symptoms it causes) or regression of the disease.
  • Therapeutic agents can be administered before, during, or after the onset of the disease or injury.
  • the treatment of an ongoing disease wherein the treatment stabilizes or reduces undesired clinical symptoms in the patient. It is preferred that such treatment be performed prior to complete loss of function of the affected tissue.
  • the treatments of the present disclosure will be administered during, and in some cases after, the symptomatic phase of the disease.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Retinal cells in this disclosure may refer to any of the cell types comprising the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; photoreceptor cells including rods and cones; Müller glial cells; astrocytes (eg, retinal astrocytes); and retinal pigment epithelial cells.
  • “Individual”, “subject” and “patient” are used interchangeably herein and include, but are not limited to, humans and non-human primates such as apes, humans and other mammals (e.g., horses, sheep, Goats, dogs, cats, and rodents (such as mice, rats, etc.)), preferably humans.
  • mammals e.g., horses, sheep, Goats, dogs, cats, and rodents (such as mice, rats, etc.)
  • rodents such as mice, rats, etc.
  • polypeptide and “protein” are used interchangeably.
  • Embodiment 1 AAV capsid transformation
  • the heparan sulfate receptor is the most important receptor that mediates AAV cell infection, and also plays an important role in the infection of retinal and optic nerve cells.
  • the amino acid sequence of AAV2 natural serotype capsid protein is in accordance with its space Structural features, there are 5 ring domains (shown in Figure 1) related to the infectivity of the virus. Among them, inserting a short peptide of 7-12 amino acids in the middle of the 587th and 588th positions of the ring domain IV will not affect the assembly of the viral capsid protein, and can greatly change the AAV capsid and cell receptor (acetyl sulfate Heparin), thus affecting the ability of AAV to infect host cells.
  • This disclosure uses a self-developed bioinformatics algorithm to insert several random amino acid sequences (7-12 amino acid length) between the 587th and 588th positions in the amino acid sequence of the AAV2 natural serotype capsid protein, and predict the capsid protein Interaction with heparan sulfate receptors. Select the sequence predicted by the algorithm that can greatly improve the infection ability for subsequent verification. According to the algorithm prediction, 6 amino acid sequences seq1 to seq6 as shown in Table 1 were selected, and the length of each sequence was 10 amino acids. At the same time, the 708th amino acid-valine (V) in the amino acid sequence of the AAV2 natural serotype capsid protein is replaced with isoleucine (I).
  • the AAV capsid protein Since the AAV capsid protein is expressed, it is composed of three monomeric proteins, VP1, VP2, and VP3, assembled in a certain proportion, and has a higher-level structure than ordinary recombinant proteins.
  • the functional impact caused by polypeptide insertion is not limited to the changes in physical and chemical properties, and it is more likely to exert more complex effects on the assembly process of the three monomeric proteins VP1, VP2, and VP3.
  • capsids with 8 sequences from SEQ ID NO: 3 to SEQ ID NO: 8, AAV2 (SEQ ID NO: 1), AAV2.7m8 (SEQ ID NO: 2), and package the plasmid pGOI respectively , the plasmid contains and can express the EGFP target gene driven by the CMV promoter.
  • AAV vectors with EGFP expression ability packaged in different AAV capsids were injected into the vitreous of mice at equal doses, and the expression of EGFP was detected by fluorescent fundus photography. Differences in tissue infection efficiency.
  • the packaging and purification steps of the rAAV vector virus include: transfecting 293T adherent cells with pHelper, pR2C9, and pGOI three-plasmid system, harvesting the cells and supernatant, purifying by iodixanol centrifugation, and then concentrating by ultrafiltration. The solution was replaced with DPBS. After the virus packaging and purification are completed, the titer (in vg) is detected by qPCR.
  • viruses expressing the EGFP target gene were diluted with DPBS buffer to the same titer of 1.03 E+12.
  • the 10-week-old C57 mice purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd.
  • intravital fluorescein fundus photography was performed every week.
  • AAV9 seq2 was obtained by inserting the "LALGDTTRPA" sequence in Table 1 into the same position (between positions 588 and 589) in the AAV9 capsid. Seven kinds of capsids, including AAV2, AAV2.7m8, AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, and AAV9 seq2, were packaged into plasmid vectors, which contained and could express the EGFP target gene driven by the CMV promoter.
  • AAV vectors with EGFP expression ability packaged in different AAV capsids were injected into the vitreous of mice at equal doses, and the expression of EGFP was detected by fluorescent fundus photography, and the effect of different AAV capsids on retinal tissue was evaluated by the difference in the intensity of EGFP signals. Differences in infection efficiency.
  • AAV2 seq2 and AAV2.7m8 were respectively packaged with a CMV promoter-driven aflibercept target plasmid vector (AAV2 seq2- aflibercept, AAV2.7m8-aflibercept), the two viruses were injected into the vitreous of New Zealand rabbits and C57 mice, and the content of aflibercept protein in the injected eye tissue was detected two weeks after the injection, so as to evaluate the effect of the corresponding AAV virus on the retina infection and its associated biological functions.
  • AAV2 seq2- aflibercept AAV2.7m8-aflibercept
  • the structure of the gene expression cassette delivered by rAAV contains from the 5' end to the 3' end (as shown in Figure 5A): CMV enhancer, promoter, 5'UTR, Kozak sequence (gccacc), VEGF inhibitor Pu, 3'UTR, WPRE and SV40polyA.
  • CMV enhancer is shown in SEQ ID NO: 28
  • base sequence of the CMV promoter is shown in SEQ ID NO: 29
  • the 5'UTR is shown in SEQ ID NO: 30
  • the Kozak sequence is shown in SEQ ID NO: 31, 3'UTR as SEQ ID NO: 32, WPRE as SEQ ID NO: 33, SV40polyA as SEQ ID NO: 34.
  • the construction of the expression vector is as follows: constructing the expression cassette, and constructing the AAV packaging plasmid expressing aflibercept through conventional molecular biology operations such as restriction enzyme digestion, ligation, transformation and clone screening identification, which in turn include: CMV enhancer, promoter, 5'UTR, Kozak sequence (gccacc), VEGF trap (Aflibercept), 3'UTR, WPRE and SV40polyA, the expression cassette is flanked by inverted terminal repeats (ITR), the schematic diagram of its structure is shown in Figure 5B, EcoRV and BSMI are enzyme cutting sites.
  • the vector was packaged and the rAAV vector virus was purified according to the method in Example 2.
  • Two weeks after the injection the mice were sacrificed, and eye tissues were collected and ground separately.
  • the left and right eyes of each injection group were subjected to tissue grinding by two different methods. Method 1 for the left eye: grind with PBS+protease inhibitors, centrifuge and take the supernatant.
  • Method 2 for the right eye grind with RIP Buffer + protease inhibitors, centrifuge and take the supernatant.
  • the aflibercept protein content in the supernatant was detected by enzyme-linked immunoassay (4 samples per injection group, and each sample was tested in triplicate). The result is shown in Figure 6.
  • the blank control is the PBS injection group, and the ordinate HRAMD/total protein is the number of ng of aflibercept protein contained in each mg of total tissue protein.
  • Figure 6 and Table 2 show that the expression level of AAV seq2-aflibercept group in mice is higher than that of AAV2.7m8-aflibercept group.
  • the vector was packaged and the rAAV vector virus was purified according to the method in Example 2.
  • Method 1 for the left eye grind with PBS+protease inhibitors, centrifuge and take the supernatant.
  • Method 2 for the right eye grind with RIP Buffer + protease inhibitors, centrifuge and take the supernatant.
  • the aflibercept protein content in the supernatant was detected by enzyme-linked immunoassay (2 samples per injection group, and each sample was tested in triplicate). The result is shown in Figure 7.
  • the blank control is the PBS injection group, and the ordinate HRAMD/total protein is the number of ng of aflibercept protein contained in each mg of total tissue protein.
  • the results in Figure 7 show that the expression levels of the AAV seq2-aflibercept group in New Zealand rabbits were higher than those of the AAV2.7m8-aflibercept group. Due to the large size of the rabbit eye, aflibercept is diluted in the interstitial fluid after the whole eye grinding process, so the concentration is much lower than that in the vitreous and aqueous humor. Since the AAV packaged vector can only successfully express the target gene protein carried by the AAV virus after successfully infecting the cells, the results in Figure 7 show that the infection efficiency of the AAV2 seq2 capsid is higher than that of the AAV2.7m8 capsid.
  • AAV2 seq2 and AAV2.7m8 capsid delivery target gene protein VEGF inhibitor
  • the experimental method is as follows: 8 New Zealand rabbits were injected into both eyes according to the dose in Table 3, and 2 rabbits were injected into each dose group, with four eyes, totally 4 groups. On the 28th day after the injection, the experimental animals were killed and the aqueous humor was extracted, and the molar concentration of the target gene protein in the aqueous humor sample was detected by ELISA method.

