WO2024002076A1 - 一种治疗新生血管相关眼底疾病的aav药物 - Google Patents

一种治疗新生血管相关眼底疾病的aav药物 Download PDF

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WO2024002076A1
WO2024002076A1 PCT/CN2023/102771 CN2023102771W WO2024002076A1 WO 2024002076 A1 WO2024002076 A1 WO 2024002076A1 CN 2023102771 W CN2023102771 W CN 2023102771W WO 2024002076 A1 WO2024002076 A1 WO 2024002076A1
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vegf
aav
expression cassette
protein
fusion protein
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PCT/CN2023/102771
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French (fr)
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谭青乔
曹曦
陈秋宇
周睿
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上海鼎新基因科技有限公司
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Definitions

  • the invention belongs to the technical field of recombinant adeno-associated virus (rAAV) gene therapy, involves the preparation of rAAV-delivered Fc-engineered VEGF receptor fusion protein or anti-VEGF antibody, and is applied to the treatment of angiogenesis-related fundus diseases, such as age-related macula. Treatment of degeneration, wet macular degeneration, diabetic retinopathy and other diseases.
  • rAAV adeno-associated virus
  • Angiogenesis can occur in intraocular tissues such as the retina, choroid, macula, optic disc, cornea, iris and ciliary body, causing pathological changes such as bleeding, exudation and proliferation of tissues in these parts, including age-related macular degeneration (Age-related Macular Degeneration). , AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), etc., seriously affect vision and are the main causes of blindness among the elderly.
  • AMD age-related macular degeneration
  • DR diabetic retinopathy
  • ROP retinopathy of prematurity
  • VEGF Vascular endothelial growth factor
  • Diabetic retinopathy is a common microvascular complication of diabetes, with a high blindness rate and complex pathogenesis.
  • the pathological characteristics of DR are retinal neovascularization and blood-retinal barrier destruction.
  • the therapeutic application of anti-VEGF drugs is also relatively common.
  • AAV recombinant adeno-associated virus
  • anti-VEGF therapy has become the first-line treatment for neovascular eye diseases.
  • Drugs approved for clinical use include antibodies and antibody fragments (Bevacizumab, Ranibizumab and Brolucizumab), fusion proteins (Aflibercept and Conbercept), and nucleic acid aptamers Pegaptanib and double-antibody Vabysmo (Faricimab-svoa).
  • Eylea is a recombinant fusion protein composed of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused with the Fc fragment of IgG1. It inhibits neovascularization and reduces vascular permeability by binding to vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor (PlGF).
  • VEGF-A and VEGF-B vascular endothelial growth factor subtypes A and B
  • PlGF placental growth factor
  • the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) respectively approved aflibercept through intravitreal injection in August 2014 for the treatment of macular edema secondary to retinal vein occlusion, wet age-related macular degeneration and 3 indications for diabetic macular edema.
  • Conbercept (Langmu, Conbercept) is a type of Chinese biological product that has received the World Health Organization's international generic name.
  • the first type of drug is a genetically engineered antibody drug. It has a similar structure to Aflibercept, but the difference is that Conbercept contains the Ig-like region of the VEGF receptor. This structure can improve the affinity with VEGF and can also block all subtypes of VEGF-A and VEGF.
  • -B and placental growth factor increase the binding rate and extend the half-life of the drug in the body, molecular weight 142KD. It has been approved by the China Food and Drug Administration for the treatment of wAMD since the end of 2013.
  • the present invention uses recombinant AAV delivery to achieve long-term stable expression of the target gene in the RPE layer through genetic modification and vector optimization, and delivers the optimized target gene sequence to the patient's fundus cells. Achieve "once administration, long-term effectiveness" and provide patients with safer, more economical, and more This convenient and excellent treatment reduces the burden of existing clinical treatments for neovascular eye diseases and meets unmet clinical needs.
  • aflibercept it can be used as a candidate molecule for gene therapy delivery with long-term expression in vivo to maintain efficacy and low risk.
  • Choroidal neovascularization is the hallmark lesion of exudative nAMD and an important cause of vision loss in the elderly.
  • laser-induced CNV animal models have been commonly used to evaluate the efficacy of AMD therapeutic drugs, including protein drugs such as bevacizumab, aflibercept, and ranibizumab.
  • transient vascular leakage and neovascular responses in the choroidal vasculature persist for 2-3 weeks (rodent) or 6-8 weeks (NHP) after laser disruption of Bruch's membrane , then subsided spontaneously.
  • DL-AAA DL- ⁇ -aminoadipic acid
  • the DL-AAA model is a chronic leakage model that has been reported in rats and rabbits, and has been routinely used for candidate drug screening in these species.
  • DL-AAA is a selective glial cytotoxin that is reported to inhibit the action of glutamine synthetase, impairing broader Müller cell retinal homeostatic function leading to glial dysfunction and death, resulting in hemoretinal Barrier breach.
  • the retinal vasculature, retinal segmentation and basal lamina boundaries, proportional abundance of retinal neuronal and glial cell subtypes, and presence of the macula are homologous in monkeys and humans.
  • Preclinical models of simultaneous chronic retinal vascular leakage and neovascularization allow screening of the efficacy of short- and long-acting anti-angiogenic compounds at multiple stages of disease development.
  • the present invention will use the NHP model of chronic vascular leakage caused by DL-AAA to verify the effect.
  • One of the technical solutions provided by the present invention is a Fc fragment mutant of human IgG1, which is obtained by at least one of the following mutations:
  • T250A L251A, M252L, I253A/D/P, S254A, T256A, L309A, H310L/V/A/D/E/Q, Q311A, L314A, M428L/I, H433L/V/A, N434L/V/A, H435L/V/A, Y436A;
  • the mutant has one of the following single mutations: H310A, H310L, H435A, H435L, I253A, I253D, I253P, H310D, H310E or H310Q;
  • the mutant has one of the following single point mutations: H310A, H310E or H435A;
  • the mutant has one of the following double mutations: M252L/M482L, M252L/M482I, M252L/M482L, T250A/H310L, L251A/H310L, I253A/H310L, S254A/H310L, T256A/H310L, L309A/ H310L, H310L/Q311A, H310L/L314A, H310L/H433A, H310L/N434A, H310L/H435A, H310L/Y436A, I253A/H310A, I253A/H435A, or H310A/H435A;
  • the mutant has the following double mutations: H310A/H435A;
  • the mutant has one of the following three mutations: M252L/H310L/M482L, M252L/H310V/M482L, M252L/M482L/H433L, M252L/M482L/H433V, M252L/M482L/N434L, or M252L/ M482L/H435V;
  • the mutant has one of the following four mutations: M252L/M482L/N434V/H435L, M252L/H310L/M482L/H433L, M252L/H310L/M482L/N434L, or M252L/H310L/M482L/H435L.
  • the Fc fragment mutant has the sequence shown in SEQ ID NO. 1, 2, 3 or 4.
  • the second technical solution provided by the present invention is a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody;
  • the VEGF receptor recombinant fusion protein is formed by fusion of domain 2 of VEGFR-1, domain 3 of VEGFR-2, and the Fc fragment mutant of IgG1 described in technical solution one;
  • domain 2 of the VEGFR-1 is shown in SEQ ID NO.5;
  • domain 3 of the VEGFR-2 is shown in SEQ ID NO.6;
  • the Fc fragment mutants of IgG1 are: H310A (shown in SEQ ID NO.1), H310E (shown in SEQ ID NO.2), H435A (shown in SEQ ID NO.3), H310A/H435A ( SEQ ID NO.4 shown).
  • amino acid sequence of the VEGF receptor recombinant fusion protein is shown in SEQ ID NO. 7, 8, 9 or 10;
  • the anti-VEGF recombinant antibody is based on the existing anti-VEGF antibody in which the Fc fragment is replaced with the Fc fragment mutant of IgG1 described in the technical solution one;
  • existing anti-VEGF antibodies include but are not limited to Bevacizumab, Ranibizumab and Brolucizumab;
  • the anti-VEGF recombinant antibody replaces the Fc fragment of bevacizumab with the Fc fragment mutant of IgG1 described in Technical Solution 1, and more preferably, has the amino acid sequence shown in SEQ ID NO. 11.
  • the third technical solution provided by the present invention is an AAV viral vector expression cassette that expresses the VEGF receptor recombinant fusion protein or the anti-VEGF recombinant antibody described in the second technical solution.
  • the expression cassette contains the following formula from the 5'-3' end. The structure described in I:
  • ITR is the inverted terminal repeat sequence
  • E1 is the promoter
  • E2 is the signal peptide
  • E3 is a nucleotide sequence encoding the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in Technical Solution 2;
  • E4 is Poly A sequence.
  • the ITR inverted terminal repeat sequence
  • the ITR sequence is selected from AAV2;
  • the promoter is a DNA sequence that can initiate the transcription of the target gene. This sequence can be recognized by RNA polymerase and start transcribing and synthesizing RNA.
  • the promoter includes but is not limited to natural, optimized or combined promoters;
  • the promoter is preferably CMV, CBA, EF1a, SV40, PGK1, Ubc, CAG, TEF1, U6 or H1 promoter;
  • the source of the signal peptide includes but is not limited to Human OSM, Gaussia luc, or Albumin (HSA);
  • Poly A sequence is selected from bGH PolyA, SV40 PolyA or hGH PolyA;
  • the above-mentioned expression cassette also includes regulatory elements, which include but are not limited to regulatory elements with the following functions: (1) for regulating the expression of the target protein, such as IRES, for initiating the translation of downstream genes; (2) ) Regulatory elements used to express miRNA and siRNA sequences; (3) Introns; (4) Positioning sequences to position and express the target protein into the nucleus, cytoplasm or various organelles, and secrete it outside the cell; (5) Regulatory elements It can also be a partial kozak sequence.
  • regulatory elements include but are not limited to regulatory elements with the following functions: (1) for regulating the expression of the target protein, such as IRES, for initiating the translation of downstream genes; (2) ) Regulatory elements used to express miRNA and siRNA sequences; (3) Introns; (4) Positioning sequences to position and express the target protein into the nucleus, cytoplasm or various organelles, and secrete it outside the cell; (5) Regulatory elements It can also be a partial kozak sequence.
  • the kozak sequence is G/NC/NC/N-ANNAUGG, such as GCCACCAUGG, etc.; (6) Enhancer, which can be from SV40 virus, CMV virus or adenovirus, etc.; (7)
  • the regulatory element can be WPRE.
  • the above-mentioned expression cassette also includes tag elements, which include but are not limited to FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc.
  • the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody is to express the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in technical solution 2 on an AAV viral vector;
  • the AAV viral vector includes but is not limited to pAAV-CMV, pX601, pX551, pAAV-MCS plasmid, etc.;
  • the AAV viral vector expression cassette expressing VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody includes: ITR-CMV promoter-CMV enhancer-intron-signal peptide-Kozak sequence-VEGF receptor recombinant fusion Protein/anti-VEGF recombinant antibody coding sequence-PolyA-ITR;
  • the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein has the sequence shown in SEQ ID NO. 12;
  • the AAV viral vector expression cassette expressing anti-VEGF recombinant antibody has the sequence shown in SEQ ID NO. 13.
  • the fourth technical solution provided by the present invention is an adeno-associated virus packaging vector system.
  • the packaging vector system includes: the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or the anti-VEGF recombinant antibody described in the third technical solution. , a vector carrying AAV rep and cap genes and a helper virus vector. The above vectors are packaged into AAV viruses;
  • vectors carrying AAV rep and cap genes include but are not limited to: AAV1, AAV2, AAV5, AAV8, AAV9, AAV-R100, AAV-NN, AAV-GL, AAV8-Y447F, AAV8-Y733F, AAV8- Y447F/Y733F, AAV-DJ or AAV7m8 vector, etc.;
  • helper virus vector is an adenovirus or herpes virus helper virus vector, preferably pHelper plasmid.
  • the fifth technical solution provided by the present invention is a packaging method for adeno-associated virus.
  • the adeno-associated virus packaging vector system described in technical solution 4 is transferred into host cells for virus packaging;
  • the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or the anti-VEGF recombinant antibody in the packaging vector system described in the fourth technical solution, the vector carrying the AAV rep, cap genes, and the helper virus vector are transferred into the host cell. Perform virus packaging;
  • the host cell is a cell line capable of viral replication and stable inheritance, including but not limited to Hela-S3, HEK-293, HEK-293T, HEK-293FT, Expi293F, A549 and Sf9 cells;
  • the host cell is HEK-293, HEK-293T cell or Expi293F cell.
