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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English (en)
French (fr)
Chinese (zh)
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张文涛
廖成
宁威
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Shanghai Regenelead Therapie Co Ltd
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Shanghai Regenelead Therapie Co Ltd
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Priority to AU2022281825A priority Critical patent/AU2022281825A1/en
Priority to BR112023024375A priority patent/BR112023024375A2/pt
Priority to CA3219898A priority patent/CA3219898A1/en
Priority to US18/564,584 priority patent/US20240252679A1/en
Priority to KR1020237043802A priority patent/KR20240014477A/ko
Priority to JP2023571788A priority patent/JP2024519888A/ja
Priority to CN202280028081.9A priority patent/CN117157309A/zh
Priority to MX2023014041A priority patent/MX2023014041A/es
Priority to EP22810641.5A priority patent/EP4349852A4/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.

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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
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