Abstract

提供了衣壳变异的重组腺相关病毒及其应用。具体地，提供了变体腺相关病毒（AAV）衣壳蛋白，包含所述变体AAV衣壳蛋白的重组腺相关病毒（rAAV）病毒粒子及其靶向细胞（例如，视网膜细胞）递送基因产物的应用。

Description

衣壳变异的重组腺相关病毒及其应用
本申请要求2021年05月28日提交的申请号为202110594986.X的中国专利申请的优先权。
技术领域
本公开涉及重组腺相关病毒(rAAV)技术领域，具体涉及一种衣壳变异的rAAV病毒粒子及其向靶细胞(例如，视网膜细胞)递送基因产物的应用。
背景技术
腺相关病毒(AAV)属于细小病毒科(Parvoviridae)的依赖病毒属(Dependovirus)，是小的(25nm)、无包膜的单链DNA病毒，核酸被二十面体衣壳(cap)所包裹。AAV含有两个开放阅读框rep和cap，rep用于编码基因组复制所必需的四个蛋白(Rep78、Rep68、Rep52、和Rep40)，cap用于编码病毒衣壳装配所需的三个结构蛋白(VP1-3)。
AAV作为载体，表现出对于遗传病、基因病的治疗的巨大潜力，可用于基因补充疗法(也称为基因增强疗法)。其通过补足缺失的基因功能来恢复由突变导致的基因功能丧失或失调，从而使目标细胞的生物学功能恢复到正常的生理状态。已有研究显示，基于AAV的载体在临床前疾病模型及在人临床试验中的表现，显示出若干疾病治疗的应用的前景，例如，可以实现将基因高效转移到视网膜细胞中并长期持续地进行表达(Boye et al.Mol Ther.2013 Mar；21(3)：509-19.Trapani et al.Prog Retin Eye Res.2014 Nov；43：108-28.)。目前用于临床的AAV载体经过基因工程改造，无辅助病毒时，呈潜伏状态，其安全性和转基因的长期表达已在啮齿类动物模型、非人灵长类动物和多项人类试验中被广泛测试(MacLaren et al.Lancet.2014 Mar 29；383(9923)：1129-37.；Maguire et al.N Engl J Med.2008 May 22；358(21)：2240-8；Simonelli et al.Mol Ther.2010 Mar；18(3)：643-50；Nathwani et al.N Engl J Med.2014 Nov 20；371(21)：1994-2004)。
AAV衣壳蛋白在组成和结构上有不同的天然存在，不同的衣壳具有不同的组织噬性。已经鉴别出了多个同源的灵长类AAV血清型和非人灵长类AAV血清型，开发了不同的工程化改造的AAV变体(也称AAV血清型)。虽然AAV载体在不同生物体和组织中均表现出一定程度的扩散感染，但多数临床试验中均采用病灶区局部注射的方式，尤其是在眼部基因治疗的应用。目前主要的眼部临床实施方式为视网膜下腔注射(即，在RPE和感光之间注射液体后形成的空腔)和玻璃体腔注射。视网膜下腔注射使得AAV对RPE和感光细胞得到充分接触，局部感染效果较好，但相对注射操作风险较高，易造成视网膜脱落。玻璃体腔注射，AAV制剂会首先均匀分布分布于玻璃体液中而后逐层对视网膜进行扩散感染。由于视网膜层致密的结构和复杂的细胞种群构成，天然血清型中对眼部浸染能力较强的AAV8、AAV2，在玻璃体腔注射时效果较差，而部分工程改造的衣壳却在实施玻璃体腔注 射时显露出较强的感染能力(如AAVDJ、AAV2.7M8等)。
本领域仍然需要新型AAV变体。本公开提供了这样的重组腺相关病毒(rAAV)病毒粒子，其具有新结构的衣壳蛋白，对于视网膜组织具有高感染噬性，对异源基因具有高表达效率，从而为临床治疗提供了更有潜力的方案。
发明内容
本公开提供变体AAV衣壳蛋白及其可携带的基因产物，含有所述衣壳蛋白的rAAV病毒粒子，药物组合物，所述rAAV病毒粒子侵染细胞(例如，视网膜细胞)、治疗和预防疾病(例如，眼部疾病)的方法和制药用途。
变体腺相关病毒(AAV)衣壳蛋白
本公开提供一种腺相关病毒(AAV)衣壳蛋白，其相对于亲本AAV衣壳蛋白包含插入的多肽，所述插入的多肽包含选自如下1)-6)任一项所示的多肽或其任意组合：
1)LAETTRP(SEQ ID NO：11)或由SEQ ID NO：11组成的多肽；
2)LGDTTRP(SEQ ID NO：12)或由SEQ ID NO：12组成的多肽；
3)LGETTRN(SEQ ID NO：13)或由SEQ ID NO：13组成的多肽；
4)KADTTKN(SEQ ID NO：14)或由SEQ ID NO：14组成的多肽；
5)KDDTTRN(SEQ ID NO：15)或由SEQ ID NO：15组成的多肽；
6)LADTTKN(SEQ ID NO：16)或由SEQ ID NO：16组成的多肽。
本公开提供一种腺相关病毒(AAV)衣壳蛋白，其相对于亲本AAV衣壳蛋白包含插入的多肽，所述多肽包含X 1X 2X 3TTX 4X 5(SEQ ID NO：35)或由SEQ ID NO：35组成，其中，X 1选自L或K，X 2选自G、D或A，X 3选自D或E，X 4选自R或K，X 5选自P或N。
一些实施方案中，前述1)-6)或SEQ ID NO：35所示的多肽在其氨基末端和/或羧基末端具有1-4个间隔氨基酸(Y 1-Y 4)。一些具体实施方案中，所述间隔氨基酸包括但不限于A、L、G、S和T。
本公开提供一种腺相关病毒(AAV)衣壳蛋白，其相对于亲本AAV衣壳蛋白包含插入的多肽，所述多肽包含Y 1Y 2X 1X 2X 3TTX 4X 5Y 3Y 4(SEQ ID NO：36)或由SEQ ID NO：36组成，其中，X 1选自L或K，X 2选自G、D或A，X 3选自D或E，X 4选自R或K，X 5选自P或N；Y 1、Y 2、Y 3、Y 4可以独立的存在或不存在，Y 1、Y 2、Y 3、Y 4可以独立的选自A、L、G、S和T。例如，Y 1为L，Y 2为A，Y 3为A，Y 4不存在。
本公开提供一种腺相关病毒(AAV)衣壳蛋白，其相对于亲本AAV衣壳蛋白包含插入的多肽，所述插入的多肽选自如下1-1)至1-6)任一项所示的多肽或其任意组合：
1-1)包含LALAETTRPA(SEQ ID NO：17)或由SEQ ID NO：17组成的多肽；
2-1)包含LALGDTTRPA(SEQ ID NO：18)或由SEQ ID NO：18组成的多肽；
3-1)包含LALGETTRNA(SEQ ID NO：19)或由SEQ ID NO：19组成的多肽；
4-1)包含LAKADTTKNA(SEQ ID NO：20)或由SEQ ID NO：20组成的多肽；
5-1)包含LAKDDTTRNA(SEQ ID NO：21)或由SEQ ID NO：21组成的多肽；
6-1)包含LALADTTKNA(SEQ ID NO：22)或由SEQ ID NO：22组成的多肽。
一些实施方案中，上述AAV衣壳蛋白为AAV2衣壳蛋白，或AAV9衣壳蛋白。
一些实施方案中，前述1)至6)、1-1)至6-1)、SEQ ID NO：35或36的多肽位于亲本AAV衣壳蛋白的GH环或IV环(环状结构域IV)中，例如在AAV衣壳蛋白的GH环或IV环的溶剂可及部分(参见van Vliet等(2006)Mol.Ther.14：809；Padron等(2005)J.Virol.79：5047；和Shen等(2007)Mol.Ther.15：1955)。“亲本AAV衣壳蛋白”指无插入的多肽的相同AAV血清型的衣壳蛋白(包括野生型AAV血清型或其变体衣壳蛋白，例如本公开的SEQ ID NO：1所示的AAV2衣壳蛋白或SEQ ID NO：37所示的AAV9衣壳蛋白，所述AAV2衣壳蛋白是可以具有或不具有V708I突变的)。
一些实施方案中，所述AAV选自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10(包括AAVrh10)。
一些实施方案中，前述插入的多肽位于亲本AAV衣壳蛋白的氨基酸残基411位至650位内、或432位至640位内、或570位至671位内、或570位至614位内、或570位至610位内、或580位至600位内、或570位至575内、或575位至580内、或580位至585内、或585位至590内、或590位至600内、或600位至614内。例如，位于亲本AAV2衣壳蛋白的氨基酸残基570位至611位内、亲本AAV1衣壳蛋白的氨基酸残基571位至612位内、亲本AAV5衣壳蛋白的氨基酸残基560位至601位内、亲本AAV6衣壳蛋白的氨基酸571位至612位内、亲本AAV7衣壳蛋白的氨基酸残基572位至613位内、亲本AAV8衣壳蛋白的氨基酸残基573位至614位内、亲本AAV9衣壳蛋白的氨基酸残基571位至612位内，或亲本AAV10(包括AAVrh10)衣壳蛋白的氨基酸残基573位至614位内。
一些实施方案中，前述插入的多肽位于亲本AAV2衣壳蛋白的氨基酸残基587位和588位之间，或亲本AAV9衣壳蛋白的氨基酸残基588位和589位之间，或其他亲本AAV血清型的衣壳蛋白的对应位置。一些实施方案中，插入的多肽位于亲本AAV2衣壳蛋白的氨基酸残基587位和588位之间，或亲本AAV9衣壳蛋白的氨基酸残基588位和589位之间，或其他亲本AAV血清型的衣壳蛋白的对应位置。其他血清型例如选自AAV1、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8或AAV10(包括AAVrh10)。不同AAV血清型中与AAV2的衣壳蛋白VP1的氨基酸570-611相对应的序列是本领域公知的(参见例如WO2012145601A的图6，以及，GenBank登记号NP_049542的AAV1；AAD13756的AAV5；AAB95459的AAV6；YP_077178的AAV7；YP_077180的AAV8；AAS99264的AAV9和AAT46337的 AAV10)。一些实施方案中，插入的多肽位于亲本AAV1衣壳蛋白的氨基酸残基590位和591位之间、亲本AAV5衣壳蛋白的氨基酸残基575位和576位之间、亲本AAV6衣壳蛋白的氨基酸残基590位和591位之间、亲本AAV7衣壳蛋白的氨基酸残基589位和590位之间、亲本AAV8衣壳蛋白的氨基酸残基590位和591位之间、亲本AAV10(包括AAVrh10)衣壳蛋白的氨基酸残基588位和589位之间。
本公开的氨基酸残基计数是相对于AAV衣壳蛋白的VP1的编码氨基酸序列从N端的自然计数。例如“插入的多肽位于AAV2衣壳蛋白的氨基酸残基587位和588位之间”是指所述多肽位于AAV2衣壳蛋白的VP1的编码氨基酸序列的第587位至588位之间，其在VP2的编码氨基酸序列中对应的是第450位至451位之间，在VP3的编码氨基酸序列中对应的是第385位至386位之间。
一些实施方案中，前述插入的多肽位于亲本AAV衣壳蛋白的氨基酸残基450位和460位之间，例如亲本AAV2的氨基酸残基453位、亲本AAV1的氨基酸残基454位、亲本AAV6的氨基酸残基454位、亲本AAV7的氨基酸残基456位、亲本AAV8的氨基酸残基456位、亲本AAV9的氨基酸残基454位、亲本AAV10(包括AAVrh10)的氨基酸残基456位之前或之后。这里全文引入WO2012145601A的图17所展示的不同AAV血清型的衣壳蛋白的氨基酸残基及其对应关系。
一些实施方案中，本公开提供变体AAV衣壳蛋白，其含有前述1)至6)、1-1)至6-1)、SEQ ID NO：35或36的多肽。
一些实施方案中，前述本公开的变体AAV衣壳蛋白还包含一个或多个氨基酸残基的点突变(包括取代、缺失和/或添加)。
一些实施方案中，所述氨基酸残基的点突变位于第1、15、34、57、66、81、101、109、144、164、176、188、196、226、236、240、250、312、363、368、449、456、463、472、484、524、535、551、593、698、708、719、721和735位的一个或其任意组合。
一些实施方案中，所述氨基酸残基的点突变(取代)选自1L、15P、34A、57D、66K、81Q、101R、109T、144K或M、164K、176P、188I、196Y、226E、236V、240T、250S、312K、363L、368H、449D、456K、463Y、472N、484C、524T、535S、551S、593E、698V、708I、719M、721L和735Q的一个或其任意组合，例如312K、449D、472N、551S、698V、735Q、273F、444F、500F、730F、708I的一个或多个，例如708I。