  • the sixth technical solution provided by the present invention is an adeno-associated virus obtained by using the packaging method described in the fifth technical solution;
  • the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein shown in SEQ ID NO. 12, the AAV8Rep-Cap plasmid, and the pHelper plasmid are transferred into the host cell Expi293F cells for virus packaging;
  • the AAV viral vector expression cassette expressing the anti-VEGF recombinant antibody shown in SEQ ID NO. 13, the AAV8Rep-Cap plasmid, and the pHelper plasmid are transferred into the host cell Expi293F for virus packaging.
  • the seventh technical solution provided by the present invention is an AAV viral vector expression cassette that expresses the VEGF receptor recombinant fusion protein or the anti-VEGF recombinant antibody described in the technical solution three, or the preparation or formula of the adeno-associated virus described in the sixth technical solution, or drug;
  • preparation or formula or medicine can be in any dosage form, including but not limited to injection dosage forms and ointment dosage forms;
  • the above-mentioned AAV viral vector expression cassette expressing VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody, or adeno-associated virus is the only active ingredient.
  • the eighth technical solution provided by the present invention is an AAV viral vector expression cassette that expresses the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in technical solution three, or the adeno-associated virus described in technical solution six is used in the preparation and treatment of neovascularization-related Fundus diseases, especially preparations or formulations or applications in medicines for age-related macular degeneration, wet maculopathy, diabetic retinopathy and other diseases;
  • the administration method of the preparation or formula or drug is unilateral eye administration or bilateral eye administration;
  • administration methods include intravitreal injection, subretinal space injection, suprachoroidal space injection and other injection methods;
  • the total dose is 1 ⁇ 10 8 -1 ⁇ 10 13 viral genomes/eye.
  • the present invention reduces the affinity to FcRn through Fc mutation, improves ocular PK behavior, and reduces blood infiltration. It does not affect stability, significantly increases accumulation in the eye and extends half-life; even if it enters the blood, its half-life in the circulation system is shorter due to reduced FcRn affinity, avoiding other systemic safety risks;
  • the rAAV vector element designed in the present invention delivers a Fc mutation-modified VEGF antagonist combination that is more durable than the control group of existing marketed protein drugs;
  • the AAV virus prepared by the present invention can alleviate or cure angiogenic fundus diseases for life through one administration, or reduce the number of protein drug administrations;
  • the same combination design can be used in multiple administration methods such as intravitreal injection, subretinal space, and suprachoroidal space.
  • each promoter and enhancer in Figure 1 is represented as follows:
  • FIG. 4 Effects of different PolyA on the expression results of different Aflibercept transiently transfected ARPE-19 cells.
  • Figure 5 The effects of different PolyA on the expression results of different Aflibercept transiently transfected HEK293 cells.
  • Figure 6 Results of 37°C thermal stability analysis of candidate Fc-engineered VEGF receptor recombinant fusion protein.
  • Figure 7 Results of thermal stability analysis at 40°C of candidate Fc-engineered VEGF receptor recombinant fusion protein.
  • FIG. 8 AAV vector expression product inhibits HUVEC cell proliferation.
  • Figure 11 The average score of fundus light spot leakage in each group of animals.
  • FIG 12 Typical fundus angiography (FFA) 4 weeks after laser modeling.
  • the present invention provides a method to deliver optimized VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody through AAV, and deliver the optimized target gene sequence to the patient's eyes through subretinal space, vitreous or suprachoroidal space injection, etc., to achieve Long-term stable expression of the target gene in the retina requires lifelong treatment with a low-dose administration.
  • Fc fusion protein refers to a new recombinant protein produced by fusing a certain biologically active functional protein molecule with the Fc fragment of immunoglobulin (IgG, IgA, etc.) using technologies such as genetic engineering. It not only retains the biological activity of functional protein molecules, but also has some properties of antibodies, such as extending half-life by binding to relevant Fc receptors and triggering antibody-dependent cell-mediated cytotoxic effects, etc., which is important in the diagnosis and treatment of diseases. meaning.
  • FcRn is an IgG antibody receptor located on the cell membrane surface. Its protein structure is similar to MHC-I molecules. It is mainly expressed in endothelial cells (can also be detected in other tissues or cells).
  • FcRn can bind to the Fc part of IgG to prevent IgG molecules from being cleaved by lysosomes. It can increase the half-life of IgG in the body and participate in the transport, maintenance and distribution metabolism of IgG in the body.
  • the Fc portion of IgG interacts with the neonatal Fc receptor (FcRn, Brambell receptor), preventing its degradation in lysosomes and enabling long-term serum persistence of antibodies.
  • FcRn neonatal Fc receptor
  • Other biological roles of FcRn include perinatal IgG transfer, antibody-mediated antigen presentation, and IgG transfer across epithelial and endothelial barriers.
  • IgG:FcRn interactions has been increasingly investigated to improve the therapeutic or diagnostic applications of antibodies.
  • the in vivo action pathway of intraocular injection of IgG or Fc fusion protein is mainly through the FcRn on the retinal RPE and endothelial cells, which crosses the blood-retina barrier and enters the systemic circulation and is gradually eliminated (elimination). Therefore, FcRn plays an important role in eye metabolism. Mediates the blood entry and clearance of IgG or Fc fusion proteins.
  • the optimized VEGF receptor recombinant fusion protein of the present invention includes human vascular endothelial growth factor (VEGF) receptor 1 Domain 2 and receptor 2 Domain 3 and human immunoglobulin Fc segment mutant recombinant protein.
  • VEGF vascular endothelial growth factor
  • multiple groups of Fc single-point or multi-point mutants are designed, and the selection of mutation sites of the present invention is
  • FcRn affinity it is preferable to consider the stability and persistence of the target protein expression of the rAAV composition in vivo and in vitro.
  • the specific mutation sites of Fc mutants include: T250A, L251A, M252L, I253A/D/P, S254A, T256A, L309A, H310L/V/A/D/E/Q, Q311A, L314A, M428L/I, H433L/V /A, N434L/V/A, H435L/V/A, Y436A, etc.
  • preferred mutation sites are single point mutations, including H310A, H310L, H435A, H435L, I253A, I253D, I253P, H310D, H310E, and H310Q.
  • preferred mutation sites are double point mutations, including M252L and M482L, M252L and M482I, M252L and M482L, T250A and H310L, L251A and H310L, I253A and H310L, S254A and H310L, T256A and H310L, L309A and H310L, H310L and Q311A, H310L and L314A, H310L and H433A, H310L and N434A, H310L and H435A, H310L and Y436A, I253A and H310A, I253A and H435A, H310A and H43 5A etc.
  • preferred mutation sites are three-point mutations, including M252L, H310L and M482L, M252L, H310V and M482L, M252L, M482L and H433L, M252L, M482L and H433V, M252L, M482L and N434L , M252L, M482L and H435V.
  • the preferred mutation sites are four point mutations, including M252L, M482L, N434V and H435L, M252L, H310L, M482L and H433L, M252L, H310L, M482L and N434L, M252L, H310L, M482L and H435L.
  • the more preferred single point mutation sites are H310A, H310E, and H435A, and the obtained Fc mutant has the amino acid sequence shown in SEQ ID NO. 1, 2, or 3.
  • a more preferred double-point mutation site is H310A/H435A, and the Fc mutant has the amino acid sequence shown in SEQ ID NO. 4.
  • the above Fc mutant is a mutation based on the following Fc wild-type fragment of human IgG1 (SEQ ID NO. 15):
  • the numbering of amino acid residues in the Fc fragment is the numbering of the immunoglobulin heavy chain, which is based on Sequences of Proteins of Immunological Interest by Kabat et al., 5th edition. Public Health Service [ EU Index in National Institutes of Health, Bethesda, MD (1991) (expressly incorporated herein by reference).
  • EU index/numbering refers to the EU coding sequence number of human IgG1 antibody constant region residues". This numbering is well known to those skilled in the art and is frequently used in the art. You can also refer to the following URL: https://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.
  • the amino acid sequence of the VEGF receptor recombinant fusion protein is shown in SEQ ID NO. 7-10.
  • the anti-VEGF recombinant antibody of the present invention is based on the existing anti-VEGF antibody in which the Fc fragment is replaced by the Fc fragment mutant of the aforementioned IgG1; in another embodiment of the present invention, the existing anti-VEGF antibody includes But it is not limited to Bevacizumab, Ranibizumab and Brolucizumab; in another embodiment of the present invention, the anti-VEGF recombinant antibody is Bevacizumab
  • the anti-Fc fragment is replaced with the Fc fragment mutant H310E of IgG1 described in Technical Scheme 1, preferably with the amino acid sequence shown in SEQ ID NO. 11.
  • the invention also provides a vector expression cassette, which contains the structure described in Formula I from the 5'-3' end:
  • ITR is the inverted terminal repeat sequence
  • E1 is the promoter (including natural, optimized or combined promoter)
  • E2 is the signal peptide
  • E3 is the nucleotide sequence encoding the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody of the present invention
  • E4 is Poly A sequence.
  • the ITR sequence (inverted terminal repeat) is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9; preferably from AAV2;
  • the nucleic acid sequence has a promoter.
  • the promoter is used to initiate the expression of the nucleic acid sequence connected to it.
  • the promoter can be constitutive or inducible.
  • the promoter can be a promoter specifically expressed in retinal pigment epithelial cells, or it can be ubiquitous. Promoter.
  • Preferred promoters include various eukaryotic promoters, for example, CMV, EF1a, CBA, SV40, PGK1, Ubc, CAG, TEF1, U6, H1, etc.
  • CMV enhancer and CMV promoter use a broad spectrum of CMV (CMV enhancer and CMV promoter), CBA (CMV enhancer and chicken 9-actin promoter), PGK promoter and EF1a promoter. More preferably, the promoter is a CMV promoter.
  • Signal peptide is a short peptide chain (5-30 amino acids in length) that guides the transfer of newly synthesized proteins to the secretory pathway. Often refers to the N-terminal amino acid sequence (sometimes not necessarily at the N-terminus) in a newly synthesized polypeptide chain that is used to guide the transmembrane transfer (positioning) of the protein. After the start codon, there is an RNA region encoding a hydrophobic amino acid sequence. This amino acid sequence is called a signal peptide sequence, which is responsible for guiding the protein to subcellular organelles containing different membrane structures in the cell.
  • a positively charged N terminus called the basic amino terminus
  • a middle hydrophobic sequence mainly neutral amino acids, which can form an ⁇ -helical structure, which is The main functional region of the signal peptide
  • a longer negatively charged C-terminus containing small amino acids which is the signal sequence cleavage site. It is also called the processing region.
  • SRP signal recognition particle
  • protein synthesis is paused or slowed down.
  • the signal recognition particle carries the ribosomes to the endoplasmic reticulum, and protein synthesis restarts.
  • newly synthesized proteins enter the lumen of the endoplasmic reticulum.
  • the signal peptide sequence is removed by the action of signal peptidase. If the termination transport sequence exists at the C-terminus of the nascent peptide chain, it may not be cleaved by the signal peptidase.
  • commonly used eukaryotic expression signal peptides are selected, SP1 ⁇ SP13 signal peptides are inserted into the N-terminus of the target gene by PCR, and homologous recombination is performed into the EcoRI and HindIII dual enzymes cut into the PTT5 vector. After transformation of E. coli and sequencing to verify that the sequence is correct, the plasmid is extracted in large quantities without endotoxin and transiently transfected into HEK293E cells using the PEI method. After culturing for 5 days in CD05 medium containing glutamine, the cell supernatant is taken to detect protein expression.
  • preferred signal peptides are from Human OSM, Gaussia luc, or Albumin (HSA). In another embodiment, a more preferred signal peptide is from Gaussia luc.
  • Polyadenylation refers to the covalent linkage of polyadenylic acid to messenger RNA (mRNA) molecules. In the process of protein biosynthesis, this is part of the way that mature mRNA is produced ready for translation. In eukaryotes, polyadenylation is a mechanism that interrupts mRNA molecules at their 3' ends.
  • the polyA tail (or polyA tail) protects mRNA from exonuclease attack and is important for transcription termination, export of mRNA from the nucleus, and translation.