一些实施方案中，所述氨基酸残基的点突变(取代)选自M1L、L15P、P34A、N57D、N66K、R81Q、Q101R、S109T、R144K、R144M、Q164K、T176P、L188I、S196Y、G226E、G236V、I240T、P250S、N312K、P363L、D368H、N449D、T456K、S463Y、D472N、R484C、A524T、P535S、N551S、A593E、I698V、V708I、V719M、S721L、L735Q、Y273F、Y444F、Y500F、Y730F的一个或其任意组合，例如N312K、N449D、D472N、N551S、I698V、L735Q、Y273F、Y444F、Y500F、Y730F、V708I 的一个或多个。
一些具体实施方案中，所述氨基酸残基的点突变(取代)为708I和/或449D，或V708I和/或N449D。一些具体实施方案中，所述氨基酸残基的突变(取代)为273F、444F、500F和/或730F，或Y273F、Y444F、Y500F和/或Y730F。
上述点突变是相对于相应亲本AAV衣壳蛋白的相应位置的，例如相对于亲本AAV2衣壳蛋白的相应位置的。
本公开全文引入WO2012145601A、WO2017197355A、WO2018022905A中衣壳蛋白的突变。
一些实施方案中，本公开的AAV衣壳是嵌合衣壳。例如，衣壳包括第一AAV血清型AAV衣壳的一部分和第二血清型AAV衣壳的一部分，所述血清型包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10(包括AAVrh10)。例如，所述AAV衣壳可以是AAV2G9，其包含来自AAV2和AAV9的序列，此处全文引入US20160017005中AAV2G9的序列。
一些实施方案中，本公开的变体AAV衣壳蛋白是经分离的，和/或经纯化的。
一些实施方案中，本公开提供变体AAV2衣壳蛋白，相对于相应亲本AAV2衣壳蛋白(例如SEQ ID NO：1所示)，在衣壳蛋白GH环或IV环(环状结构域IV)中包含多肽，所述多肽选自前述1)至6)、1-1)至6-1)、SEQ ID NO：35或36。一些具体实施方案中，多肽包含或为SEQ ID NO：12或18。一些具体实施方案中，多肽位于亲本AAV2衣壳蛋白的VP1的第587位和588位氨基酸残基之间。
一些实施方案中，本公开提供变体AAV9衣壳蛋白，相对于相应亲本AAV9衣壳蛋白(例如SEQ ID NO:37所示)，在衣壳蛋白GH环或IV环(环状结构域IV)中包含多肽，所述多肽选自前述1)至6)、1-1)至6-1)、SEQ ID NO：35或36。一些具体实施方案中，多肽包含或为SEQ ID NO：12或18。一些具体实施方案中，多肽位于亲本AAV9衣壳蛋白的VP1的第588位和589位氨基酸残基之间。
一些实施方案中，本公开提供变体AAV衣壳蛋白，其氨基酸序列为SEQ ID NO：3-9、23-27任一所示或与之具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％的序列同一性。
一些实施方案中，本公开提供的变体AAV衣壳蛋白具有如下的特性：
(a)与包括对应的亲本AAV衣壳蛋白的AAV病毒粒子的眼组织(例如，视网膜细胞)的感染性相比，增加的眼组织(例如，视网膜细胞)的感染性，特别是增加的视网膜神经细胞层感染性；
(b)与包括对应的亲本AAV衣壳蛋白的AAV病毒粒子的趋向性相比，改变的细胞趋向性；
(c)与包括对应的亲本AAV衣壳蛋白的AAV病毒粒子相比，增加的结合和/或穿过内界膜(ILM)的能力；和/或
(d)与包括对应的亲本AAV衣壳蛋白的AAV病毒粒子相比，在眼组织(例如， 视网膜组织、房水、玻璃体)中具有对其包裹或携带的基因产物具有增加的表达量。
重组腺相关病毒(rAAV)病毒粒子
本公开提供重组腺相关病毒(rAAV)病毒粒子，其包含：
(a)前述本公开任意的变体AAV衣壳蛋白；可选地，包含
(b)异源多核苷酸。
一些实施方案中，所述异源多核苷酸包含表达或编码基因产物的多核苷酸。一些实施方案中，所述基因产物是相对于AAV是异源的。一些实施方案中，所述基因产物是相对于靶细胞是异源的或内源的。一些实施方案中，所述基因产物为一个或多个(例如，2、3、4个)。
一些实施方案中，所述异源多核苷酸中包含调控序列，所述调控序列调控基因产物的表达或编码。
关于基因产物：
一些实施方案中，所述基因产物是针对疾病、病症治疗性的或预防性的。
一些实施方案中，所述基因产物选自干扰RNA(RNAi)、适配体、多肽。
一些具体实施方案中，所述基因产物是RNAi，例如减少、降低细胞中凋亡因子或血管生成因子的水平的RNAi。例如，RNAi可以是降低细胞中诱导或促凋亡的基因产物的水平的shRNA或siRNA，所述促凋亡基因产物包含例如Bax、Bid、Bak和Bad基因产物(参见US7,846,730，并全文引入)。又例如，RNAi可以是针对血管生成产物的，例如VEGF(例如Cand5，参见US2011/0143400、US2008/0188437，并全文引入)、VEGFR1(例如Sirna-027，参见Kaiser等(2010)Am.J.Ophthalmol.150：33；和Shen等(2006)Gene Ther.13：225，并全文引入)或VEGFR2(例如Kou等(2005)Biochem.44：15064，并全文引入)。
一些具体实施方案中，所述基因产物是适配体，例如是针对VEGF的特异性适配体(例如5'-cgcaaucagugaaugcuuauacauccg-3'，参见Ng等(2006)Nat.Rev.Drug Discovery5：123，和Lee等(2005)Proc.Natl.Acad.Sci.USA 102：18902，并全文引入)、针对PDGF的特异性适配体(例如E10030，参见Ni和Hui(2009)Ophthalmologica 223：401，和Akiyama等(2006)J.CellPhysiol.207：407，并全文引入)。
一些具体实施方案中，所述基因产物是多肽。
一些具体实施方案中，所述基因产物是神经保护多肽、抗血管生成多肽或增强视网膜细胞功能的多肽。
一些具体实施方案中，所述多肽可以增强视网膜细胞的功能，例如，增强视杆或视锥感光细胞、视网膜神经节细胞、Muller细胞双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞的功能。
一些具体实施方案中，所述多肽包含或选自：神经保护多肽(例如，GDNF、CNTF、NT4、NGF和NTN)；抗血管生成多肽(例如，可溶性血管内皮生长因子(VEGF)受体、抗VEGF抗体或其抗原结合片段、内皮抑素(endostatin)、肿瘤抑素(tumstatin)、 血管抑素(angiostatin)、可溶性Flt多肽及其与Fc区的融合蛋白(参见Lai等(2005)Mol.Ther.12：659，Pechan等(2009)Gene Ther.16：10)、色素上皮衍生因子(PEDF)、可溶性Tie-2受体等)；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)等。
一些具体实施方案中，所述多肽包括但不限于：表皮生长因子、视紫红质、X连锁凋亡抑制蛋白。
一些具体实施方案中，所述多肽包括但不限于：视网膜劈裂蛋白(retinoschisin)、视网膜色素变性GTP酶调节剂(RGPR)相互作用蛋白-1(GenBank Accession NO.Q96KN7、Q9EPQ2、Q9GLM3)、外周蛋白-2(Prph2)(GenBank Accession NO.NP_000313)、视网膜色素上皮特异性蛋白(RPE65)(GenBank Accession NO.AAC39660)。
一些具体实施方案中，所述多肽包括但不限于：缺损或缺失时，引发无脉络膜的多肽，如CHM(脉络膜缺损(choroidermia)(Rab护送蛋白1))(Donnelly等(1994)Hum.Mol.Genet.3：1017)；缺损或缺失时，引发利伯氏先天性黑内障的多肽和色素性视网膜炎的多肽，如碎屑同源物1(CRB1)(GenBank Accession NO.CAM23328)；缺损或缺失时，引发色盲的多肽，如杆状感光cGMP-门控通道亚基α(CNGA3)(GenBank Accession NO.NP_001289)、杆状感光cGMP-门控通道β亚基(CNGB3)、鸟嘌呤核苷酸结合蛋白(G蛋白)、α转导活性多肽2(GNAT2)(ACHM4)、ACHM5、L-视蛋白、M-视蛋白和S-视蛋白。
一些具体实施方案中，所述基因产物提供用于对基因功能进行位点特异性敲减的位点特异性内切核酸酶，例如，其中内切核酸酶敲除了与视网膜疾病相关的等位基因。例如，在显性等位基因对当属于野生型时是视网膜结构蛋白和/或提供正常的视网膜功能的基因的缺陷副本进行编码的情况下，位点特异性内切核酸酶可以靶向到缺陷等位基因并且敲除缺陷等位基因。所述位点特异性内切核酸酶例如为：锌指核酸酶(ZFNs)，以及转录激活因子样效应物核酸酶(TALENs)，其中这种位点特异性内切核酸酶是非天然存在的并且被修饰以靶向特定基因。
此外，本公开全文引入WO2012145601A、WO2017197355A、WO2018022905A中的前述基因产物的序列、来源。
一些具体实施方案中，本公开的基因产物为抗血管生成剂，包括抗血管生成多肽，例如，抗VEGF抗体或其抗原结合片段；又例如VEGF拮抗剂(例如VEGF-A、B、C拮抗剂)或PDGF拮抗剂。
一些具体实施方案中，VEGF拮抗剂包括但不限于兰尼单抗(ranibizumab)、贝伐单抗(bevacizumab)、阿柏西普(aflibercept)、KH902VEGF受体-Fc融合蛋白、2C3抗体、ORA102、哌加他尼钠(pegaptanib)、贝伐西尼(bevasiranib)、SIRNA-027、紫花前胡素(decursin)、紫花前胡醇(decursinol)、苦鬼臼脂素(picropodophyllin)、没药甾酮(guggulsterone)、PLG101、类二十烷酸LXA4、PTK787、帕唑帕尼(pazopanib)、 阿西替尼(axitinib)、CDDO-Me、CDDO-Imm、紫草素(shikonin)、β-羟异戊酰紫草素(beta-hydroxyisovalerylshikonin)、或神经节苷脂GM3、DC101抗体、Mab25抗体、Mab73抗体、4A5抗体、4E10抗体、5F12抗体、VA01抗体、BL2抗体、VEGF相关蛋白、sFLT01、sFLT02、肽B3、TG100801、索拉非尼(sorafenib)、舒尼替尼(sunitnab)、G6-31抗体，或其药学上可接受的盐。此处全文引入WO2018160686A中的VEGF拮抗剂信息。
兰尼单抗
Figure PCTCN2022095422-appb-000001
的序列信息参见US7,060,269(其图1)，贝伐单抗
Figure PCTCN2022095422-appb-000002
的序列信息参见US6,054,297(其图1)，阿柏西普的
Figure PCTCN2022095422-appb-000003
的序列信息参见Do等人(Br J Ophthalmol.2009,93：144-9)，此处通过全文引用的方式并入。
一些实施方案中，VEGF拮抗剂包含或为天然存在的蛋白质sFlt-1，或其功能片段(例如，sFlt-1结构域2，参见US5,861,484中的sFlt-1序列信息、US2013/0323302中的sFlt-1结构域2序列信息，全文引入)。
一些实施方案中，VEGF拮抗剂为VEGF结合融合蛋白，此处全文引入US7,635,474中结合VEGF的融合蛋白的序列信息。
一些具体实施方案中，阿柏西普(aflibercept)的氨基酸序列如SEQ ID NO：38所示，并提供编码SEQ ID NO：38的多核苷酸序列，例如，经密码子优化的多核苷酸序列，其如SEQ ID NO：39-41任一所示。
关于调控序列：
一些实施方案中，前述多核苷酸包含如下(a)-(h)的任一项的多核苷酸(调控序列)或任意组合：
(a)5’反向末端重复(5’ITR)和/或3’反向末端重复(3’ITR)；
(b)5’非翻译区(5’UTR)和/或3’非翻译区(3’UTR)；
(c)启动子；
(d)增强子；
(e)内含子(intron)；
(f)转录后调控元件；
(g)多聚腺苷酸化信号(polyA)；
(h)Kozak序列。
一些实施方案中，AAV ITR不需要具有野生型核苷酸序列并且可以通过核苷酸的插入、删除或取代来改变，或者AAV ITR可以从几种AAV血清型中的任何一种得到，例如AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9和AAV10。一些具体实施方案中，5’ITR和3’ITR为AAV2的5’ITR和3’ITR。
一些实施方案中，编码基因产物的核苷酸序列与组织特异性或细胞类型特异性调控元件可操作地连接，例如感光特异性调控元件(例如，感光特异性启动子)可操作地连接，又例如赋予感光细胞内可操作地连接的基因选择性表达的调控元件。
一些具体实施方案中，(a)-(h)的任意组合能够满足使得基因产物(例如，抗血管生成剂(或抗血管生成多肽)，又例如，抗VEGF抗体或其抗原结合片段、阿柏西普)表达的功能，例如在受试者眼组织(例如房水、视网膜组织)表达的功能。
一些具体实施方案中，所述(a)-(h)的任一项的多核苷酸或任意组合与前述编码基因产物的多核苷酸可操作地连接。
一些具体实施方案中，所述5’ITR和/或3’ITR源自AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV9.47、AAV9(hu14)、AAV10、AAV11、AAV12、AAVrh8、AAVrh10、AAV-DJ或AAV-DJ8；例如，源自AAV2、AAV9。
一些具体实施方案中，所述5’UTR和/或3’UTR源自血红素结合蛋白(hemopexin，HPX)、β珠蛋白(hemoglobin subunit beta，HBB)、HSPB1、CCL13、爪蟾球蛋白(Xenopus globin)。例如，5’UTR的序列如SEQ ID NO：30所示，3’UTR的序列如SEQ ID NO：32所示。
一些具体实施方案中，所述启动子可以是组成型启动子或诱导型启动子。
一些具体实施方案中，所述启动子选自巨细胞病毒(CMV)启动子、劳斯氏肉瘤病毒(RSV)启动子、UB6启动子、鸡β-肌动蛋白启动子、CAG启动子、RPE65启动子、CBh启动子、EFS启动子、EF1(例如EF-1α)启动子、PGK启动子、SV40启动子、Ubi启动子、视蛋白启动子或其任意组合。所述视蛋白启动子包括但不限于视紫红质启动子、视紫红质激酶启动子(Young等(2003)Ophthalmol.Vis.Sci.44：4076)、β磷酸二酯酶基因启动子(Nicoud等(2007)J.Gene Med.9：1015)、色素性视网膜炎基因启动子(Nicoud等(2007)同上)、感光间维甲酸结合蛋白(IRBP)基因启动子(Yokoyama等(1992)Exp Eye Res.55：225)。例如，启动子为CMV启动子，其序列例如SEQ ID NO：29所示。
一些具体实施方案中，所述增强子选自Ubi、CMV、RSV、IRBP基因增强子(Nicoud等(2007)J.Gene Med.9：1015)或其任意组合。例如，增强子为CMV增强子，其序列例如SEQ ID NO：28所示。
一些具体实施方案中，所述内含子选自MVM、SV40、βGlobin、EF1(例如EF-1α)、hybrid内含子，或其任意组合。
一些具体实施方案中，所述polyA选自PA75polyA、SV40polyA、hGH polyA、BGH polyA、rbGlob polyA或其任意组合。例如，polyA为SV40polyA，其序列例如SEQ ID NO：34所示。
一些具体实施方案中，所述转录后调控元件选自WPRE、HPRE或其组合。例如，转录后调控元件为WPRE，其序列例如SEQ ID NO：33所示。
一些实施方案中，前述多核苷酸包含如下(a)-(g)的任一项的多核苷酸(调控序列)或任意组合：
(a)源自AAV2的5’ITR和/或3’ITR；
(b)源自Xenopus globin的5’UTR和/或3’UTR；
(c)CMV启动子；
(d)CMV增强子；
(e)WPRE；
(f)Kozak序列；
(g)SV40polyA。
示例性的，多核苷酸从5’端和3’端的顺序为(括号内为可选的调控序列)：
5’ITR-增强子-启动子-5’UTR-(Kozak序列)-基因产物-3’UTR-(WPRE)-polyA-3’ITR，或
增强子-启动子-5’UTR-(Kozak序列)-基因产物-3’UTR-(WPRE)-polyA；
启动子-(5’UTR)-(Kozak序列)-基因产物-(3’UTR)-(WPRE)-polyA。
关于rAAV病毒粒子的效果：
一些实施方案中，rAAV病毒粒子具有感染性；另一些实施方案中，rAAV病毒粒子不具有感染性。
一些实施方案中，与包含相应亲本AAV衣壳蛋白的AAV病毒粒子对细胞的感染性相比，前述本公开的rAAV病毒粒子表现出增强至少2倍、至少3倍、至少4倍、至少5倍、至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的细胞感染性，所述细胞选自：
1)视网膜细胞；
2)感光细胞；
3)RPE细胞；
4)双极细胞；
5)无长突细胞；和/或
6)水平细胞。
一些实施方案中，与包含相应亲本AAV衣壳蛋白的AAV病毒粒子穿过内界膜(ILM)的能力相比，前述本公开的rAAV病毒粒子表现出增强至少2倍、至少3倍、至少4倍、至少5倍、至少10倍、至少15倍、至少20倍、至少25倍、至少50倍或50倍以上的穿过ILM的能力。
一些实施方案中，前述本公开的rAAV病毒粒子选择性感染视网膜细胞，例如，前述本公开的rAAV病毒粒子以比非视网膜细胞(例如非眼组织细胞)强2倍、3倍、4倍、5倍、10倍、15倍、20倍、25倍、50倍或50倍以上的特异性感染视网膜细胞。
一些实施方案中，与包含AAV2.7m8衣壳蛋白的rAAV病毒粒子相比，前述本公开rAAV病毒粒子(例如衣壳蛋白包含seq2的rAAV病毒粒子)，对基因产物(例如阿柏西普)在全眼组织的表达量强2倍、3倍、4倍、5倍、10倍、15倍、20倍、25倍、50倍或50倍以上。
一些实施方案中，与包含AAV2.7m8衣壳蛋白的rAAV病毒粒子相比，前述本公开rAAV病毒粒子(例如衣壳蛋白包含seq2的rAAV病毒粒子)，对基因产物(例如阿柏西普)在房水的表达量强2倍、3倍、4倍、5倍、10倍、15倍、20倍、25倍、50倍或50倍以上。
一些实施方案中，与包含AAV2.7m8衣壳蛋白的rAAV病毒粒子相比，前述本公开rAAV病毒粒子(例如衣壳蛋白包含seq2的rAAV病毒粒子)，对基因产物(例如阿柏西普)在玻璃体的表达量强2倍、3倍、4倍、5倍、10倍、15倍、20倍、25倍、50倍或50倍以上。
一些实施方案中，与包含AAV2.7m8衣壳蛋白的rAAV病毒粒子相比，前述本公开rAAV病毒粒子(例如衣壳蛋白包含seq2的rAAV病毒粒子)，对基因产物(例如阿柏西普)在视网膜组织的表达量强2倍、3倍、4倍、5倍、10倍、15倍、20倍、25倍、50倍或50倍以上。
一些实施方案中，上述rAAV病毒粒子的给药方法是经玻璃体内注射。
多核苷酸和(表达)载体
本公开提供编码阿柏西普(aflibercept)的多核苷酸，其编码的氨基酸序列如SEQ ID NO：38所示。
一些实施方案中，本公开提供编码阿柏西普(aflibercept)的经密码子优化的多核苷酸，如SEQ ID NO：39-41任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性。
本公开提供编码前述本公开的任意基因产物的多核苷酸。
本公开提供编码前述本公开任意的AAV衣壳蛋白的多核苷酸。
一些实施方案中，上述多核苷酸可以为RNA、DNA或cDNA。
一些实施方案中，上述多核苷酸是分离的多核苷酸。
本公开的多核苷酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒、YAC或病毒载体。载体可例如为表达载体，即可提供多核苷酸在体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本公开的多核苷酸，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。
本公开的多核苷酸可通过已知的方式(例如通过自动DNA合成和/或重组DNA技术)制备或获得，和/或可从适合的天然来源加以分离。
本公开提供载体，其包含：
(a)分离的多核苷酸，其编码前述本公开任意的变体AAV衣壳蛋白(如SEQ ID NO：3-9和23-27中任一所示或与之具有至少90％或95％的序列同一性)；
(b)分离的多核苷酸，其编码前述本公开任意的变体AAV衣壳蛋白(如SEQ ID NO：3-9和23-27中任一所示或与之具有至少90％或95％的序列同一性)；和/或前述编码基因产物的异源多核苷酸(如编码SEQ ID NO：38所示阿柏西普(aflibercept) 的多核苷酸、SEQ ID NO：39-41中任一所示或与之具有至少90％或95％序列同一性的多核苷酸)。
一些实施方案中，(b)中编码变体AAV衣壳蛋白的多核苷酸和编码基因产物的异源多核苷酸在不同的载体中。
宿主细胞
本公开提供宿主细胞，其包含前述本公开任意的多核苷酸或(表达)载体。
一些实施方案中，所述宿主细胞可以是分离的细胞，例如来自体外细胞培养物的细胞。这种细胞用于产生前述本公开任意的rAAV衣壳蛋白、基因产物或rAAV病毒粒子，又称生产细胞。
一些具体实施方案中，生产细胞为细菌细胞、真菌细胞或哺乳动物细胞。示例性生产细胞包含但不限于HeLa、CHO、293(包括293T)、Vero、NIH 3T3、Huh-7、BHK、PC12、COS(包括COS-7)、RAT1、HepG2细胞等。示例性哺乳动物细胞包含但不限于293(293T)、COS、HeLa、Vero、3T3、C3H10T1/2、CHO细胞。生产细胞也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达蛋白、病毒粒子的任何其他细胞。示例性昆虫细胞包括但不限于草地贪夜蛾、果蝇(Drosophila)细胞系，或蚊细胞系，如白纹伊蚊(Aedesalbopictus)衍生细胞系，包括但不限于Se301、SeIZD2109、SeUCR1、Sf9、Sf900+、Sf21、BTI-TN-5B1-4、MG-1、Tn368、HzAm1、Ha2302、Hz2E5、HighFive(Invitrogen，CA，USA)、AO38和BM-N细胞。
rAAV病毒粒子的生产、制备方法
生产rAAV病毒粒子的方法是本领域常规的。本公开全文引入WO200028004、WO200123001、WO2004112727、WO 2005005610、WO2005072364、WO2013123503、WO2015191508和US20130195801中的rAAV病毒粒子生产、制备方法。所述rAAV病毒粒子可以具有增强递送效率的特性，能进行有效包装，能以高频率和最小的毒性成功地感染靶细胞(例如哺乳动物或人细胞)。
本公开提供生产、制备rAAV病毒粒子的方法，包括将本公开任意多核苷酸包装至AAV衣壳中。
一些实施方案中，提供生产、制备rAAV病毒粒子的方法，包括：将前述本公开包含编码基因产物的多核苷酸或其(表达)载体，编码前述本公开任意AAV衣壳蛋白的多核苷酸或其(表达)载体，和辅助功能质粒(例如，pHelper)，引入生产细胞(例如293细胞)，包装、纯化，获得rAAV病毒粒子。
一些实施方案中，提供生产、制备rAAV病毒粒子的生产方法，包括：
1)将本公开任意包含编码基因产物的多核苷酸或(表达)载体，表达Rep和Cap基因的载体(例如，pR2C9)，以及辅助载体(以实现辅助功能，例如pHelper)同时共转染哺乳动物细胞(例如293细胞)，所述表达Rep和Cap基因的载体含有编码前述本公开任意AAV衣壳蛋白的多核苷酸；
2)收获、纯化包含编码基因产物的多核苷酸的rAAV病毒粒子。
一些实施方案中，提供rAAV病毒粒子生产系统，用于生产前述本公开任意的rAAV病毒粒子，包含：
1)编码AAV衣壳蛋白的多核苷酸；
2)本公开任意的包含编码基因产物的异源多核苷酸或其(表达)载体(例如，pGOI质粒)；和
3)辅助元件，其具有足够的AAV rep功能和辅助功能，以将2)中包含编码基因产物的异源多核苷酸包装到AAV衣壳中。
一些具体实施方案中，足够的AAV rep功能和辅助功能通过包装细胞或pHelper、pR2C9、pGOI三质粒提供，所述包装细胞可以包含pHelper、pR2C9、pGOI这三种质粒。