  • the main choices for PolyA sequences are bGH PolyA (derived from pCMV3), SV40 PolyA (derived from pCGS3) sequences. Different PolyA sequences may interact with other expression elements to increase transcription levels. In one embodiment of the present invention, three different PolyA sequences bGH PolyA, SV40 PolyA and hGH PolyA were tested to obtain optimal gene expression and virus production. In another embodiment of the invention, preferred PolyA sequences are bGH PolyA and hGH PolyA. In another embodiment of the invention, a preferred PolyA sequence is bGH PolyA.
  • the above-mentioned expression cassette also includes regulatory elements, which include, but are not limited to, regulatory elements with the following functions: (1) for regulating the expression of the target protein, such as IRES, for initiating the expression of downstream genes. Translation; (2) Regulatory elements used to express miRNA and siRNA sequences; (3) Intron, also known as spacer sequence, refers to a gene or mRNA molecule that has no coding function Fragment; (4) Positioning sequence to localize and express the target protein into the nucleus, cytoplasm or various organelles, and secrete it out of the cell; (5)
  • the regulatory element can also be a partial Kozak sequence, which is located in eukaryotic mRNA 5 A nucleic acid sequence behind the 'end cap structure, usually GCCACCAUGG, which can bind to translation initiation factors and mediate translation initiation of mRNA containing a 5' cap structure, corresponding to the SD sequence of prokaryotes and present in eukaryotic mRNA A sequence that plays an important role
  • the above-mentioned expression cassette also includes a tag element, which includes but is not limited to, for example, FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc.
  • a tag element which includes but is not limited to, for example, FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc.
  • the invention discloses a recombinant viral vector comprising the following elements: (a) first AAV2 inverted terminal repeat (ITR) sequence, (b) CMV enhancer and promoter, (c) embedded Synthetic intron, (d) Kozak sequence, (e) signal peptide sequence, (f) VEGF receptor recombinant fusion protein/anti-VEGF recombinant antibody coding sequence, (g) WPRE sequence, (h) bGH polyA sequence, and ( i) Second AAV2ITR.
  • the above-mentioned optimized VEGF receptor recombinant fusion protein/anti-VEGF recombinant antibody is expressed on an AAV viral vector, which includes but is not limited to pAAV-CMV and pAAV-MCS plasmids; preferably Land, the AAV viral vector is pAAV-CMV.
  • the AAV virus packaged by the above-mentioned vector expressing the optimized VEGF receptor fusion protein/anti-VEGF recombinant antibody is also provided.
  • AAV viruses can be prepared using standard methods that have been published in the field, see "AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction".
  • Replication-defective recombinant AAV can be produced by cotransfection of three plasmids into cell lines infected with a human helper virus (e.g., adenovirus): containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions.
  • a human helper virus e.g., adenovirus
  • ITR inverted terminal repeat
  • the resulting AAV recombinant virus is then purified by standard techniques.
  • the recombinant adeno-associated virus is a single-chain AAV.
  • the optimized gene fragment encoding the VEGF receptor recombinant fusion protein/anti-VEGF recombinant antibody is packaged into viral particles (for example, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV serotype virus particles such as AAV13, AAV14, AAV15, AAV16, AAV7m8, AAV PHP.eB, etc.).
  • the present disclosure includes recombinant viral particles containing any of the vectors described herein.
  • the recombinant adeno-associated virus serotype is AAV2, AAV8. More preferably, the recombinant adeno-associated virus serotype is AAV8.
  • AAV vectors such as self-complementary AAV (scAAV), single-chain AAV (ssAAV), pAAV-CMV, pAAV-MCS wait.
  • scAAV self-complementary AAV
  • ssAAV single-chain AAV
  • pAAV-CMV pAAV-MCS wait.
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV7m8, AAV PHP.eB) or any other Modified serotypes can all achieve selective targeting.
  • the host cells used in the present invention to assemble the above-mentioned virus are used for AAV vector-transduced cells to express Fc-engineered VEGF receptor recombinant fusion proteins and anti-VEGF antibodies.
  • the host cells are mammalian cells (preferably cells derived from human bodies, more preferably human optic nerve cells or photoreceptor cells), which increase the expression of VEGF receptor fusion proteins and anti-VEGF antibodies and embody the AAV vector molecules of the present invention. specific expression.
  • the host cells include but are not limited to Hela-S3, HEK-293, HEK-293T, HEK-293FT, Expi293F, A549 and Sf9 cells; in some embodiments of the invention, preferred host cells are HEK-293, HEK-293T cells or Expi293F cells.
  • the invention discloses the following recombinant adeno-associated virus: AAV8-Afli Fc Variant10.
  • the recombinant virus is an AAV8 serotype virus.
  • the vector carrying the transgene has the sequence described in SEQ ID NO: 12.
  • the transgene carried is the VEGF receptor recombinant fusion protein encoding gene.
  • H310E was carried out at position 310 of the Fc region of the Aflibercept fusion protein gene. Mutational transformation.
  • the invention discloses the following recombinant virus: AAV8-Bevacizumab Fc Variant10.
  • the recombinant virus is an AAV8 serotype virus.
  • the vector carrying the transgene has the sequence described in SEQ ID NO:13.
  • the transgene carried is the mutated gene of the Bevacizumab monoclonal antibody, which is modified by the H310E mutation at position 310 of the Fc region of the Bevacizumab gene. .
  • the recombinant virus constructed in the present invention is suitable for use in vivo or ex vivo, and is preferably suitable for use in patients with angiogenesis-related fundus diseases (including age-related macular degeneration, wet macular degeneration, and diabetic macular edema). Use once in the vitreous body, subretinal space or suprachoroidal space.
  • angiogenesis-related fundus diseases including age-related macular degeneration, wet macular degeneration, and diabetic macular edema.
  • the safe and effective dose for mouse administration is in the range of 1 ⁇ 10 6 -1 ⁇ 10 11 viral genomes/eye
  • the safe and effective dose for NHP administration is The dosage range is 1 ⁇ 10 8 -1 ⁇ 10 13 viral genomes/eye
  • the safe and effective dosage range for human administration is 1 ⁇ 10 8 -1 ⁇ 10 13 viral genomes/eye (viral genome, genomic copies, referred to as GC).
  • the invention provides a composition comprising a polynucleotide or viral vector or adeno-associated virus of the invention.
  • the invention provides a method for preparing a composition comprising a polynucleotide or viral vector or adeno-associated virus of the invention.
  • the invention provides for administration into the subretinal space of a composition comprising a polynucleotide or viral vector or adeno-associated virus of the invention.
  • the invention provides for the administration of a composition comprising a polynucleotide or a viral vector or an adeno-associated virus of the invention by intravitreal injection.
  • the invention provides for suprachoroidal administration of a composition comprising a polynucleotide or viral vector or adeno-associated virus of the invention.
  • the present invention provides methods for observing mice/NHP/humans after administration, including optical coherence tomography (OCT) and fluorescein fundus angiography (FFA).
  • OCT optical coherence tomography
  • FFA fluorescein fundus angiography
  • the present invention provides a method for detecting the expression of aflibercept protein in eyeball tissue of mice after administration.
  • the present invention provides a preparation or composition, which contains the adeno-associated virus vector or virus of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical preparation is used to treat eye diseases.
  • the pharmaceutical preparation is used to treat eye diseases related to VEGF.
  • the pharmaceutical preparation is used to treat macular degeneration and/or diabetic retinopathy. More preferably, for the treatment of wet age-related macular degeneration or wet macular degeneration.
  • the pharmaceutical composition of the present invention can be filled in sealed vials or prefilled syringes.
  • the vial or prefilled syringe can be packaged in a pharmaceutical packaging box to facilitate storage and use.
  • the pharmaceutical preparations and compositions of the present invention are stored in an ultra-low temperature refrigerator of ⁇ -60°C, and are transported by dry ice-cold chain during transportation.
  • the dosage form of the pharmaceutical preparation or preparation composition may be liquid or solid, such as powder, gel or paste.
  • the composition is a liquid, more preferably an injectable.
  • the titer of the AAV vector preparation provided by the present invention is 1 ⁇ 10 10 -1 ⁇ 10 14 GC/ml, and the preferred titer is 1 ⁇ 10 12 -1 ⁇ 10 13 GC/ml.
  • the formulations include pharmaceutically acceptable excipients.
  • the excipients include surfactants or stabilizers.
  • the surfactant is selected from polysorbate, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethyl oxide gum, polyethoxylated alcohol, polyoxyethylene sorbitol, Octinol, Brij, pluronic and polyoxyl caster oil.
  • the pharmaceutically acceptable excipients include phenol, mannitol, sorbitol, sucrose, or sodium chloride.
  • the “active ingredient” in the pharmaceutical composition of the present invention refers to the vector or AAV virus of the present invention.
  • the "active ingredients”, formulations and/or compositions of the present invention may be used to treat eye diseases.
  • Safe and effective amount refers to the amount of active ingredients that is sufficient to significantly improve the condition or symptoms without causing serious side effects.
  • “Pharmaceutically acceptable carrier or excipient (excipient)” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must be of sufficient purity and sufficient Low toxicity.
  • “Compatibility” here refers to the ability of each component of the composition to be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.
  • the present invention provides a method for treating VEGF-mediated neovascular eye disease, the method comprising introducing a virus or viral vector comprising a nucleic acid sequence encoding an Fc-engineered VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody into the eye. .
  • the method may include injecting a nucleic acid vector targeting RPE cells or photoreceptor cells into the subretinal space, intravitreal space, or suprachoroidal space.
  • the present invention provides a nucleic acid vector used in a method for treating neovascular eye disease by delivering a nucleic acid sequence encoding a therapeutic protein to retinal cells, the nucleic acid vector comprising a recombinant fusion protein encoding an Fc-engineered VEGF receptor or an anti-VEGF recombinant protein.
  • Antibody nucleic acid sequence The composition of the present invention can be administered alone or in combination with other therapeutic drugs (eg, formulated in the same pharmaceutical composition).
  • treating a disease means administering a nucleic acid or vector or virus as described herein to ameliorate or alleviate one or more symptoms of the disease, including reducing vascular leakage, reducing neovascularization, and the like.
  • the method of the invention includes introducing a nucleic acid sequence encoding an Fc-engineered VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody into the subretinal space of the eye.
  • the method of the invention comprises contacting the cell with a vector (preferably a virus, more preferably an adeno-associated virus) comprising a nucleic acid sequence encoding an Fc-engineered VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody.
  • a vector preferably a virus, more preferably an adeno-associated virus
  • the cells are retinal cells, preferably pigment epithelial cells, cones, rods, bipolar cells, horizontal cells, ganglion cells and/or amacrine cells.
  • the recombinant adeno-associated virus vector of the present invention can effectively inhibit the expression of VEGF in animal eyes, reduce leakage and angiogenesis caused by laser damage, and can also reduce retinal vasculopathy caused by DL-AAA, and therefore can be used to prepare drugs for treating VEGF-related diseases. , to treat human eye diseases related to VEGF, and has broad market prospects.
  • the clinical dosage is 1 ⁇ 10 8 -1 ⁇ 10 13 GC virus, and the eye injection volume is 50 to 300 ⁇ l.
  • Routes of administration are subretinal, intravitreal, or suprachoroidal injection.
  • mutant names and mutant information involved in the present invention and examples are as follows:
  • mutants Variant1-12 in the table are based on Eylea (Aflibercept) shown in SEQ ID NO. 14, and the Aflibercept mutants were obtained by carrying out corresponding mutations on the Fc fragment.
  • CMV enhancer and CMV promoter use broad-spectrum CMV (CMV enhancer and CMV promoter), CBA (CMV enhancer and chicken- ⁇ actin promoter).
  • CBA CMV enhancer and chicken- ⁇ actin promoter
  • Optimized retina Cell-specific promoters may express the protein of interest more efficiently in specific tissues.
  • CBA, CMV+TPL-MLP the TPL-MLP sequence is derived from the pTT5 vector
  • RPE65 GenBank: AF304008.1
  • IRBPe/GNAT2 GenBank: X53044.1, GenBank: U66698.1
  • the amino acid sequence of Aflibercept comes from the DrugBank database (accession number DB08885).
  • the codons are optimized according to the codon usage preference of Homo sapiens, and the sequence is cloned into the pTT5 expression vector.
  • the specific experimental steps are as follows: 1) The aflibercept sequence is optimized and synthesized, and the RPE65 and IRBPe/GNAT2 promoter sequences are synthesized; 2) The RPE65 and IRBPe/GNAT2 promoter sequences are amplified by PCR and combined with NcoI and SacI, MluI and SacI in sequence. The digested pAAV-MCS vector was subjected to homologous recombination. After transformation into E. coli, sequencing and digestion verification were performed to obtain pAAV-RPE65-MCS and pAAV-IRBPe/GNAT2-MCS vectors.