一些实施方案中，Rep基因编码调节功能的非结构蛋白，例如AAV基因组的复制，可以选自Rep78、Rep68、Rep52、Rep40。Rep78和Rep68通常从p5启动子转录，而Rep52和Rep40通常从p19启动子转录。Cap基因编码组装形成病毒壳体壳体的结构蛋白VP1、VP2和/或VP3。Cap基因通常从p40启动子转录。
一些实施方案中，提供上述AAV生产方法或生产系统生产的rAAV病毒粒子。
药物组合物
本公开提供药物组合物，其含有：
(a)前述本公开任意的AAV衣壳蛋白或rAAV病毒粒子；和
(b)一种或多种药学上可接受的载剂、稀释剂、赋形剂或缓冲液。
本公开提供药物组合物，其含有：
(a)预防或治疗有效量的活性成分；和
(b)一种或多种药学上可接受的载剂、稀释剂、赋形剂或缓冲液；
所述活性成分例如选自：前述本公开任意的rAAV病毒粒子，编码SEQ ID NO：39-41或其至少80％、85％、90％、95％、96％、97％、98％、99％同一性序列的基因产物的多核苷酸。
一些实施方案中，所述药物组合物单位剂量中可含有0.01至99重量％的多核苷酸(例如VEGF抑制剂，又例如阿伯西普的编码多核苷酸)或rAAV病毒粒子。一些具体实施方案中，药物组合物单位剂量中含基因产物(例如VEGF抑制剂，又例如阿伯西普)拷贝量为0.1至10×10 13。一些具体实施方案中，药物组合物中的rAAV病毒粒子浓度为每毫升1×10 8个或更多，通常不多于每毫升1×10 15个。
一些实施方案中，可以用前述本公开任意的rAAV病毒粒子转染细胞，而后将所述细胞转移或移植入受试者。
一些实施方案中，药物组合物中含有前述本公开任意的rAAV病毒粒子，所述rAAV病毒粒子包裹有多核苷酸，所述多核苷酸为SEQ ID NO：39-41任一所示或与之具有至少80％、85％、90％、95％、96％、97％、98％、99％同一性序列。
治疗、预防疾病的方法和制药用途
本公开提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸用于治疗、缓解或改善疾病或症状的方法或用途。
一些实施方案中，提供向有需要的受试者递送基因产物的方法，包括向所述 受试者施用有效量的前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸。
一些实施方案中，提供向靶细胞递送基因产物的方法，其包括使所述靶细胞与前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸接触。一些具体实施方案中，靶细胞选自肝细胞、胰腺细胞、骨骼肌细胞、心肌细胞、成纤维细胞、视网膜细胞、滑膜关节细胞、肺细胞、T细胞、神经元、神经胶质细胞、干细胞、内皮细胞或癌细胞。一些具体实施方案中，靶细胞是体外的；另一些具体实施方案中，靶细胞是体内的。同时提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸制备向靶细胞递送异源核酸的制药用途。
一些实施方案中，提供特异性感染视网膜细胞的方法，包括向眼内施用预防或治疗有效量的前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸，例如通过玻璃体内注射或视网膜下注射。同时提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸制备特异性感染视网膜细胞药物的制药用途。
一些具体实施方案中，提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸用于治疗选自由以下组成的组的视网膜细胞的疾病或病症：感光细胞、视网膜神经节细胞、Muller细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞。在一些情况下，视网膜细胞是感光细胞，例如视杆细胞或视锥细胞。
一些具体实施方案中，提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸用于治疗选自由以下组成的组的视网膜细胞的疾病或病症：急性黄斑神经视网膜病变；白塞氏病；脉络膜新生血管；糖尿病性葡萄膜炎；组织胞浆菌病；黄斑变性，如急性黄斑变性、非渗出性年龄相关性黄斑变性和渗出性年龄相关性黄斑变性；水肿，如黄斑水肿、黄斑囊样水肿和糖尿病性黄斑水肿；多灶性脉络膜炎；眼外伤，其影响眼睛后部位点或位置；眼部肿瘤；视网膜病症，如视网膜中央静脉阻塞、糖尿病性视网膜病变(包含增生性糖尿病视网膜病变)、增生性玻璃体视网膜病变(PVR)、视网膜动脉闭塞性疾病、视网膜脱离和葡萄膜炎性视网膜疾病；交感性眼炎；伏格特-小柳-原田三氏(Vogt Koyanagi-Harada)(VKH)综合征；葡萄膜扩散；由眼部激光治疗引起的或受其影响的眼后病状；由光动力学治疗引起或受其影响的眼后病状；光凝固法、放射性视网膜病变；视网膜前膜病症；视网膜分支静脉阻塞；前部缺血性视神经病变；非视网膜病变糖尿病视网膜功能障碍；视网膜劈裂症；视网膜色素变性；青光眼；乌谢尔综合征、锥体-杆体营养不良；斯图加特氏疾病(眼底黄色斑点症)；遗传性黄斑变性；脉络膜视网膜变性；莱伯先天性黑朦；先天性静止性夜盲；无脉络膜；巴比二氏综合征；黄斑毛细血管扩张症；莱伯遗传性视神经病变；早 产儿视网膜病变；以及色觉病症，包含全色盲、红色盲、绿色盲和蓝色盲，遗传性视网膜色素变性。
一些实施方案中，提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸用于治疗眼部疾病的方法或用途，所述眼部疾病包括但不限于：年龄相关性黄斑变性(AMD)、湿性AMD、干性AMD、视网膜新生血管、脉络膜新生血管、糖尿病性视网膜病变、增生性糖尿病视网膜病变、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、糖尿病性黄斑水肿、糖尿病性视网膜缺血、缺血性视网膜病变或糖尿病性视网膜水肿。可选地，是通过玻璃体内注射或视网膜下注射。
一些实施方案中，提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸治疗视网膜相关疾病的方法或用途。
同时提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸制备治疗或预防上述疾病的药物的制药用途。
给药方法
本公开提供前述本公开任意的AAV衣壳蛋白、rAAV病毒粒子、药物组合物、编码阿柏西普(aflibercept)的多核苷酸用于治疗前述眼部疾病(例如视网膜相关疾病)的给药方法，其是通过眼内注射施用。
一些实施方案中，眼内注射包括通过玻璃体内注射、通过视网膜下注射、通过脉络膜上注射或通过将导致rAAV病毒粒子递送至眼睛的任何其它方便的施用模式或途径。其它方便的施用模式或途径包含但不限于静脉内、动脉内、眼周、前房内、结膜下和眼球囊下注射和局部施用和鼻内施用。
附图说明
图1为AAV2衣壳蛋白序列及VP1、VP2、VP3的起始位点，环状结构域I-V的示意图。
图2为本公开变体AAV衣壳蛋白在587到588位之间插入肽段，具有708位点突变的示意图。
图3为本公开的rAAV载体病毒AAV2 seq1、AAV2 seq2、AAV2 seq3、AAV2 seq4、AAV2 seq5、AAV2 seq6与AAV2、AAV2.7m8感染小鼠视网膜的荧光信号检测结果图。
图4A和图4B为本公开的rAAV载体病毒AAV2 seq1、AAV2 seq2、AAV2 seq3、AAV2 seq4、AAV9 seq2与AAV2、AAV2.7m8感染小鼠视网膜荧光信号检测结果图，图4A为第一周眼底镜检测结果，图4B为第四周眼底镜检测结果。
图5A为含有表达VEGF Trap(阿柏西普)的核酸分子的载体结构示意图；图5B 为AAV包装质粒示意图。
图6为rAAV载体病毒AAV2 seq2和AAV2.7m8在小鼠眼球表达目的基因(阿柏西普)的检测结果。
图7为rAAV载体病毒AAV2 seq2和AAV 2.7m8在兔眼球中表达目的基因蛋白的检测结果，其是病毒注射14天后，对房水中目的基因(阿柏西普)蛋白含量的检测。
图8为rAAV载体病毒AAV2 seq2和AAV 2.7m8在兔眼球中表达目的基因蛋白的检测结果，其是病毒注射28天后，对房水中目的基因蛋白摩尔浓度的检测。
具体实施方式
为了更容易理解本公开，以下具体定义了一些技术和科学。除非在本文中另有明确定义，本文使用的所有其它技术和科学都具有本公开所属领域的一般技术人员通常理解的含义。
“AAV”是腺相关病毒的缩写，且可用于指代病毒本身或其衍生物。除非另有所指，否则该涵盖所有AAV亚型以及天然存在形式和重组形式。“AAV”包含但不限于AAV 1型(AAV1)、AAV 2型(AAV2)、AAV 3型(AAV3)、AAV 4型(AAV4)、AAV 5型(AAV5)、AAV 6型(AAV6)、AAV 7型(AAV7)、AAV 8型(AAV8)、AAV 9型(AAV9)、AAV10型(AAV10)、AAVrh10型(AAVrh10)，以及不同种属的禽类AAV、牛类AAV、犬类AAV、马类AAV、灵长类AAV、非灵长类AAV和绵羊类AAV。
AAV是由在非包膜二十面体衣壳内的4.7kb单链DNA基因组构成的非致病性细小病毒，基因组含有侧接有用作病毒复制起点和包装信号的反向末端重复(ITR)的三个开放阅读框(ORF)。Rep ORF编码四个非结构蛋白，这些蛋白在病毒复制、转录调控、位点特异性整合、及病毒粒子装配中发挥作用。Cap ORF编码三个结构蛋白(VP1-3)，这些蛋白装配形成60聚体病毒衣壳。最终，作为cap基因内的替代阅读框存在的ORF产生装配活化蛋白(AAP)，这是一种将AAV衣壳蛋白定位在细胞核中并在衣壳装配过程中发挥作用的病毒蛋白。
各种血清型AAV的基因组序列以及天然末端重复(ITR)序列、Rep蛋白序列和衣壳亚基序列是本领域已知的。这种序列可以在文献或如GenBank等公共数据库中找到。参见例如GenBank登录号NC_002077.1(AAV1)、AF063497.1(AAV1)、NC_001401.2(AAV2)、AF043303.1(AAV2)、J01901.1(AAV2)、U48704.1(AAV3)、NC_001729.1(AAV3)、NC_001829.1(AAV4)、U89790.1(AAV4)、NC_006152.1(AAV5)、AF085716.1(AAV5)、AF028704.1(AAV6)、NC_006260.1(AAV7)、AF513851.1(AAV7)、AF513852.1(AAV8)NC_006261.1(AAV8)、以及AY530579.1(AAV9)。以及，Srivistava等人(1983)J.Virology45：555；Chiorini等人(1998)J.Virology71：6823； Chiorini等人(1999)J.Virology73：1309；Bantel-Schaal等人(1999)J.Virology73：939；Xiao等人(1999)J.Virology73：3994；Muramatsu等人(1996)Virology221：208；Shade等人,(1986)J.Virol.58：921；Gao等人(2002)Proc.Nat.Acad.Sci.USA99：11854；Moris等人(2004)Virology33：375-383；以及，WO00/28061、WO99/61601、WO98/11244、US6156303、WO2012145601A、WO2017197355A、WO2018022905A。以上内容全文引入本公开。
关于AAV衣壳的GH环或环IV，参见例如van Vliet等人(2006)Mol.Ther.14:809；Padron等人(2005)J.Virol.79:5047；和Shen等人(2007)Mol.Ther.15:1955。
“AAV病毒颗粒”或“AAV病毒粒子”是指由至少一种AAV衣壳蛋白和衣壳化的AAV多核苷酸构成的病毒颗粒。
“rAAV”是指重组腺相关病毒，应用于多核苷酸的“重组”是指多核苷酸是克隆、限制或连接步骤的各种组合的产物、以及导致不同于在自然中找到的的多核苷酸的构建体的其它过程。重组病毒是包括重组多核苷酸的病毒颗粒。
如果“AAV病毒粒子”包括异源多核苷酸(即除野生型AAV基因组以外的多核苷酸，例如待递送到靶细胞的基因产物(例如转基因、RNAi等)，它通常被称为“重组AAV(rAAV)病毒粒子”或“rAAV病毒颗粒”或“rAAV载体病毒”。通常，异源多核苷酸的两侧是至少一个，并且通常是两个AAV反向末端重复序列(ITR)。
“rAAV载体”涵盖rAAV病毒粒子，其包含rAAV多核苷酸；并且还涵盖对rAAV进行编码的多核苷酸(例如，对rAAV进行编码的单链多核苷酸(ss-rAAV)；对rAAV进行编码的双链多核苷酸(ds-rAAV)，例如对rAAV进行编码的质粒；等等)。
“AAV变体”、“AAV突变体”或“衣壳变异的rAAV”是指由以下构成的病毒颗粒：(a)变体AAV衣壳蛋白，其中变体AAV衣壳蛋白相对于对应的亲本AAV衣壳蛋白包括至少一个氨基酸差异(例如氨基酸取代、插入或缺失)，其中AAV衣壳蛋白与天然存在的AAV衣壳蛋白的氨基酸序列不同或不对应；以及任选地，(b)包括编码异源基因产物的异源多核苷酸，其中与包括对应的亲本AAV衣壳蛋白的AAV病毒粒子的结合相比，变体AAV衣壳蛋白赋予了与类肝素或硫酸类肝素蛋白多糖的增加结合。
“包装”是指导致AAV颗粒进行组装和衣壳化的一系列细胞内事件。
“rep”和“cap”基因是指对腺相关病毒的复制和衣壳化蛋白进行编码的多核苷酸序列。AAV rep和cap在本文中被称为AAV“包装基因”。
“辅助病毒”是指允许AAV(例如野生型AAV)被哺乳动物细胞复制和包装的病毒。AAV的各种辅助病毒是本领域已知的，包含腺病毒、疱疹病毒和如牛痘等痘病毒。腺病毒涵盖许多不同的亚群，但是最常用的是C亚群中的腺病毒5型； 疱疹家族的病毒包含例如单纯疱疹病毒(HSV)和艾巴氏病毒(EBV)以及巨细胞病毒(CMV)和假狂犬病病毒(PRV)。人类、非人类哺乳动物和禽类来源的许多腺病毒、疱疹病毒等是已知的并且可从如ATCC等机构获得。
“辅助病毒功能”或“辅助功能”是指在辅助病毒基因组中编码的允许AAV复制和包装(结合对本文所述的复制和包装的其它需求)的功能。本公开中，“辅助病毒功能”可以呈许多方式提供，包括通过提供辅助病毒或提供例如编码必需功能的多核苷酸序列至在转运中的生产细胞。
“感染性”病毒或病毒颗粒是包括适合地组装的病毒衣壳并且能够将多核苷酸组分递送到病毒种类具有趋向性的细胞中的病毒或病毒颗粒，不必然暗示病毒有任何复制能力。本领域公知对感染性病毒颗粒进行计数的测定。病毒感染性可以表达为感染性病毒颗粒与总病毒颗粒之比。确定感染性病毒颗粒与总病毒颗粒之比的方法是本领域已知的。参见例如Grainger等人(2005)《分子疗法(Mol.Ther.)》11：S337(描述了TCID 50感染滴度测定)；Zolotukhin等人(1999)《基因疗法(Gene Ther.)》6：973。
“趋向性”或“特异性”是指病毒(例如，AAV)优先靶向特定宿主物种的细胞或宿主物种内的特定细胞类型。例如，相对于仅可能感染肺和肌肉细胞的病毒，可以感染心脏、肺、肝和肌肉细胞的病毒具有更宽(即增加)的趋向性。趋向性还可以包含病毒对宿主的特定类型的细胞表面分子的依赖性。例如，一些病毒只可能感染具有表面糖胺聚糖的细胞，而其它病毒只可能感染具有唾液酸的细胞(这种依赖性可以使用缺乏特定类别分子的各种细胞系作为病毒感染的潜在宿主细胞进行测试)。在某些情况下，病毒的趋向性描述了病毒的相对偏好。例如，第一种病毒可能能够感染所有细胞类型，但在用表面糖胺聚糖感染这些细胞方面更成功。如果第二种病毒也更喜欢相同的特征(例如，第二种病毒在用表面糖胺聚糖感染这些细胞方面也更成功)，则可以认为第二种病毒与第一种病毒具有相似(或一致)的趋向性，即使绝对转导效率不相似。例如，第二种病毒在感染每种测试的给定细胞类型时可能比第一种病毒更有效，但如果相对偏好相似(或一致)，则第二种病毒仍可被认为具有与第一种病毒相似(或一致)的趋向性。在一些实施例中，相对于天然存在的病毒粒子，包括本公开变体AAV衣壳蛋白的病毒粒子的趋向性没有改变。在一些实施例中，相对于天然存在的病毒粒子，包括本公开变体AAV衣壳蛋白的病毒粒子的趋向性被扩展(即，变宽)。在一些实施例中，相对于天然存在的病毒粒子，包括本公开变体AAV衣壳蛋白的病毒粒子的趋向性降低。
“多核苷酸”是指任何长度的核苷酸的聚合形式或其类似物，包含脱氧核糖核苷酸或核糖核苷酸。多核苷酸可以包括经过修饰的核苷酸，如甲基化核苷酸和核苷酸类似物，并且可以被非核苷酸组分打断。对核苷酸结构的修饰可以在组装聚合物之前或之后进行。多核苷酸可互换地指双链分子和单链分子，除非另外指明，否则本公开的多核苷酸涵盖双链形式以及构成双链形式的两种互补的单链形 式。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同核苷酸或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被相同核苷酸占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。序列相似性可以许多不同的方式测定。为了测定序列同一性，可使用方法和计算机程序比对序列，计算机程序包括可由因特网址ncbi.nlm.nih.gov/BLAST/获得的BLAST。另一种比对算法是FASTA，可由Madison,Wisconsin,USA,a wholly owned subsidiary of Oxford Molecular Group,Inc的Genetics Computing Group(GCG)包装中获得。其他比对技术描述于Enzymology,第266卷：Computer Methods for Macromolecular Sequence Analysis(1996),Doolittle编著,Academic Press,Inc.,adivision of Harcourt Brace&Co.,SanDiego,California,USA的方法中。特别关注的是容许序列中的缺口的对比程序。Smith-Waterman是容许序列比对中的缺口的一种类型的算法。参见Meth.Mol.Biol.70：173-187(1997)。使用Needleman和Wunsch对比方法的GAP程序也可用于比对序列。参见J.Mol.Biol.48：443-453(1970)。
“基因”是指含有至少一个开放读码框的能够在转录并且有时翻译后对特定基因产物进行编码的多核苷酸。“基因”或“编码序列”是指对基因产物进行编码的体外或体内核苷酸序列。一些情况下，基因由编码序列组成或基本上由编码序列组成，所述编码序列即对基因产物进行编码的序列。另一些情况下，基因包括另外的非编码序列。例如，基因可以包含或可以不包含在编码区域之前和之后的区域(例如5'UTR、3'UTR以及各个编码区段(外显子)之间的插入序列(内含子))。
“基因产物”是由特定基因的表达产生的分子，例如多肽、适配体、干扰RNA、mRNA等。一些实施方案中，“基因产物”是多肽、肽、蛋白质或干扰RNA，包含短干扰RNA(siRNA)、miRNA或小发夹RNA(shRNA)。一些具体实施方案中，基因产物是治疗性基因产物，例如治疗性多肽。所述治疗性基因产物对其所在的细胞、组织或哺乳动物赋予了有益效果，所述有益效果包含改善病状或疾病的体征或症状、预防或抑制病状或疾病或者赋予期望的特性。当基因编码多肽时，“基因产物”和“目的基因表达的产物”可互用。
“RNA干扰剂”或“RNAi试剂”包括可用于改变基因(如上所定义)表达的任何试剂(或编码这种试剂的多核苷酸)。本领域普通技术人员已知的RNAi试剂的实例包括但不限于(i)siRNA试剂(“小干扰”或“短干扰RNA”(或siRNA))；(ii)反义RNA；(iii)CRISPR试剂；(iv)锌指核酸酶试剂；以及(v)转录激活因子样效应子核酸酶(TALEN)试剂。这里全文引入WO2017197355A对(i)-(v)的定义。
其中，siRNA试剂是靶向所关注基因(“靶基因”)的核苷酸的RNA双链体。 “RNA双链体”是指由RNA分子的两个区域之间的互补配对形成的结构，从而形成双链RNA的区域(dsRNA)。siRNA“靶向”基因是因为siRNA的双链体部分的核苷酸序列与靶基因的核苷酸序列互补。在一些实施方案中，siRNA的双链体长度小于30个核苷酸。在一些实施方案中，双链体可以是29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11或10个核苷酸长度。在一些实施方案中，双链体的长度是19-25个核苷酸长度。siRNA的RNA双链体部分可以是发夹结构的一部分。含有发夹的siRNA试剂也可被称为“shRNA(短发夹RNA)试剂”。除双链体部分之外，发夹结构还可含有位于形成双链体的两条序列之间的环部分。环在长度方面可变化。在一些实施方案中，环的长度是5、6、7、8、9、10、11、12或13个核苷酸。发夹结构还可含有3'或5'突出端部分。在一些实施方案中，突出端是0、1、2、3、4或5个核苷酸长度的3'或5'突出端。一般说来，靶基因的表达产物(例如，mRNA、多肽等等)水平通过siRNA试剂(例如，siRNA、shRNA等等)而降低，所述siRNA试剂含有至少与靶基因转录产物的19-25个核苷酸长度的区段(例如，20-21个核苷酸序列)互补的特定双链核苷酸序列，包括5'非翻译(UTR)区、ORF、或3'UTR区。在一些实施方案中，短干扰RNA的长度为约19-25nt。参见，例如PCT申请WO0/44895、WO99/32619、WO01/75164、WO01/92513、WO01/29058、WO01/89304、WO02/16620、及WO02/29858；以及美国专利公布号20040023390(关于siRNA技术的描述)。siRNA和/或shRNA可由核酸序列编码，且该核酸序列还可包括启动子。该核酸序列还可包括聚腺苷酸化信号。在一些实施方案中，聚腺苷酸化信号是合成的最小聚腺苷酸化信号。
其中，反义RNA是与基因表达产物互补的RNA。举例来说，靶向特定mRNA的反义RNA是与mRNA互补的基于RNA的试剂(或可以是修饰的RNA)，其中反义RNA与mRNA的杂交改变mRNA的表达(例如，经由改变RNA的稳定性、改变RNA的翻译，等等)。编码反义RNA的核酸也包括在“反义RNA”中。
“VEGF”是指诱导血管生成或血管生成过程的血管内皮生长因子，包含通过例如替代性地剪接VEGF-A/VPF基因产生的各种亚型的VEGF(也称为血管通透因子(VPF)和VEGF-A)(参见美国专利申请公开第20120100136号的图2(A)和(B))，包含VEGF121、VEGF165和VEGF189。进一步地，“VEGF”包含VEGF相关血管生成因子，如PIGF(胎盘生长因子)、VEGF-B、VEGF-C、VEGF-D和VEGF-E，所述VEGF相关血管生成因子通过同源VEFG受体(即VEGFR)起作用以诱导血管生成或血管生成过程。
“sFlt-1”或“sFlt-1蛋白”在本文中是指与天然存在的人sFLT-1序列至少90％或更高同源，使得sFlt-1蛋白或多肽与VEGF和/或VEGF受体结合的多肽序列或其功能片段。
“多肽”、“肽”和“蛋白质”是指任何长度的氨基酸的聚合物，还包含被修饰的氨基酸聚合物，例如二硫键形成、糖基化、脂化、磷酸化或与标记组分缀 合。
“调控元件”或“调控序列”是参与分子的相互作用的核苷酸序列，所述相互作用有助于对多核苷酸进行功能调节，包含多核苷酸的复制、重复、转录、剪接、翻译或降解。调节会影响过程的频率、速度或特异性并且在本质上会具有增强或抑制性。已知的控制元件包含例如转录调节序列，如启动子和增强子。启动子是在某些条件下能够结合RNA聚合酶并且启动对通常位于启动子下游(沿3'方向)的编码区进行转录的DNA区域。
“表达载体”是包括对目的基因产物进行编码的区域的载体，并且用于在预期靶细胞中实现基因产物的表达，所述载体包括对关注的基因产物进行编码的多核苷酸。表达载体还包括与编码区域操作性地连接的以促进基因产物在靶标中表达的控制元件。控制元件例如启动子、增强子、UTR、miRNA靶向序列等和与其可操作地连接以供表达的一个或多个基因的组合有时被称为“表达盒”。许多表达盒在本领域中是已知且可获得的，或者可以由本领域中可获得的组分容易构建的。
“操作性地连接”或“可操作地连接”是指遗传元件的并列，其中元件处于允许其按预期方式操作的关系中。例如，如果启动子帮助启动编码多核苷酸序列的转录，则启动子与编码多核苷酸序列操作性地连接，在启动子与编码多核苷酸序列之间可能有插入核酸残基，只要维持此功能关系即可。