  • Target protein in cell culture supernatant by affinity chromatography. Quantification of protein was detected by BCA method (Pierce TM Rapid Gold BCA protein assay kit). Obtain the preferred signal peptide sequence based on the expression level of the target protein. This study compared the effects of 13 different signal peptides SP1 to SP13 on the expression of the target protein, while using Gaussia luc as a control. The results are shown in Figure 2. The results showed that the SP1 signal peptide had the highest protein expression.
  • Variant1, 2, 6 and 10 mutants did not bind to FcRn, and the protein expression was not significantly different from that before mutation.
  • Variant12 is an affinity positive mutant of the Fc region, which increases its affinity to FcRn.
  • Afli Select the preferred Fc-mutated Aflibercept mutant molecules and unmutated Aflibercept molecules (referred to as "Afli"), insert these target gene fragments into the pAAV-MCS vector (containing hGH PolyA), and replace the PolyA sequence with bGH PolyA respectively. , SV40PolyA sequence.
  • Each constructed AAV vector was transiently transfected into ARPE-19 cells and HEK293 cells using lipofectamine2000 transfection reagent, and the expression level of the target protein was detected 72 hours later.
  • Afli the control group Aflibercept
  • the drug-time curve was drawn based on the mean concentration of serum samples against time points, and the non-compartmental model was used to calculate the drug exposure AUC of each protein molecule after entering the blood through the eye (see Table 4).
  • the average AUC of Variant10 was 74.2% lower than Afli, and Variant2 The average AUC is 54.9% lower than Afli.
  • the amino acid sequence of Aflibercept comes from the DrugBank database (accession number DB08885, SEQ ID NO.14). Using the codon-optimized synthesized Aflibercept as a template, the Aflibercept fragment was amplified by PCR, and a signal peptide derived from Gaussia luc ( The sequence information is shown in Table 1), and the fragment was inserted into the PTT5 vector digested by NotI and BamHI. After sequencing and verification, the Aflibercept-PTT5 vector was obtained.
  • Variant10-PTT5 Using Variant10-PTT5 as a template, the Variant10 coding fragment was amplified by PCR and inserted into the pAAV-MCS plasmid double-digested by EcoRI and HindIII by homologous recombination. After transformation into E. coli, clones were selected and sequenced to verify the sequence. At the same time, the correctly sequenced vector was digested with SmaI to verify whether the ITR was missing, and the correct vector was named Afli Fc Variant10.
  • the above-mentioned double-digested vector and the bGHpolyA obtained by PCR After the fragments were purified, homologous recombination was performed and the clones were selected and sequenced to verify the sequence after transformation into E. coli. At the same time, the correctly sequenced vector was digested with SmaI to verify whether the ITR was deleted. After the verification is completed, the target plasmid Variant 10-pAAV (shown as SEQ ID NO. 12) is obtained.
  • the AAV virus was packaged by co-transfecting Expi293F cells with three plasmids: auxiliary packaging plasmid, AAV8rep-cap plasmid, and transgenic plasmid.
  • the Variant 10-pAAV vector, AAV8rep-cap plasmid and pHelper helper plasmid constructed in step 1 need to be extracted in large quantities.
  • the concentration is greater than 1 ⁇ g/ ⁇ L.
  • A260/280 is between 1.8-2.0 to package the virus.
  • Expi293F cells were cultured in serum-free suspension to a cell density of 1E+6 cells/ml. Mix the three extracted plasmids in a molar ratio of 1:1:1, then mix the total mass of plasmid DNA and PEIpro transfection reagent in a mass ratio of 1:2, incubate at room temperature for 20 minutes and then slowly add to the cells. Mix well in the suspension (the ratio of cells to three plasmids is 1mL:1 ⁇ g). Incubate in a 37°C, 8% CO2 shaker for 3 days, and collect the cell suspension.
  • the composition of the dialysis buffer is: 0.001% Pluronic F68 in PBS, pH 7.2.
  • the harvested post-dialysis sample is the purified AAV virus AAV8-Afli Fc Variant10, which can be used for in vitro and in vivo analysis and testing.
  • the purified virus is stored in a -80°C ultra-low temperature refrigerator.
  • the AAV viruses used in the present invention are all prepared by the above method, and specific AAV vectors, expression elements, and VEGF receptor recombinant fusion protein mutants or VEGF recombinant antibodies can be selected as needed. Among them, Aflibercept was replaced with Bevacizumab, and the H310E mutation of the Fc fragment occurred.
  • the plasmid constructed using the same method as above is shown in SEQ ID NO. 13.
  • the AAV virus constructed with this plasmid using the same method as above was named AAV8-Bevacizumab. Fc Variant10.
  • Verification of the biological activity of candidate vector molecule expression products is an important consideration in the pharmaceutical evaluation of gene therapy.
  • AAV8-Afli Fc Variant 10 and AAV8 empty vector prepared in Example 7 were used to infect HEK293T cells at an MOI of 2E+5, and the cell supernatant was collected after 72 hours.
  • 6E+3HUVEC cells were seeded in a 96-well plate (50 ⁇ L per well) and cultured in basal medium containing 100ng/ml VEGF165. After 4-6 hours, add 50 ⁇ L of virus-infected cell supernatant and continue culture medium for 72 hours.
  • mice SPF grade C57BL/6J male mice aged 8 to 10 weeks were raised in the laboratory for 3 to 5 days. Before grouping, the animals were subjected to general ophthalmic examination, fundus photography (FP) and fundus fluorescein angiography (FFA), and qualified animals were screened for use in the experiment. The qualified mice were randomly grouped according to body weight before administration and divided into 4 groups. The AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 was used as the test product. The animal grouping and administration treatment are shown in Table 7 below:
  • the solvent used in the examples of the present invention is PBS containing 0.001% (W/V) Pluronic F68, pH 7.2.
  • test sample should be used according to dosage requirements and diluted with solvent if necessary.
  • Subretinal space injection cover the cornea with a cover glass, use carbomer eye drops to remove the air, use a disposable insulin needle to puncture the sclera and choroid at a small angle with the sclera to form a channel; withdraw the insulin needle and replace the microscope The syringe is inserted from the channel port to the subretinal area for injection.
  • Intravitreal injection cover the cornea with a cover glass, remove the air with carbomer eye drops, puncture the sclera and choroid with a disposable insulin needle at a small angle to the sclera to form a channel; withdraw the insulin needle and replace the microinjector Enter the vitreous cavity from the channel port for injection.
  • Suprachoroidal space injection Use a disposable insulin needle to puncture the sclera to form a channel; withdraw the insulin needle, replace the microinjector and enter the suprachoroidal space from the channel opening for injection.
  • the deep-well microplate is coated with VEGF165 protein.
  • HRP-conjugated goat anti-human IgG (Fc-specific) antibody is added to detect aflibercept captured on the plate.
  • TMB substrate solution is added.
  • HRP catalyzes the substrate to undergo a color reaction; finally, the stop solution is added to terminate the reaction, and the solution changes from blue to yellow.
  • the color depth is positively related to the concentration of aflibercept protein in the sample.
  • Use a microplate reader to detect the solution after the color development reaction, and calculate the amount of aflibercept protein in the eyeball tissue homogenate supernatant.
  • mice SPF grade C57BL/6J male mice aged 8 to 10 weeks were raised in the laboratory for 3 to 5 days. Before grouping, the animals were subjected to general ophthalmic examination, fundus photography (FP) and fundus fluorescein angiography (FFA), and qualified animals were screened for use in the experiment. Screen 28 qualified mice and randomly group them according to body weight before administration, and divide them into 7 groups.
  • the AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 is used as the test product.
  • the animal grouping and administration treatment are shown in Table 8 below. :
  • Each mouse was administered a single dose to both eyes, using the same subretinal space injection administration method and post-treatment method as in Example 9, and the expression of aflibercept protein in the eyeballs was detected at different time points after administration.
  • the results of aflibercept protein detection are shown in Table 9, and the time expression curve is shown in Figure 9.
  • the candidate carrier molecule has sustained high expression for up to 12 weeks at the dose of 3E+8vg/eye.
  • the protein drug Eylea can only maintain 4-8 weeks after administration.
  • Example 11 AAV vector molecule dose escalation test in mice
  • Each mouse was given a single administration to both eyes, and the vehicle and different doses of AAV8-Afli Fc Variant10 test products were injected into the subretinal space respectively.
  • the administration method was the same as in Example 9.
  • Example 12 Candidate carrier molecule laser-induced CNV model in mice - drug efficacy test
  • mice Purchase 8-10 week old SPF grade C57BL/6J male mice and raise them in the laboratory for 3-5 days. Before grouping, the animals were subjected to general ophthalmic examination, fundus photography (FP) and fundus fluorescein angiography (FFA), and qualified animals were screened for use in the experiment. Thirty qualified mice were screened and randomly divided into 6 groups according to body weight before administration, using AAV virus AAV8-Afli Fc Variant10 prepared in Example 7, and The protein drug is the test product. The animal grouping and administration treatment are shown in Table 11 (the administration volume of each group is 1 ⁇ L/eye):
  • Protein drugs can take effect quickly after administration, so they should be administered after modeling;
  • AAV8-Afli Fc Variant10 reaches a stable expression level about 1-2 weeks after administration and can maintain long-term high expression for more than 12 weeks. , so administer the drug before modeling.
  • protein drugs are administered via intravitreal injection.
  • the administration method was the same as in Example 9.
  • the second group is the protein control group, with 80 ⁇ g/eye injected into the vitreous body.
  • the bilateral fundus laser-induced choroidal neovascularization model was performed on D17, and the planned number of laser burns in each eye was 4. The specific steps are:
  • FFA fundus fluorescein angiography
  • Analysis indicators Compare the early (within 1.5 min) and late (more than 3 min) images of fundus fluorescein angiography, determine whether there is choroidal neovascularization and leakage based on whether there is fluorescence leakage in the animal's fundus, and rate the degree of fluorescence leakage. , calculate the ratio of leakage spots at each level and the average spot leakage score.
  • the rating standards for spot fluorescence leakage are: Level 0 (no fluorescence leakage), Level 1 (mild fluorescence leakage, the leakage area is 1% to 50% of the laser spot size), Level 2 (moderate fluorescence leakage, The leakage area is 50% to 100% of the laser spot size), level 3 (severe fluorescence leakage, the leakage area is larger than the laser spot size).
  • Average score of light spot leakage [(number of light spots of level 0 ⁇ 0) + (number of light spots of level 1 ⁇ 1) + (number of light spots of level 2 ⁇ 2) + (number of light spots of level 3 ⁇ 3)] ⁇ total number of 4 types of light spots (i.e. Number of effective spots), where the effective spot refers to a spot that does not have severe preretinal or subretinal hemorrhage nearby and can be completely displayed in fluorescence imaging.
  • the average spot leakage scores are shown in Figure 11. On D31, the average scores of the AAV8-Afli Fc Variant10 group were lower than those of the vehicle control and Protein control group. And compared with D24, the average spot leakage scores of each dose group of AAV8-Afli Fc Variant10 were reduced, and The average spot leakage score of the protein control group increased, indicating that AAV8-Afli Fc Variant10 can be continuously and stably expressed in each dose group, and compared with Protein medicine is more effective and lasts longer.
  • Non-human primates have eye structures similar to humans, and cynomolgus monkeys are a commonly used non-rodent animal model for non-clinical research on biopharmaceuticals.
  • Laser photocoagulation was used to establish a choroidal neovascularization (CNV) model in cynomolgus monkeys, and the inhibitory effect of the AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 on CNV in cynomolgus monkeys after a single subretinal injection was examined. .
  • CNV choroidal neovascularization
  • a single dose was administered into the subretinal space on D0, and iodophor was used to disinfect the ocular surface.
  • a 30G disposable injection needle was used to puncture the cynomolgus monkey's sclera inside the corneoscleral limbus, and a microinjection tube with a 35G flat needle was used.
  • the sample device enters along the puncture port and bypasses the lens before reaching the vitreous body, avoiding the main blood vessels, and then gradually inserts the needle into the subretinal space, and slowly injects the needle to a point above the fovea.
  • a cotton swab with iodophor to press the needle entrance for 5 seconds.
  • Grade III-IV spots are leakage lesions.
  • the target protein content in the aqueous humor was continuously detected after administration, and its expression continued to be stable for 12 weeks, and is still being monitored (while Protein pills only last 4-8 weeks).