“施用”或“引入”是指将用于重组基因或蛋白表达的载体递送到细胞、受试者的细胞和/或器官或者受试者。这种施用或引入可以在体内、在体外或离体地发生。用于表达基因产物的载体可以通过以下引入到细胞中：转染，其通常意指通过物理手段(例如，磷酸钙转染、电穿孔、微注射或脂质转染)将异源DNA插入到细胞中；感染，其通常是指通过感染剂即病毒引入；或者转导，其通常意指用细胞稳定感染细胞或将来自一个微生物的基因材料通过病毒剂(例如细菌噬菌体)转移到另一个微生物。“转化”通常用于指包括异源DNA的细菌或表达致癌基因并且已经转换为连续生长模式的细胞，如肿瘤细胞。用于“转化”细胞的载体可以是质粒、病毒或其它媒剂。根据用于将异源DNA(即载体)施用、引入或插入到细胞中的方式，细胞通常被称为“转导”、“感染”、“转染”或“转化”。“转导”、“转染”和“转化”可以在本文中可互换地使用，不考虑异源DNA的引入方法。
“宿主细胞”是指已经用载体转导、感染、转染或转化的细胞，涵盖最初转导、感染、转染或转化的细胞和其子代。载体可以是质粒、病毒颗粒、噬菌体等。如温度、pH等培养条件对于本领域技术人员而言将是显而易见的。
“治疗”通常用于意指获得期望的药理学和/或生理学效果。效果在完全或部分地预防疾病或其症状方面可以是预防性的，例如降低受试者体内发生疾病或其症状的可能性，和/或在部分或完全治愈疾病和/或由疾病引起的不良反应方面可以 是治疗性的。“治疗”覆盖对哺乳动物的疾病的任何治疗，并且包含：(a)防止疾病在可能倾向于患有疾病但尚未被诊断为患有疾病的受试者身上发生；(b)抑制或中止疾病发展；或者(c)缓解疾病(或其引起的症状)或使疾病消退。可以在疾病或损伤发作之前、期间或之后施用治疗剂。特别是对发展中的疾病的治疗，其中所述治疗稳定或减少了患者的不期望的临床症状。一些优选方案是，在受影响组织的功能完全丧失之前执行这种治疗。一些优选方案是，将在疾病的症状期期间并且在一些情况下在疾病的症状期之后施用本公开的治疗。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“视网膜细胞”在本公开中可以指包含视网膜的细胞类型中的任一种，例如视网膜神经节细胞；无长突细胞；水平细胞；双极细胞；包括视杆和视锥的感光细胞；米勒胶质细胞(Müller glial cell)；星形细胞(例如视网膜星形细胞)；和视网膜色素上皮细胞。
“个体”、“受试者”和“患者”在本文中可互换地使用，包含但不限于人类和非人类灵长类动物，如猿猴、人类和其他哺乳类动物(例如马、绵羊、山羊、狗、猫，以及啮齿动物(例如小鼠、大鼠等))，优选为人。
本公开中，“多肽”和“蛋白”可互换使用。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，则该试剂可自任意分子生物学试剂的供应商以用于分子生物学应用的质量/纯度而获得。
实施例1.AAV衣壳改造
分子动力学模拟和蛋白质指纹技术在蛋白质工程、抗体亲和性改造中被广泛应用，通过对抗体和受体的理化性质的动力学模拟，在一定程度上可以对氨基酸改变造成的局部亲和力影响进行预测。
已证实硫酸乙酰肝素受体为介导AAV细胞感染的最主要受体，在视网膜和是视神经细胞的感染中也起到重要的作用，研究证实在AAV2天然血清型衣壳蛋白氨基酸序列依照其空间结构特征，有5个环状结构域(图1所示)与病毒的感染能力相关。其中，环状结构域IV的第587和588位点中间插入7-12个氨基酸的短肽不会影响病毒衣壳蛋白的组装，同时能够大幅度的改变AAV衣壳与细胞受体(硫酸乙酰肝素)的相互作用方式，从而影响AAV对宿主细胞的感染能力。
本公开利用自主研发的生物信息学算法，在AAV2天然血清型衣壳蛋白氨基酸序列中的第587和588位点之间插入若干随机氨基酸序列(7-12个氨基酸长度)，并预测衣壳蛋白与硫酸乙酰肝素受体间的相互作用。选取算法中预测能够较大提升感染能力的序列进行后续验证。根据算法预测，选取如表1所示的6个氨基酸序列seq1至seq6，每个序列的长度均为10个氨基酸。同时，将AAV2天然血清型衣壳蛋白氨基酸序列中第708位氨基酸-缬氨酸(V)替换为异亮氨酸(I)。587和588位之间的插入方式和708位的氨基酸替换方式如图2所示，获得经工程改造的AAV衣壳蛋白序列。同时，制备获得seq2插入AAV9相应位置的衣壳。
表1.AAV衣壳的第587和588位之间插入的氨基酸序列及其对应编号
Figure PCTCN2022095422-appb-000004
(注：下划线为连接子。)
由于AAV衣壳蛋白表达后是由VP1、VP2、VP3三种单体蛋白按一定比例组装而成，相比于普通的重组蛋白具有更高一级的的结构，因此发生在AAV衣壳蛋白的多肽插入造成的功能影响不只限于其带来的理化性质改变，更有可能在VP1、VP2、VP3三种单体蛋白的组装过程中施加更为复杂的影响。
实施例2.rAAV载体病毒感染小鼠模型视网膜能力检测
为验证功能，我们将具有SEQ ID NO：3至SEQ ID NO：8、AAV2(SEQ ID NO：1)、AAV2.7m8(SEQ ID NO：2)这8种序列的衣壳，分别包装质粒pGOI，所述质粒含有并能表达CMV启动子驱动的EGFP目的基因。将上述不同AAV衣壳包装的具有EGFP表达能力的AAV载体对小鼠玻璃体进行等剂量注射，通过荧光眼底照相来检测EGFP的表达，以EGFP荧光信号的强度差异来评估不同AAV衣壳的对视网膜组织感染效率的差异。
rAAV载体病毒的包装和纯化步骤包括：将pHelper、pR2C9、pGOI三质粒系统转染293T贴壁细胞，收获细胞和上清，通过碘克沙醇离心法进行纯化，再经超滤浓缩，将缓冲液置换为DPBS。病毒包装纯化完成后，通过qPCR检测滴度(单位vg)。将表达EGFP目的基因的8种病毒(衣壳分别为AAV2、AAV2.7m8、AAV2 seq1、AAV2 seq2、AAV2 seq3、AAV2 seq4、AAV2 seq5、AAV2 seq6)，分别用 DPBS缓冲液稀释至同一滴度1.03E+12。对周龄为10周的C57小鼠(购于北京维通利华实验技术有限公司)进行单侧眼玻璃体注射，注射体积为1μL，每种AAV载体注射3只(n＝3)，注射后1周至4周，每周进行活体荧光眼底照相。
部分结果如图3所示，其中AAV2组、AAV2 seq5组、AAV2 seq6组，荧光信号强度明显弱于其他组。而AAV2 seq1组、AAV2 seq2组、AAV2 seq3组、AAV2 seq4组荧光信号强度与AAV2.7m8较为相似，显示其感染视网膜组织的效率较高。
将表1中的“LALGDTTRPA”序列插入AAV9衣壳中的同一位置(第588和589位之间)，获得AAV9 seq2。将AAV2、AAV2.7m8、AAV2 seq1、AAV2 seq2、AAV2 seq3、AAV2 seq4、AAV9 seq2共7种衣壳，分别包装质粒载体，所述质粒载体含有并能表达CMV启动子驱动的EGFP目的基因。将上述不同AAV衣壳包装的具有EGFP表达能力的AAV载体对小鼠玻璃体进行等剂量注射，通过荧光眼底照相来检测EGFP的表达，以EGFP信号的强度差异来评估不同AAV衣壳的对视网膜组织感染效率的差异。
使用上述方法对AAV病毒进行载体包装和纯化。将表达EGFP目的基因的7种病毒(衣壳分别为AAV2、AAV2.7m8、AAV2 seq1、AAV2 seq2、AAV2 seq3、AAV2 seq4、AAV2 seq5、AAV9 seq2)，分别用DPBS缓冲液稀释至同一滴度1.03E+12。对周龄为10周的C57小鼠进行单侧眼玻璃体注射，注射体积为1μL，每种AAV载体注射3只(n＝3)，注射后1周至4周，每周进行活体荧光眼底照相。
结果如图4A、图4B所示，其中AAV2组、AAV2 seq2组、AAV2 seq3组，荧光信号强度相较其他组明显较强，且与AAV2.7m8相似。
实施例3.rAAV载体病毒对小鼠模型视网膜感染的生物学功能检测
为进一步验证seq2衣壳对视网膜组织的感染能力和其所关联的生物学功能，将AAV2 seq2和AAV2.7m8分别包装含有CMV启动子驱动的阿柏西普(aflibercept)目的质粒载体(AAV2 seq2-aflibercept，AAV2.7m8-aflibercept)，两种病毒分别对新西兰兔和C57小鼠进行玻璃体注射，并于注射两周后检测注射眼组织中的阿柏西普蛋白含量，以评估对应AAV病毒对视网膜的感染和其关联的生物学功能。
其中，rAAV递送的基因表达盒的结构从其从5’端至3’端含有(如图5A)：CMV增强子、启动子、5’UTR、Kozak序列(gccacc)、VEGF抑制剂阿柏西普、3’UTR、WPRE以及SV40polyA。其中，CMV增强子的碱基序列如SEQ ID NO：28所示，CMV启动子碱基序列如SEQ ID NO：29所示，5’UTR如SEQ ID NO：30所示，Kozak序列如SEQ ID NO：31所示、3’UTR如SEQ ID NO：32所示、WPRE如SEQ ID NO：33所示、SV40polyA如SEQ ID NO：34所示。
表达载体的构建为：构建表达盒，通过酶切、连接、转化和克隆筛选鉴定等常规分子生物学操作构建了表达阿柏西普的AAV包装质粒，其依次包括：CMV增强子、启动子、5’UTR、Kozak序列(gccacc)、VEGF trap(阿柏西普)、3’UTR、 WPRE以及SV40polyA，表达盒两侧是反向末端重复序列(ITR)，其结构图示意图见图5B，EcoRV和BSMI为酶切位点。
按照实施例2中的方法包装载体和纯化rAAV载体病毒。
将纯化后的AAV seq2-aflibercept和AAV2.7m8-aflibercept这两种病毒分别用DPBS缓冲液稀释至同一滴度9E+12vg/mL，对周龄为8-10周的C57小鼠进行双眼玻璃体注射，注射体积为1μL，每种rAAV载体病毒注射4只(n＝4)。注射后2周，处死小鼠，取眼组织分别研磨，每个注射组左眼和右眼分别用两种不同的方法进行组织研磨处理。左眼使用方法1：PBS+蛋白酶抑制剂研磨，离心后取上清。右眼使用方法2：RIP Buffer+蛋白酶抑制剂研磨，离心后取上清。使用酶联免疫分析法检测上清中的阿柏西普蛋白含量(每个注射组4个样本，每个样本检测三重复)。结果如图6所示。其中空白对照为PBS注射组，纵坐标HRAMD/总蛋白为每mg组织总蛋白中含有多少ng阿柏西普蛋白。
表2.小鼠眼组织中目的基因蛋白(阿柏西普)的表达量
组织 空白对照 AAV2 seq2 AAV2.7m8
左眼 0.01±0.01 1.10±0.59 0.39±0.17
右眼 0 1.66±1.08 0.44±0.11
图6、表2结果显示，小鼠中AAV seq2-aflibercept组的表达量高于AAV2.7m8-aflibercept组。
实施例4.rAAV载体病毒对兔模型视网膜感染的生物学功能检测
按照实施例2中的方法包装载体和纯化rAAV载体病毒。
将纯化后的AAV seq2-aflibercept和AAV2.7m8-aflibercept这两种病毒分别用DPBS缓冲液稀释至4E+9vg/mL,对周龄为8-10周的新西兰兔(购自北京维通利华实验技术有限公司)进行双眼玻璃体注射，注射体积为50μL，每种rAAV载体病毒注射2只(n＝2)。注射后2周，处死兔，取房水、玻璃体和全眼组织，分别研磨，每个注射组左眼和右眼分别用两种不同的方法进行组织研磨处理。左眼使用方法1：PBS+蛋白酶抑制剂研磨，离心后取上清。右眼使用方法2：RIP Buffer+蛋白酶抑制剂研磨，离心后取上清。使用酶联免疫分析法检测上清中的阿柏西普蛋白含量(每个注射组2个样本，每个样本检测三重复)。结果如图7所示。其中空白对照为PBS注射组，纵坐标HRAMD/总蛋白为每mg组织总蛋白中含有多少ng阿柏西普蛋白。
图7结果显示，新西兰兔中AAV seq2-aflibercept组的表达量均高于AAV2.7m8-aflibercept组。由于兔眼体积较大，阿柏西普在全眼研磨处理后被稀释在组织液中，因此浓度远低于玻璃体和房水。由于AAV包装的载体只有在AAV病毒成功感染细胞后才会成功表达所携带的目的基因蛋白，因此图7结果表明，AAV2 seq2衣壳对细胞的感染效率要高于AAV2.7m8衣壳。
此外，我们还比对了AAV2 seq2和AAV2.7m8衣壳递送目的基因蛋白(VEGF抑制剂)的表达量进行了检测。实验方法为：新西兰兔8只，按照表3剂量分别进行双眼注射，每个剂量组注射2只兔，四只眼，共计4组。注射后第28天处死实验动物并抽取房水，使用ELISA方法检测房水样本中的目的基因蛋白的摩尔浓度。
结果如图8所示，等剂量注射下，AAV2 seq2所表达的目的基因蛋白的摩尔浓度明显高于AAV2.7m8。
表3.