  • the typical fundus angiography (FFA) pattern 4 weeks after laser modeling is shown in Figure 12.
  • DL-AAA DL- ⁇ -aminoadipic acid
  • the DL-AAA model is a chronic leakage model that has been described in rats and rabbits and has been routinely used for candidate drug screening in these species.
  • DL-AAA is a selective glial cytotoxin that is reported to inhibit the action of glutamine synthetase, impairing broader Müller cell retinal homeostatic function leading to glial dysfunction and death, resulting in hemoretinal Barrier breach.
  • a novel DL-AAA-induced NHP model of chronic vascular leakage, a preclinical model of chronic retinal vascular leakage and neovascularization, allows for efficacy screening of short- and long-acting anti-angiogenic compounds at multiple stages of disease development check.
  • PRNV retinal neovascularization
  • AAV8-Afli Fc Variant10 test product has exceeded the relief time of CNV laser modeling, reflecting the advantages of this model in observing the long-term efficacy of AAV.
  • Statistical analysis found that both the leakage area and leakage intensity of the test product were significantly different from the vehicle group, indicating that AAV8-Afli Fc Variant10 adeno-associated virus has obvious curative effect in PRNV model non-human primates.

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Abstract

本发明属于重组腺相关病毒(rAAV)基因治疗技术领域，涉及rAAV递送Fc工程化改造的VEGF受体融合蛋白或抗VEGF抗体的制备，应用于血管新生相关的眼底疾病的治疗，如年龄相关黄斑变性、湿性黄斑病变、糖尿病视网膜病变等疾病的治疗。本发明以阿柏西普表达基因为基础，通过基因改造和载体优化，用重组AAV递送实现目的基因在RPE层的长期稳定表达，将优化的目的基因序列递送至患者眼底细胞。

Description

一种治疗新生血管相关眼底疾病的AAV药物 技术领域：
本发明属于重组腺相关病毒(rAAV)基因治疗技术领域，涉及rAAV递送Fc工程化改造的VEGF受体融合蛋白或抗VEGF抗体的制备，应用于血管新生相关的眼底疾病的治疗，如年龄相关黄斑变性、湿性黄斑病变、糖尿病视网膜病变等疾病的治疗。
背景技术：
血管新生可以在视网膜、脉络膜、黄斑、视盘、角膜、虹膜睫状体等眼内组织中出现，引起这些部位组织出血、渗出及增生等病理改变，包括年龄相关黄斑变性(Age-related Macular Degeneration，AMD)、糖尿病视网膜病变(diabetic retinopathy，DR)、早产儿视网膜病变(retinopathy of prematurity，ROP)等，严重影响视力，是老年人群中致盲的主要原因。
AMD分为两个主要亚型：非新生血管型(干性AMD)和新生血管型(湿性AMD)。血管内皮生长因子(Vascular endothelial growth factor,VEGF)是促进血管生成的重要蛋白质之一。目前，抗VEGF药物治疗(包括Ranibizumab，Aflibercept，Bevacizumab，Conbercept，和Brolucizumab等)已成为湿性AMD治疗的标准治疗方案。
糖尿病视网膜病变(DR)是糖尿病常见的微血管并发症，致盲率高，发病机制复杂。DR的病理特征为视网膜新生血管形成和血视网膜屏障破坏。抗VEGF药物治疗应用也较为普遍。
近年来，随着基因递送系统和编辑技术的不断进步，基因治疗领域迅速发展。目前，重组AAV(rAAV，recombinant adeno-associated virus)已经成为体内基因治疗递送的主要平台。rAAV包装的基因组删除了AAV蛋白编码序列，同时添加治疗性基因表达盒。唯一的病毒来源序列是ITR，它们是载体生产过程中指导基因组复制和包装所必需的。由于AAV具有宿主范围广、安全性高、免疫原性低、组织嗜性、可长期稳定表达等优点，已被广泛地应用于基础研究和临床试验中。
目前抗VEGF治疗已成为新生血管型眼病的一线治疗方法。已批准在临床上使用的药物包括抗体和抗体片段(Bevacizumab、Ranibizumab和Brolucizumab)，融合蛋白(Aflibercept和Conbercept)以及核酸适配体Pegaptanib，双抗Vabysmo(Faricimab-svoa)。
Eylea(阿柏西普，Aflibercept)是一种重组融合蛋白，由VEGFR-1的结构域2和VEGFR-2的结构域3与IgG1的Fc片段融合构成。它通过与血管内皮生长因子亚型A和B(VEGF-A和VEGF-B)和胎盘生长因子(PlGF)结合，抑制新生血管形成并降低血管通透性。美国食品药品监督管理局(FDA)和欧洲药物管理局(EMA)分别于2014年8月前相继批准了阿柏西普通过玻璃体注射治疗视网膜静脉阻塞继发黄斑水肿、湿性年龄相关性黄斑变性和糖尿病性黄斑水肿3个适应症。
Conbercept(朗沐，康柏西普)是一类获得世界卫生组织国际通用名称的中国生物制品。该药一类是基因工程抗体药物，与Aflibercept结构类似，但区别在于Conbercept包含了VEGF受体Ig样区域，这样的结构能提高与VEGF的亲和力，还可阻断VEGF-A所有亚型、VEGF-B和胎盘生长因子，提高结合速率，延长药物在体内的半衰期，分子量142KD。自2013年底经中国国家食品药品管理总局批准用于治疗wAMD。
尽管阿柏西普等蛋白药治疗方法是目前治疗新生血管型眼病的标准治疗，但长期的抗VEGF治疗可能增加RPE萎缩、脉络膜萎缩及地图样萎缩的发生率。多中心临床研究发现：部分患者在抗VEGF治疗七年后，其视力降至基线甚至基线水平以下，甚至伴随着黄斑萎缩和纤维化的发生。
而且每4-8周一次的标准治疗实际上很难维持，重复注射可以在一些患者中增加炎症、感染和其他副作用的风险，治疗成本也高，患者接受度较差。
因此，为了改善新生血管型眼病患者的治疗效果，必须开发出替代或补充治疗，减少给药次数，甚至一次给药终生受益，提高患者的接受度。
发明内容：
本发明以阿柏西普表达基因为基础，通过基因改造和载体优化，用重组AAV递送实现目的基因在RPE层的长期稳定表达，将优化的目的基因序列递送至患者眼底细胞。实现“一次给药，长期有效”，为患者提供更安全，更经济，和更 方便的优异的治疗方式，减轻现有新生血管型眼病临床治疗的负担，满足临床上尚未满足的需求。
考虑到阿柏西普在临床上的良好表现，其作为基因治疗递送候选分子在体内长期表达维持疗效同时风险低。
脉络膜新生血管(CNV)是渗出性nAMD的标志性病变，也是老年人视力丧失的一个重要原因。目前激光诱导CNV动物模型已经普遍用于AMD治疗用药的药效评价，包括贝伐珠单抗、阿柏西普和雷珠单抗等蛋白药物。在啮齿动物和NHP激光诱导的nAMD的CNV模型中，脉络膜脉管系统的短暂血管渗漏和新血管反应在激光破坏Bruch膜后持续2-3周(啮齿动物)或6-8周(NHP)，此后自发地消退。
建立一个持续性和复发性血管渗漏和新生血管的模型，将大大有助于并加速对长效干预措施的评估，以解决病理性血管不稳定和新血管形成的多种临床表现。DL-α-氨基己二酸(DL-AAA)模型是一种已经在大鼠和兔中报告的慢性渗漏模型，并且已经在这些物种中进行常规候选药物筛选。DL-AAA是一种选择性神经胶质细胞毒素，据报道可抑制谷氨酰胺合成酶的作用，损害更广泛的Müller细胞视网膜稳态功能而导致神经胶质功能障碍和死亡，从而导致血视网膜屏障破坏。注射D-LAAA后2个月大鼠视网膜下血-视网膜屏障破坏，并增加血管的渗漏和迂曲。最近有几组研究表明，在兔IVT给予DL-AAA后2-36个月，血管渗漏和RNV升高，并且抗VEGF药物贝伐单抗，雷珠单抗，阿柏西普和DARPins靶向VEGF-A165抑制了此类病理。大鼠、兔和人类的视网膜血管和神经元解剖结构不同，但猴和人类之间基本上是相同的。猴和人类的视网膜脉管系统，视网膜分割和基底层边界，视网膜神经元和神经胶质细胞亚型的比例丰度以及黄斑的存在是同源的。同时慢性视网膜血管渗漏和新血管形成的临床前模型允许在疾病发展的多个阶段对短效和长效抗血管生成化合物进行疗效筛查。本发明将使用DL-AAA引起的慢性血管渗漏NHP模型进行效果验证。
本发明提供的技术方案之一，是一种人源IgG1的Fc片段突变体，所述突变体是发生以下突变中的至少一种获得的：
T250A，L251A，M252L，I253A/D/P，S254A，T256A，L309A，H310L/V/A/D/E/Q，Q311A，L314A，M428L/I，H433L/V/A，N434L/V/A，H435L/V/A，Y436A；
其中的编号由EU索引指示；
优选地，所述突变体具有以下单突变中的一种：H310A、H310L、H435A、H435L、I253A、I253D、I253P、H310D、H310E或H310Q；
更优选地，所述突变体具有如下单点突变中的一种：H310A，H310E或H435A；
优选地，所述突变体具有以下双突变中的一种：M252L/M482L、M252L/M482I、M252L/M482L、T250A/H310L、L251A/H310L、I253A/H310L、S254A/H310L、T256A/H310L、L309A/H310L、H310L/Q311A、H310L/L314A、H310L/H433A、H310L/N434A、H310L/H435A、H310L/Y436A、I253A/H310A、I253A/H435A、或H310A/H435A；
更优选地，所述突变体具有如下双突变：H310A/H435A；
优选地，所述突变体是具有以下三突变中的一种：M252L/H310L/M482L、M252L/H310V/M482L、M252L/M482L/H433L、M252L/M482L/H433V、M252L/M482L/N434L、或M252L/M482L/H435V；
优选地，所述突变体具有以下四突变中的一种：M252L/M482L/N434V/H435L、M252L/H310L/M482L/H433L、M252L/H310L/M482L/N434L、或M252L/H310L/M482L/H435L。
更优选地，所述Fc片段突变体具有SEQ ID NO.1、2、3或4所示的序列。
本发明提供的技术方案之二，是一种VEGF受体重组融合蛋白或抗VEGF重组抗体；
所述VEGF受体重组融合蛋白由VEGFR-1的结构域2，VEGFR-2的结构域3，以及技术方案一所述的IgG1的Fc片段突变体融合而成；
进一步地，所述VEGFR-1的结构域2如SEQ ID NO.5所示；
进一步地，所述VEGFR-2的结构域3如SEQ ID NO.6所示；
优选地，所述IgG1的Fc片段突变体为：H310A(SEQ ID NO.1所示)、H310E(SEQ ID NO.2所示)、H435A(SEQ ID NO.3所示)、H310A/H435A(SEQ ID NO.4所示)。
更优选地，所述VEGF受体重组融合蛋白的氨基酸序列如SEQ ID NO.7、8、9或10所示；
所述抗VEGF重组抗体是在现有抗VEGF抗体的基础上将其中的Fc片段替换为技术方案一所述的IgG1的Fc片段突变体；
进一步地，现有抗VEGF抗体包括但不限于贝伐单抗(Bevacizumab)、雷珠单抗(Ranibizumab)和帕普利珠单抗(Brolucizumab)；
更近一步地，所述抗VEGF重组抗体是将贝伐单抗Fc片段替换为技术方案一所述的IgG1的Fc片段突变体，更优选地，具有SEQ ID NO.11所示的氨基酸序列。
本发明提供的技术方案之三，是一种表达技术方案二所述VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，所述表达盒从5’-3’端包含如下式I所述的结构：
ITR-E1-E2-E3-E4-ITR式(I)，其中：
ITR为反向末端重复序列；
E1为启动子；
E2为信号肽；
E3为编码技术方案二所述VEGF受体重组融合蛋白或抗VEGF重组抗体的核苷酸序列；
E4为Poly A序列。
进一步地，所述ITR(反向末端重复序列)来自于AAV1，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8或AAV9，优选地，所述ITR序列选自AAV2；
进一步地，所述启动子是可以启动目的基因转录的DNA序列，该序列可以被RNA聚合酶所识别，并开始转录合成RNA，所述启动子包括但不限于天然、优化或组合启动子；
更进一步地，所述启动子优选CMV、CBA、EF1a、SV40、PGK1、Ubc、CAG、TEF1、U6或H1启动子；
进一步地，所述信号肽来源包括但不限于Human OSM、Gaussia luc、或者Albumin(HSA)；
进一步地，所述Poly A序列选自bGH PolyA、SV40PolyA或hGH PolyA；
进一步地，上述表达盒还包括调控元件，所述表达调控元件包括但不限于以下功能的调控元件：(1)用于调控目的蛋白的表达，例如IRES，用于启动下游基因的翻译；(2)用于表达miRNA和siRNA序列的调控元件；(3)内含子；(4)定位序列，将目的蛋白定位表达到细胞核，细胞质或各类细胞器，以及分泌到细胞外；(5)调控元件还可以是部分的kozak序列，kozak序列为 G/N-C/N-C/N-ANNAUGG，例如GCCACCAUGG等；(6)增强子，增强子可以来自SV40病毒，CMV病毒或腺病毒等；(7)调控元件可以是WPRE。
进一步地，上述表达盒还包括标签元件，所述标签元件包括但不限于FLAG，HA，MYC，荧光蛋白，荧光素酶，SUMO蛋白，泛素蛋白，GST等。
优选地，所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，是将技术方案二所述VEGF受体重组融合蛋白或抗VEGF重组抗体在AAV病毒载体上进行表达；
进一步地，所述AAV病毒载体包括但不限于pAAV-CMV、pX601、pX551、pAAV-MCS质粒等；
优选地，所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒包括：ITR-CMV启动子-CMV增强子-内含子-信号肽-Kozak序列-VEGF受体重组融合蛋白/抗VEGF重组抗体编码序列-PolyA-ITR；
更优选地，所述表达VEGF受体重组融合蛋白的AAV病毒载体表达盒具有SEQ ID NO.12所示的序列；
更优选地，所述表达抗VEGF重组抗体的AAV病毒载体表达盒具有SEQ ID NO.13所示的序列。
本发明提供的技术方案之四，是一种腺相关病毒包装载体系统，所述包装载体系统包括：技术方案三所述的表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，携带AAV rep、cap基因的载体以及辅助病毒载体，上述载体通过包装成为AAV病毒；
进一步地，所述携带AAV rep、cap基因的载体包括但不限于：AAV1，AAV2，AAV5，AAV8，AAV9，AAV-R100，AAV-NN，AAV-GL，AAV8-Y447F，AAV8-Y733F，AAV8-Y447F/Y733F，AAV-DJ或AAV7m8载体等；
进一步地，所述辅助病毒载体为腺病毒或疱疹病毒辅助病毒载体，优选pHelper质粒。
本发明提供的技术方案之五，是一种腺相关病毒的包装方法，将技术方案四所述腺相关病毒包装载体系统转入宿主细胞中进行病毒包装；
进一步地，将技术方案四所述包装载体系统中的表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒与携带AAV rep、cap基因的载体以及辅助病毒载体转入宿主细胞中进行病毒包装；
进一步地，所述宿主细胞是能够进行病毒复制且稳定遗传的细胞系，包括但不限于Hela-S3，HEK-293，HEK-293T，HEK-293FT，Expi293F，A549和Sf9等细胞；
优选地，所述宿主细胞为HEK-293、HEK-293T细胞或Expi293F细胞。
本发明提供的技术方案之六，是采用技术方案之五所述的包装方法获得的腺相关病毒；
优选地，是将SEQ ID NO.12所示的表达VEGF受体重组融合蛋白的AAV病毒载体表达盒、AAV8Rep-Cap质粒、pHelper质粒转入宿主细胞Expi293F细胞中进行病毒包装获得的；
优选地，是将SEQ ID NO.13所示的表达抗VEGF重组抗体的AAV病毒载体表达盒、AAV8Rep-Cap质粒、pHelper质粒转入宿主细胞Expi293F中进行病毒包装获得的。
本发明提供的技术方案之七，是包含技术方案三所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或技术方案之六所述腺相关病毒的制剂或配方或药物；
进一步地，所述制剂或配方或药物可以是任何剂型，包括但不限于注射剂型和软膏剂型；
进一步地，所述制剂或配方或药物中，上述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或腺相关病毒为唯一活性成分。
本发明提供的技术方案之八，是技术方案三所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或技术方案之六所述腺相关病毒在制备治疗新生血管相关眼底疾病，特别是年龄相关黄斑变性、湿性黄斑病变、糖尿病视网膜病变等疾病的制剂或配方或药物中的应用；
进一步地，所述制剂或配方或药物的给药方法为单侧眼给药或双侧眼给药；
更进一步地，给药方式为玻璃体注射、视网膜下腔注射、脉络膜上腔注射等注射方式；
更进一步地，终身单次给药或多次给药，给药总剂量为1×108-1×1013个病毒基因组/眼。
有益效果：
1、本发明通过Fc突变，降低与FcRn的亲和力，改善眼部PK行为，减少入血， 且不影响稳定性，大幅增加在眼部的蓄积并延长半衰期；即使入血，因降低了FcRn亲和力，在循环系统的半衰期更短，避免引起其他系统性安全风险；
2、通过Fc突变和载体元件组合设计，实现更低的AAV病毒剂量在体内持续稳定表达，跟现有同类的AAV产品体内蛋白表达数据相比具有明显优势；
3、本发明设计的rAAV载体元件递送Fc突变改造的VEGF拮抗剂组合比现有上市蛋白药对照组药效更持久；
4、本发明制备的AAV病毒可通过一次给药终身缓解或治愈血管新生型眼底疾病的机会，或者减少蛋白药给药次数；
5、同样的组合设计可以采用玻璃体注射、视网膜下腔、脉络膜上腔等多种给药方式。
附图说明：
图1启动子优化实验结果
其中，图1中各启动子、增强子表示如下：
·CMV:CMV enhancer+CMV promoter；
·GNAT2:IRBP enhancer+GNAT2promoter；
·CBA:CMV enhancer+chicken-βactin promoter；
·MPL:CMV enhancer+CMV promoter+TPL-MLP；
·RPE65:CMV enhancer+RPE65promoter。
图2不同信号肽对表达量的影响。
图3 Fc突变蛋白上清表达定量结果统计。
图4不同PolyA对不同Aflibercept瞬转ARPE-19细胞表达结果的影响。
图5不同PolyA对不同Aflibercept瞬转HEK293细胞表达结果的影响。
图6候选Fc工程化改造VEGF受体重组融合蛋白37℃热稳定性分析结果。
图7候选Fc工程化改造VEGF受体重组融合蛋白40℃热稳定性分析结果。
图8 AAV载体表达产物对HUVEC细胞增殖抑制功能。
图9小鼠注射AAV后不同时间点蛋白表达量。
图10 FFA(眼底血管荧光造影)图谱(晚期)。
图11各组动物眼底光斑渗漏平均分。
图12激光造模后4周眼底血管造影(FFA)典型图谱。
图13 PRNV造模给药组荧光渗漏基线变化。
具体实施方式：
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本专利，并不用于限定本发明。
本发明提供一种通过AAV递送经优化的VEGF受体重组融合蛋白或抗VEGF重组抗体，通过视网膜下腔、玻璃体或脉络膜上腔注射等方式，将优化的目的基因序列递送至患者眼部，实现目的基因在视网膜中的长期稳定表达，一次低剂量给药终身治疗。为患者提供更安全、方便、优异的治疗方式，减轻现有血管新生相关眼底疾病临床治疗的负担；并且解决患者依从性差和坚持性差的状况，改善患者治疗结局。
Fc融合蛋白是指利用基因工程等技术将某种具有生物活性的功能蛋白分子与免疫球蛋白(IgG、IgA等)的Fc片段融合而产生的新型重组蛋白。其不仅保留了功能蛋白分子的生物学活性，还具有一些抗体的性质，如通过结合相关Fc受体延长半衰期和引发抗体依赖细胞介导的细胞毒性效应等，在疾病的诊断与治疗方面有重要的意义。FcRn是一种位于细胞膜表面的IgG抗体受体，其蛋白结构和MHC-I分子类似，主要在内皮细胞中表达(在其它组织或细胞中也能检测到)，其结构包含由α链和β2微球蛋白组成的异二聚体。FcRn可以和IgG的Fc部分结合，阻止IgG分子被溶酶体裂解，可以起到增长IgG体内半衰期的作用，参与到IgG的体内转运、维持和分布代谢过程中。IgG的Fc部分与新生儿Fc受体相互作用(FcRn，Brambell受体)，防止其在溶酶体中降解，并使抗体长期血清持续。FcRn的其他生物学作用包括围产期IgG转移、抗体介导的抗原呈现和IgG跨越上皮和内皮屏障的转移。改变IgG：FcRn相互作用已被越来越多的研究用于改善抗体的治疗或诊断应用。眼内注射IgG或Fc融合蛋白的体内作用途径，主要是通过视网膜RPE及内皮细胞上的FcRn介导穿过血-视网膜屏障，进入系统循环后逐步清除(elimination)，因此在眼部代谢中FcRn介导IgG或Fc融合蛋白的入血及清除。
本发明所述的经优化的VEGF受体重组融合蛋白，包括人血管内皮生长因子(VEGF)受体1Domain 2和受体2Domain 3与人免疫球蛋白Fc段突变体重组蛋白。在本发明的一个实施例中，设计多组Fc单点或多点突变体，本发明突变位点的选 择除了考虑FcRn亲和力，优选地考虑rAAV组合物在体内外目标蛋白表达的稳定性和持续性。Fc突变体具体突变位点包括：T250A，L251A，M252L，I253A/D/P，S254A，T256A，L309A，H310L/V/A/D/E/Q，Q311A，L314A，M428L/I，H433L/V/A，N434L/V/A，H435L/V/A，Y436A等。在本发明的一个实施例中，优选的突变位点是单点突变，包括H310A，H310L，H435A，H435L，I253A，I253D，I253P，H310D，H310E，H310Q。在本发明的另一个实施例中，优选的突变位点是双点突变，包括M252L和M482L，M252L和M482I，M252L和M482L，T250A和H310L，L251A和H310L，I253A和H310L，S254A和H310L，T256A和H310L，L309A和H310L，H310L和Q311A，H310L和L314A，H310L和H433A，H310L和N434A，H310L和H435A，H310L和Y436A，I253A和H310A，I253A和H435A，H310A和H435A等。在本发明的另一个实施例中，优选的突变位点是三点突变，包括M252L、H310L和M482L，M252L、H310V和M482L，M252L、M482L和H433L，M252L、M482L和H433V，M252L、M482L和N434L，M252L、M482L和H435V。在本发明的另一个实施例中，优选的突变位点是四点突变，包括M252L、M482L、N434V和H435L，M252L、H310L、M482L和H433L，M252L、H310L、M482L和N434L，M252L、H310L、M482L和H435L。在本发明的另一个实施例中，更优选的单点突变位点是H310A，H310E，H435A，获得的Fc突变体具有SEQ ID NO.1、2或3所示的氨基酸序列。在本发明的另一个实施例中，更优选的双点突变位点是H310A/H435A，Fc突变体具有SEQ ID NO.4所示的氨基酸序列。