组别 病毒滴度 注射体积 vg/眼
AAV2 seq2(高) 6E12vg/mL 100μL 6E11vg/眼
AAV2 seq2(低) 2E12vg/mL 100μL 2E11vg/眼
AAV2.7m8(高) 6E12vg/mL 100μL 6E11vg/眼
AAV2.7m8(低) 2E12vg/mL 100μL 2E11vg/眼
以下为本公开的部分序列：
>AAV2衣壳氨基酸序列
Figure PCTCN2022095422-appb-000005
>AAV2.7m8衣壳氨基酸序列
Figure PCTCN2022095422-appb-000006
Figure PCTCN2022095422-appb-000007
>AAV2 seq1衣壳氨基酸序列
Figure PCTCN2022095422-appb-000008
>AAV2 seq2衣壳氨基酸序列
Figure PCTCN2022095422-appb-000009
>AAV2 seq3衣壳氨基酸序列
Figure PCTCN2022095422-appb-000010
Figure PCTCN2022095422-appb-000011
>AAV2 seq4衣壳氨基酸序列
Figure PCTCN2022095422-appb-000012
>AAV2 seq5衣壳氨基酸序列
Figure PCTCN2022095422-appb-000013
>AAV2 seq6衣壳氨基酸序列
Figure PCTCN2022095422-appb-000014
Figure PCTCN2022095422-appb-000015
>AAV9 seq2衣壳氨基酸序列
Figure PCTCN2022095422-appb-000016
>EGFP氨基酸序列
Figure PCTCN2022095422-appb-000017
>seq1(无连接子)
Figure PCTCN2022095422-appb-000018
>seq2(无连接子)
Figure PCTCN2022095422-appb-000019
>seq3(无连接子)
Figure PCTCN2022095422-appb-000020
>seq4(无连接子)
Figure PCTCN2022095422-appb-000021
>seq5(无连接子)
Figure PCTCN2022095422-appb-000022
>seq6(无连接子)
Figure PCTCN2022095422-appb-000023
>seq1
Figure PCTCN2022095422-appb-000024
Figure PCTCN2022095422-appb-000025
>seq2
Figure PCTCN2022095422-appb-000026
>seq3
Figure PCTCN2022095422-appb-000027
>seq4
Figure PCTCN2022095422-appb-000028
>seq5
Figure PCTCN2022095422-appb-000029
>seq6
Figure PCTCN2022095422-appb-000030
>AAV2 seq2衣壳氨基酸序列(不包含V708I)
Figure PCTCN2022095422-appb-000031
>AAV2 seq3衣壳氨基酸序列(不包含V708I)
Figure PCTCN2022095422-appb-000032
>AAV2 seq4衣壳氨基酸序列(不包含V708I)
Figure PCTCN2022095422-appb-000033
>AAV2 seq5衣壳氨基酸序列(不包含V708I)
Figure PCTCN2022095422-appb-000034
>AAV2 seq6衣壳氨基酸序列(不包含V708I)
Figure PCTCN2022095422-appb-000035
>CMV增强子序列
Figure PCTCN2022095422-appb-000036
Figure PCTCN2022095422-appb-000037
>CMV启动子序列
Figure PCTCN2022095422-appb-000038
>5’UTR序列
Figure PCTCN2022095422-appb-000039
>Kozak序列
Figure PCTCN2022095422-appb-000040
>3’UTR序列
Figure PCTCN2022095422-appb-000041
>WPRE序列
Figure PCTCN2022095422-appb-000042
>SV40polyA序列：
Figure PCTCN2022095422-appb-000043
>AAV9衣壳氨基酸序列
Figure PCTCN2022095422-appb-000044
>阿柏西普蛋白序列
Figure PCTCN2022095422-appb-000045
>阿柏西普核酸序列1
Figure PCTCN2022095422-appb-000046
>阿柏西普核酸序列2
Figure PCTCN2022095422-appb-000047
>阿柏西普核酸序列3
Figure PCTCN2022095422-appb-000048

Claims (21)

  1. 一种变体腺相关病毒(AAV)衣壳蛋白，其相对于亲本AAV衣壳蛋白包含插入的多肽，所述插入的多肽包含选自如下1)-6)任一项所示的多肽或其任意组合：
    1)LGDTTRP(SEQ ID NO：12)或LALGDTTRPA(SEQ ID NO：18)；
    2)LAETTRP(SEQ ID NO：11)或LALAETTRPA(SEQ ID NO：17)；
    3)LGETTRN(SEQ ID NO：13)或LALGETTRNA(SEQ ID NO：19)；
    4)KADTTKN(SEQ ID NO：14)或LAKADTTKNA(SEQ ID NO：20)；
    5)KDDTTRN(SEQ ID NO：15)或LAKDDTTRNA(SEQ ID NO：21)；
    6)LADTTKN(SEQ ID NO：16)或LALADTTKNA(SEQ ID NO：22)。
  2. 如权利要求1所述的变体AAV衣壳蛋白，所述1)-6)任一项的多肽或其任意组合位于所述AAV衣壳蛋白的GH环中；
    优选地，位于亲本AAV2衣壳蛋白的VP1编码氨基酸序列的第570位至611位之间的任一个氨基酸残基之后；
    更优选地，位于亲本AAV2衣壳蛋白的VP1编码氨基酸序列的第587位和588位之间，或亲本AAV9衣壳蛋白的VP1编码氨基酸序列的第588位和589位之间，或其他亲本AAV血清型衣壳蛋白的相应位置。
  3. 如权利要求1或2所述的变体AAV衣壳蛋白，其进一步包含选自1L、15P、34A、57D、66K、81Q、101R、109T、144K、144M、164K、176P、188I、196Y、226E、236V、240T、250S、312K、363L、368H、449D、456K、463Y、472N、484C、524T、535S、551S、593E、698V、708I、719M、721L和735Q中的一个或多个氨基酸残基的点突变，所述突变的位置基于SEQ ID NO：1所示的AAV2的衣壳蛋白，或其他AAV血清型衣壳蛋白的相应位置；
    优选地，所述点突变是708I。
  4. 如权利要求1-3中任一项所述的变体AAV衣壳蛋白，其氨基酸序列为SEQ ID NO：3-9和23-27中任一所示或与之具有至少90％或95％的序列同一性。
  5. 一种重组腺相关病毒(rAAV)病毒粒子，其包含：
    (a)权利要求1-4任一项所述的变体AAV衣壳蛋白，和
    (b)异源多核苷酸。
  6. 如权利要求5所述的rAAV病毒粒子，其中所述异源多核苷酸包含编码基因产物的多核苷酸，所述基因产物选自多肽、干扰RNA或适配体；
    所述多肽优选为抗体或其抗原结合片段、融合蛋白。
  7. 如权利要求6所述的rAAV病毒粒子，其中所述异源多核苷酸进一步包含选自以下(a)至(g)的一个或多个：
    (a)5’反向末端重复(5’ITR)和/或3’反向末端重复(3’ITR)，
    (b)5’非翻译区(5’UTR)和/或3’非翻译区(3’UTR)，
    (c)启动子，
    (d)增强子，
    (e)转录后调控元件，
    (f)多聚腺苷酸化信号(polyA)，和
    (g)Kozak序列；
    优选地，所述异源多核苷酸进一步包含选自以下(a)至(g)的一个或多个：
    (a)源自AAV2的5’ITR和/或3’ITR，
    (b)源自Xenopus globin的5’UTR和/或3’UTR，
    (c)CMV启动子，
    (d)CMV增强子，
    (e)WPRE，
    (f)SV40 polyA，和
    (g)Kozak序列。
  8. 如权利要求7所述的rAAV病毒粒子，其中所述异源多核苷酸从5’端到3’端包含如下可操作地连接并按如下(a)至(h)顺序排列的多核苷酸：
    (a)增强子，
    (b)启动子，
    (c)5’UTR，
    (d)Kozak序列,
    (e)编码基因产物的多核苷酸，
    (f)3’UTR，
    (g)WPRE，
    (h)polyA。
  9. 如权利要求6-8中任一项所述的rAAV病毒粒子，所述多肽为神经保护多肽、抗血管生成多肽、增强视网膜细胞功能的多肽；
    优选地，所述抗血管生成多肽为VEGF拮抗剂；
    更优选地，所述VEGF拮抗剂为阿柏西普(aflibercept)。
  10. 如权利要求5-9中任一项所述的rAAV病毒粒子，所述异源多核苷酸包含 编码SEQ ID NO：38所示的阿柏西普(aflibercept)的多核苷酸，
    优选地，所述异源多核苷酸包含SEQ ID NO：39-41中任一所示或与之具有至少90％或95％序列同一性的多核苷酸。
  11. 分离的多核苷酸，其编码权利要求1-4中任一项所述的变体AAV衣壳蛋白。
  12. 载体，其包含：
    (a)权利要求11所述的分离的多核苷酸，或
    (b)权利要求11所述的分离的多核苷酸和权利要求5-10中任一项所述的异源多核苷酸。
  13. 宿主细胞，其包含权利要求12所述的载体。
  14. 药物组合物，其含有
    (a)权利要求5-10中任一项所述rAAV病毒粒子；和
    (b)一种或多种药学上可接受的载剂、稀释剂、赋形剂或缓冲液。
  15. 特异性感染视网膜细胞的方法，所述方法包括眼内注射有效量的根据权利要求5-10中任一项所述的rAAV病毒粒子、权利要求5-10中任一项所述的异源多核苷酸或权利要求14所述的药物组合物的步骤；优选通过玻璃体内注射或视网膜下注射。
  16. 治疗眼部相关疾病的方法，包括向有需要的受试者施用预防或治疗有效量的权利要求5-10中任一项所述的rAAV病毒粒子、权利要求5-10中任一项所述的异源多核苷酸或权利要求14所述的药物组合物；优选通过玻璃体内注射或视网膜下注射。
  17. 如权利要求16所述的方法，其中所述眼部相关疾病为视网膜细胞疾病；优选地，所述视网膜细胞疾病选自感光细胞、视网膜神经节细胞、Muller细胞、双极细胞、无长突细胞、水平细胞或视网膜色素上皮细胞的疾病。
  18. 如权利要求16或17所述的方法，其中所述眼部相关疾病选自：视网膜色素变性、黄斑变性、湿性AMD、干性AMD、视网膜新生血管、脉络膜新生血管、糖尿病性视网膜病变、增生性糖尿病视网膜病变、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、糖尿病性黄斑水肿、糖尿病性视网膜缺血、缺血性视网膜病变、糖尿病性视网膜水肿、视网膜劈裂症、青光眼、利伯氏先天性 黑朦和/或色盲。
  19. 在体外和/或体内向靶细胞递送异源多核苷酸的方法，其包括使所述靶细胞与根据权利要求5-10中任一项的rAAV病毒粒子、权利要求5-10中任一项所述的异源多核苷酸或权利要求14所述的药物组合物接触。
  20. rAAV病毒粒子生产系统，包含：
    (a)如权利要求11所述的分离的多核苷酸；
    (b)编码基因产物的异源多核苷酸；和
    (c)辅助元件，其具有足够的AAV rep功能和辅助功能，以将(b)的编码基因产物的异源多核苷酸包装到(a)的变体AAV衣壳中。
  21. 生产或制备权利要求5-10中任一项所述rAAV病毒粒子的方法，包括：将权利要求12所述的载体、包含辅助元件的载体引入生产细胞中，包装、纯化所述rAAV病毒粒子。
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CN116789738A (zh) * 2022-12-08 2023-09-22 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN116789738B (zh) * 2022-12-08 2023-12-19 广州派真生物技术有限公司 腺相关病毒突变体及其应用
CN116836237A (zh) * 2023-06-16 2023-10-03 广州译码基因科技有限公司 提高视网膜靶向性的aav病毒衣壳蛋白突变体及其应用
CN116836237B (zh) * 2023-06-16 2023-12-05 广州译码基因科技有限公司 提高视网膜靶向性的aav病毒衣壳蛋白突变体及其应用

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