上述Fc突变体是在以下人源IgG1的Fc野生型片段的基础上发生的突变(SEQ ID NO.15)：
本发明中，Fc片段中的氨基酸残基编号是免疫球蛋白重链的编号，其是根据Kabat等人的Sequences of Proteins of Immunological Interest[免疫学目的蛋白质序列]，第5版.Public Health Service[公共卫生署]，National Institutes of Health[国立卫生研究院]，贝塞斯达，马里兰州(1991)(通过引用明确地并入本文)中的EU索引。本文的“如EU索引/编号”是指人IgG1抗体恒定区残基的EU编码序号”。 该编号是本领域技术人员众所周知的，并且经常在本领域中使用。也可参考以下网址：https://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html。
更优选地，在本发明的另一个实施例中，所述VEGF受体重组融合蛋白的氨基酸序列如SEQ ID NO.7-10所示。
本发明所述的抗VEGF重组抗体是在现有抗VEGF抗体的基础上将其中的Fc片段替换为前述IgG1的Fc片段突变体；在本发明的另一个实施例中，现有抗VEGF抗体包括但不限于贝伐单抗(Bevacizumab)、雷珠单抗(Ranibizumab)和帕普利珠单抗(Brolucizumab)；在本发明的另一个实施例中，所述抗VEGF重组抗体是将贝伐单抗Fc片段替换为技术方案一所述的IgG1的Fc片段突变体H310E，优选地，具有SEQ ID NO.11所示的氨基酸序列。
本发明还提供了一种载体表达盒，所述表达盒从5’-3’端包含式I所述结构：
ITR-E1-E2-E3-E4-ITR(I)，其中：
ITR为反向末端重复序列；E1为启动子(包括天然、优化或组合启动子)；E2为信号肽；E3为本发明编码VEGF受体重组融合蛋白或抗VEGF重组抗体的核苷酸序列；E4为Poly A序列。
在一些实施方案里，所述ITR序列(反向末端重复序列)来自于AAV1，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8或AAV9；优选地来自AAV2；
在一些实施方案里，核酸序列具有启动子。所述启动子用于启动与其连接的核酸序列的表达，启动子可以是组成型的或是诱导型的，启动子可以是在视网膜色素上皮细胞特异性表达的启动子，也可以是普遍存在的启动子。优选的启动子包括各种真核启动子，例如，CMV、EF1a、CBA、SV40、PGK1、Ubc、CAG、TEF1、U6、H1等。大部分重组AAV载体启动子使用的是广谱的CMV(CMV增强子和CMV启动子)，CBA(CMV增强子和chicken 9-actin启动子)，PGK启动子和EF1a启动子。更优选地，启动子为CMV启动子。
信号肽是引导新合成的蛋白质向分泌通路转移的短肽链(长度5-30个氨基酸)。常指新合成多肽链中用于指导蛋白质的跨膜转移(定位)的N-末端的氨基酸序列(有时不一定在N端)。在起始密码子后，有一段编码疏水性氨基酸序列的RNA区域，该氨基酸序列就被称为信号肽序列，它负责把蛋白质引导到细胞含不同膜结构的亚细胞器内。包括三个区：一个带正电的N末端，称为碱性氨基末端；一个中间疏水序列，以中性氨基酸为主，能够形成一段α螺旋结构，它是 信号肽的主要功能区；一个较长的带负电荷的C末端，含小分子氨基酸，是信号序列切割位点.也称加工区。当信号肽序列合成后，被信号识别颗粒(SRP)所识别，蛋白质合成暂停或减缓，信号识别颗粒将核糖体携带至内质网上，蛋白质合成重新开始。在信号肽的引导下，新合成的蛋白质进入内质网腔。而信号肽序列则在信号肽酶的作用下被切除。如终止转运序列存在于新生肽链的C端，也可以不被信号肽酶切除。
在本发明的一些实施例中，选取常用的真核生物表达信号肽(见表1)，PCR法将SP1～SP13信号肽插入到目的基因的N端，并同源重组到EcoRI和HindIII双酶切的PTT5载体中。大肠杆菌转化后测序验证序列正确后，质粒去内毒素大量抽提并PEI法瞬时转染HEK293E细胞，利用含谷氨酰胺谷氨酰胺的CD05培养基培养5天后，取细胞上清检测蛋白表达量，同时利用Protein A亲和层析细胞培养上清中的目的蛋白。蛋白的定量通过二喹啉甲酸(Bicinchoninic acid，BCA)方法进行。根据目的蛋白的表达量获得优选的信号肽序列。
在另一个实施例中，优选的信号肽来自Human OSM、Gaussia luc、或者Albumin(HSA)。在另一个实施例中，更优选的信号肽来自Gaussia luc。
多腺苷酸化(Polyadenylation)是指多聚腺苷酸与信使RNA(mRNA)分子的共价链接。在蛋白质生物合成的过程中，这是产生准备作翻译的成熟mRNA的方式的一部份。在真核生物中，多聚腺苷酸化是一种机制，令mRNA分子于它们的3'端中断。多聚腺苷酸尾(或聚A尾)保护mRNA，免受核酸外切酶攻击，并且对转录终结、将mRNA从细胞核输出及进行翻译都十分重要。
在某些实施例中，PolyA序列的主要选择是bGH PolyA(来源于pCMV3)、SV40PolyA(来源于pCGS3)序列。不同的PolyA序列和其他表达元件相互作用可能提高转录水平。本发明的一个实施例中测试了三种不同的PolyA序列bGH PolyA、SV40PolyA和hGH PolyA，以获得最佳的基因表达与病毒生产。在本发明的另一个实施例中，优选的PolyA序列是bGH PolyA和hGH PolyA。在本发明的另一个实施例中，优选的PolyA序列是bGH PolyA。
在某些实施例中，上述表达盒还包括调控元件，所述表达调控元件包括但不限于以下功能的调控元件：(1)用于调控目的蛋白的表达，例如IRES，用于启动下游基因的翻译；(2)用于表达miRNA和siRNA序列的调控元件；(3)内含子，内含子(Intron)又称间隔顺序，指一个基因或mRNA分子中无编码作用的 片段；(4)定位序列，将目的蛋白定位表达到细胞核，细胞质或各类细胞器，以及分泌到细胞外；(5)调控元件还可以是部分的Kozak序列，Kozak序列是位于真核生物mRNA 5’端帽子结构后面的一段核酸序列，通常是GCCACCAUGG，它可以与翻译起始因子结合而介导含有5’帽子结构的mRNA翻译起始，对应于原核生物的SD序列，存在于真核生物mRNA的一段序列，其在翻译的起始中有重要作用。Kozak序列为G/N-C/N-C/N-ANNAUGG，例如GCCACCAUGG；(6)增强子，增强子可以来自SV40病毒，CMV病毒或腺病毒等；(7)调控元件可以是WPRE。
在某些实施例中，上述表达盒还包括标签元件，所述标签元件包括但不限于例如FLAG，HA，MYC，荧光蛋白，荧光素酶，SUMO蛋白，泛素蛋白，GST等。
在一个实施方案中，本发明公开了一种重组病毒载体，其包含以下元件:(a)第一AAV2反向末端重复(ITR)序列，(b)CMV增强子和启动子，(c)嵌合内含子，(d)Kozak序列，(e)信号肽序列，(f)VEGF受体重组融合蛋白/抗VEGF重组抗体编码序列，(g)WPRE序列，(h)bGH polyA序列，和(i)第二AAV2ITR。
在一个实施方案中，将上述经优化的VEGF受体重组融合蛋白/抗VEGF重组抗体在AAV病毒载体上进行表达，所述的AAV病毒载体包括但不限于pAAV-CMV、pAAV-MCS质粒；优选地，所述AAV病毒载体为pAAV-CMV。
在本发明中，还提供由上述表达经优化的VEGF受体融合蛋白/抗VEGF重组抗体的载体包装而成的AAV病毒。AAV病毒可采用本领域已公开的标准方法制备，可参见《AAV Production Everywhere:A Simple,Fast,and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction》。
复制缺陷重组AAV可通过三质粒共转进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，辅助包装质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒，然后通过标准技术纯化所产生的AAV重组病毒。优选地，所述重组腺相关病毒为单链AAV。
在一些实施例中，将经优化的VEGF受体重组融合蛋白/抗VEGF重组抗体的编码基因片段包装到病毒颗粒(例如，包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、 AAV13、AAV14、AAV15、AAV16、AAV7m8、AAV PHP.eB等的AAV血清型病毒颗粒)中。因此，本公开包括含有本文所述的任何载体的重组病毒颗粒。优选地，所述重组腺相关病毒血清型是AAV2，AAV8。更优选地，所述重组腺相关病毒血清型是AAV8。
用于递送编码VEGF受体重组融合蛋白/抗VEGF重组抗体的核酸序列的优选的病毒载体是AAV载体，例如自身互补的AAV(scAAV)，单链AAV(ssAAV)，pAAV-CMV、pAAV-MCS等。
使用特定的AAV血清型(AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15、AAV16、AAV7m8、AAV PHP.eB)或任何一个其他修饰的血清型都可以实现选择性靶向。
本发明进行上述病毒组装采用的宿主细胞，用于AAV载体转导细胞表达Fc工程化改造的VEGF受体重组融合蛋白、抗VEGF抗体。优选地，所述宿主细胞为哺乳动物细胞(优选为人体来源的细胞，更优选为人视神经细胞或感光细胞)，提高VEGF受体融合蛋白、抗VEGF抗体的表达量，体现本发明的AAV载体分子的特异性表达。所述宿主细胞包括但不限于Hela-S3，HEK-293，HEK-293T，HEK-293FT，Expi293F，A549和Sf9等细胞；在本发明的一些实施方案中，优选的宿主细胞为HEK-293、HEK-293T细胞或Expi293F细胞。
在一个实施方案中，本发明公开了以下重组腺相关病毒：AAV8-Afli Fc Variant10。该重组病毒是AAV8血清型病毒，携带转基因的载体具有SEQ ID NO:12所述的序列，携带的转基因是VEGF受体重组融合蛋白编码基因，在Aflibercept融合蛋白基因Fc区第310位进行了H310E突变改造。
在一个实施方案中，本发明公开了以下重组病毒：AAV8-Bevacizumab Fc Variant10。该重组病毒是AAV8血清型病毒，携带转基因的载体具有SEQ ID NO:13所述的序列，携带的转基因是Bevacizumab单克隆抗体的突变基因，是在Bevacizumab基因Fc区第310位进行了H310E突变改造。
本发明中构建的重组病毒适于在体内(in vivo)或体外(ex vivo)使用，优选地适于在血管新生相关眼底疾病(包括年龄相关黄斑变性、湿性黄斑变性、糖尿病黄斑水肿)患者的玻璃体、视网膜下腔或脉络膜上腔一次给药使用。优选地，小鼠给药的安全有效量范围1×106-1×1011病毒基因组/眼，NHP给药的安全有效 量范围1×108-1×1013病毒基因组/眼，人给药的安全有效量范围1×108-1×1013病毒基因组/眼(病毒基因组，genomic copies，简称GC)。
在一个实施方案中，本发明提供了包含本发明的多核苷酸或病毒载体或腺相关病毒的组合物。
在一个实施方案中，本发明提供了包含本发明的多核苷酸或病毒载体或腺相关病毒的组合物的制备方法。
在一个实施方案中，本发明提供了包含本发明的多核苷酸或病毒载体或腺相关病毒的组合物视网膜下腔的给药方式。
在一个实施方案中，本发明提供了包含本发明的多核苷酸或病毒载体或腺相关病毒的组合物玻璃体注射的给药方式。
在一个实施方案中，本发明提供了包含本发明的多核苷酸或病毒载体或腺相关病毒的组合物脉络膜上腔的给药方式。
在一个实施方案中，本发明提供了小鼠/NHP/人给药后观察的方法，包括学相干断层扫描(OCT)和荧光素眼底血管造影(FFA)。
在一个实施方案中，本发明提供了小鼠给药后检测眼球组织表达阿柏西普蛋白的方法。
制剂和组合物
本发明提供一种制剂或组合物，所述制剂或组合物含有本发明所述的腺相关病毒载体或病毒，以及药学上可接受的载体或赋形剂。
在另一优选例中，所述药物制剂用于治疗眼部疾病，优选地，所述药物制剂用于治疗跟VEGF相关眼部疾病，优选地，用于治疗黄斑变性和/或糖尿病视网膜病变，更优选地，用于治疗湿性年龄相关性黄斑变性或湿性黄斑病变。
为了便于临床应用，本发明的药物组合物可以灌装在密封的西林瓶或者预填充式注射器里。所述的西林瓶或预填充式注射器可以包装在药品包装盒中，以方便储存、使用。本发明的药物制剂及组合物保存在＜-60℃超低温冰箱，运输时采用干冰冷链运输。
药物制剂或制剂组合物的剂型可以是液体或固体，例如粉末、凝胶或糊剂。优选地，组合物是液体，更优选地为注射剂。
本发明所提供的AAV载体制剂滴度在1×1010-1×1014GC/ml，优选的滴度为1×1012-1×1013GC/ml。
所述制剂包含药学上可接受的赋形剂。在一些情况下，所述赋形剂包括表面活性剂或稳定剂。在本发明中，所述表面活性剂选自聚山梨醇醋、十二炕基硫酸纳、月桂基硫酸纳、月桂基二甲基氧化胶、聚乙氧基化醇、聚氧乙烯山梨醇、辛苯酚聚酣、Brij、普洛尼克(pluronic)和聚氧乙烯菌麻油(polyoxyl caster oil)。在一些情况下，所述药学上可接受的赋形剂包括苯酚、甘露醇、山梨醇、蔗糖或氯化钠。
本发明所述药物组合物中的“活性成分”是指本发明所述的载体或AAV病毒。本发明所述的“活性成分”、制剂和/或组合物可用于治疗眼部疾病。“安全有效量”指的是：活性成分的量足以明显改善病情或症状，而不至于产生严重的副作用。“药学上可接受的载体或赋形剂(excipient)”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
治疗方法
本发明提供了治疗VEGF介导的新生血管性眼病的方法，所述方法包括将包含编码Fc工程化改造的VEGF受体重组融合蛋白或抗VEGF重组抗体核酸序列的病毒或病毒载体引入到眼睛内。所述方法可包括向视网膜下腔、玻璃体内或脉络膜上腔注射核酸载体，所述核酸载体靶向RPE细胞或感光受体细胞。
本发明提供了用于向视网膜细胞递送编码治疗蛋白核酸序列的治疗新生血管性眼病的方法中使用的核酸载体，所述核酸载体包含编码Fc工程化改造的VEGF受体重组融合蛋白或抗VEGF重组抗体的核酸序列。本发明组合物可以单独给药，或者与其他治疗药物联合给药(如配制在同一药物组合物中)。
如本发明所述，治疗疾病意指施用如本文描述的核酸或载体或病毒以改善或减轻疾病的一种或多种症状，包括减少血管渗漏、减少新生血管生成等。
优选地，本发明的方法包括将编码Fc工程化改造的VEGF受体重组融合蛋白或抗VEGF的重组抗体的核酸序列引入到眼睛的视网膜下腔内。
优选地，本发明的方法包括使细胞与包含编码Fc工程化改造的VEGF受体重组融合蛋白或抗VEGF的重组抗体的核酸序列的载体(优选地为病毒，更佳地为腺相关病毒)接触。优选地，细胞是视网膜细胞，优选地色素上皮细胞、视锥细胞、视杆细胞、双极细胞、水平细胞、神经节细胞和/或无长突细胞。
本发明的重组腺相关病毒载体可以有效抑制动物眼内VEGF表达，减轻激光损伤导致的渗漏和血管新生，也可以减轻DL-AAA引起的视网膜血管病变，因此可用于制备治疗VEGF相关疾病的药物，治疗人体跟VEGF相关的眼部疾病，具有广泛的市场前景。
本发明提供的包含有效量的核酸序列的病毒载体用于人体跟VEGF相关的眼部疾病治疗时，临床施用剂量1×108-1×1013GC病毒，眼部注射体积为50～300μl，给药途径是视网膜下腔、玻璃体内或脉络膜上腔注射。
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明及实施例中涉及的突变体名称及突变体信息如下表：
突变体对照表
注：表中突变体Variant1-12，是在SEQ ID NO.14所示的Eylea(阿柏西普，Aflibercept)的基础上，对其中的Fc片段进行相应的突变获的得Aflibercept突变体。
实施例1基因表达盒的启动子筛选
大部分重组AAV载体启动子使用的是广谱的CMV(CMV增强子和CMV启动子)，CBA(CMV增强子和chicken-βactin启动子)。据文献报道优化的视网膜 细胞特异性启动子可能会更高效地在特定组织表达目的蛋白。实验中我们以pAAV-MCS质粒为基础，更换启动子元件。选择了CBA，CMV+TPL-MLP(TPL-MLP序列来源于pTT5载体)，RPE65(GenBank:AF304008.1)和IRBPe/GNAT2(GenBank:X53044.1，GenBank:U66698.1)启动子开展研究。Aflibercept的氨基酸序列来自于DrugBank数据库(登录号DB08885)，按照Homo sapiens的密码子使用偏好进行密码子优化，将序列克隆到pTT5表达载体中。
具体实验步骤如下：1)阿柏西普序列优化合成，RPE65和IRBPe/GNAT2启动子序列合成；2)PCR法扩增获得RPE65、IRBPe/GNAT2启动子序列，依次与NcoI和SacI、MluI和SacI酶切后的pAAV-MCS载体进行同源重组，大肠杆菌转化后测序和酶切验证获得pAAV-RPE65-MCS、pAAV-IRBPe/GNAT2-MCS载体。3)EcoRI和HindIII双酶切pAAV-RPE65-MCS、pAAV-IRBPe/GNAT2-MCS、pAAV-MCS，PCR法扩增获得阿柏西普的目的基因序列后，同源重组法将阿柏西普的序列插入到酶切后的上述载体中，大肠杆菌转化后测序和酶切验证获得pRPE65-AAV-Afli YB、pGNAT2-AAV-Afli和pAAV-Afli YB载体。4)PCR法扩增CBA和阿柏西普序列，overlap PCR后将插入序列同源重组到NcoI和HindIII酶切后的pAAV-MCS载体中，大肠杆菌转化后测序和酶切验证获得pCBA-AAV-Afli YB载体。5)PCR法扩增CMV+TPL/MLP和阿柏西普序列，overlap PCR后将插入序列同源重组到pAAV-MCS载体中，大肠杆菌转化后测序和酶切验证获得pMPL-AAV-Afli YB载体。6)步骤3，4和5获得的质粒去内毒素大量抽提后瞬时转染ARPE-19细胞，72小时后ELISA检测细胞上清中Aflibercept的表达量。实验结果如图1启动子优化实验结果所示。结果表明含CMV启动子序列的蛋白表达量明显高于其他启动子序列。
实施例2信号肽序列的筛选
选取常用的真核生物表达信号肽(见表1)，PCR法将SP1～SP13信号肽插入到SEQ ID NO.14所示Aflibercept目的基因的N端，并同源重组到EcoRI和HindIII双酶切的PTT5载体中。大肠杆菌转化后测序验证序列正确后，质粒去内毒素大量抽提并PEI法瞬时转染HEK293E细胞，利用含谷氨酰胺的CD05培养基培养5天后，取细胞上清ELISA检测Aflibercept的表达量，同时利用Protein A 亲和层析细胞培养上清中的目的蛋白。蛋白的定量通过BCA法(PierceTMRapid Gold BCA蛋白测定试剂盒)检测。根据目的蛋白的表达量获得优选的信号肽序列。本研究比较了13种不同的信号肽SP1～SP13对目的蛋白表达量的影响，同时以Gaussia luc作为对照，结果如图2所示。结果表明，SP1信号肽的蛋白表达量最高。
表1：真核生物表达信号肽
实施例3：目标蛋白Fc段突变体的筛选
实验中以Aflibercept为基础(SEQ ID NO.14)，设计多组Fc单点和多点突变体，通过设计定点突变引物后PCR法获得各个突变体片段Variant1-12的编码基因。PCR产物纯化后使用内切酶DpnI消化30分钟。消化产物大肠杆菌转化后挑选克隆测序验证序列。
测序正确的克隆扩大培养后进行去内毒素大量抽提质粒。然后PEI法瞬时转染Expi293F细胞，利用含谷氨酰胺的CD05培养基培养5天后，取细胞上清ELISA检测Aflibercept的表达量，同时利用Protein A亲和层析纯化细胞培养上清中的 目的蛋白。蛋白的定量通过BCA方法进行。对突变分子上清表达量、FcRn亲和力进行考察，结果如图3和表2所示。Aflibercept分子经Fc突变改造后，Variant1、2、6和10突变体与FcRn不结合，蛋白表达量与突变前比无显著差异。其中，Variant12是Fc区域的亲和力正向突变体，增加其与FcRn的亲和力。
表2 Fc突变蛋白FcRn亲和力测定结果
注：ND表示未检出
实施例4多聚腺苷酸化序列(PolyA)的筛选
为了实现高效的前mRNA处理，需要构建高效的多聚腺苷酸化序列包含在转基因后面，在RNA的3’端形成一个合适的PolyA尾。rAAV载体中PolyA序列的主要选择是bGH PolyA、SV40PolyA序列。不同的PolyA序列和其他表达元件相互作用可能提高转录水平。因此，实验中测试了三种不同的PolyA序列以获得最佳的基因表达与病毒生产。
选取优选的Fc突变的Aflibercept突变体分子以及未突变Aflibercept分子(简称为“Afli”)，将这些目的基因片段插入到pAAV-MCS载体(含有hGH PolyA)中，同时将PolyA序列分别更换为bGH PolyA、SV40PolyA序列。构建好的各个AAV载体使用lipofectamine2000转染试剂瞬时转染ARPE-19细胞和HEK293细胞，72h后检测目的蛋白的表达量。
测试将3种不同PolyA序列插入到4个分子Afli、Variant2、6和10中，对目的蛋白表达量的影响，结果如图4(瞬转ARPE-19细胞表达结果)和图5(瞬转HEK293细胞表达结果)所示。结果表明，PolyA对不同变体分子的表达效率存在差异，不同分子优选不同PolyA元件。
实施例5融合蛋白热稳定性分析
本研究测试Aflibercept突变体蛋白分子在不同加热条件下的热稳定性，SEC分析柱选用Thermo fisher的MAbPac SEC-1。结果如图6(候选Fc工程化改造VEGF受体重组融合蛋白37℃热稳定性分析结果)和图7(候选Fc工程化改造VEGF受体重组融合蛋白40℃热稳定性分析结果)所示。结果表明，在37℃和40℃保存一定时间后，各蛋白分子的SEC纯度均出现下降趋势，除Variant12外，各蛋白分子的热稳定性无明显差异。
实施例6候选蛋白单次玻璃体注射药代动力学
考察不同工程化改造的Variant2、Variant10和对照组Aflibercept(以下简称Afli，SEQ ID NO.14)在兔眼玻璃体双眼单次注射给药后的PK行为，为下一步AAV病毒包装载体基因的设计和优化提供数据支持。蛋白剂量均为1.25mg/眼，给药体积为50μL/眼。新西兰兔经戊巴比妥钠麻醉后，用聚维酮碘溶液消毒待注射眼，双眼均以50μL/眼的体积按所设剂量经眼玻璃体分别注射给予候选蛋白。于待注射眼滴1～2滴盐酸奥布卡因滴眼液进行表面麻醉后再进行注射操作。玻璃体注射后，各组兔双眼均加约1～2滴氧氟沙星眼膏以保持角膜湿润及抗感染。给药后1h-672h，设置不同时间点取眼组织，分离视网膜/脉络膜丛、房水和玻璃体，组织匀浆取上清；并且采集静脉血液样本；用ELISA方法检测眼组织和血清中的蛋白药物浓度。
玻璃体、房水和视网膜脉络膜丛3种组织样本，以每组动物浓度均值对时间点绘制药时曲线，采用一房室模型进行PK分析，结果见表3。比较各分子的消除半衰期t1/2和药时曲线下面积AUC0-t，Variant10分子半衰期t1/2为Afli的1.03-1.76倍，暴露量AUC为Afli的1.19-1.84倍；Variant2分子半衰期t1/2为Afli的1.15-1.40倍，暴露量AUC为Afli的1.02-1.78倍。以血清样本浓度均值对时间点绘制药时曲线，采用非房室模型计算各蛋白分子经眼部入血后的药物暴露量AUC(见表4)，Variant10的平均AUC较Afli降低74.2％，Variant2的平均AUC较Afli降低54.9％。
表3各突变体分子眼部组织PK分析参数(一房室模型)

表4各突变体分子血清暴露量AUC汇总(非房室模型)
实施例7 AAV8-Afli Fc Variant10AAV病毒样品制备
1、Variant 10载体构建：
(1)Aflibercept的氨基酸序列来自于DrugBank数据库(登录号DB08885，SEQ ID NO.14)，以密码子优化合成的Aflibercept为模板，PCR法扩增Aflibercept片段，同时添加源于Gaussia luc的信号肽(序列信息见表1)，并将该片段插入到NotI和BamHI双酶切的PTT5载体中，测序验证正确后获得Aflibercept-PTT5载体。
(2)以Aflibercept-PTT5载体为模板，设计Fc片段的H310E的定点突变引物后PCR法获得Variant10突变体编码片段，PCR产物纯化后使用内切酶DpnI消化30分钟。消化产物大肠杆菌转化后挑选克隆测序验证序列。测序验证正确的为Variant10-PTT5载体。
(3)以Variant10-PTT5为模板，PCR法扩增Variant10编码片段后同源重组法插入到EcoRI和HindIII双酶切的pAAV-MCS质粒中，大肠杆菌转化后挑选克隆测序验证序列。同时对测序正确的载体进行SmaI酶切验证ITR是否有缺失，验证正确的载体命名为Afli Fc Variant10。
(4)将步骤(3)中验证正确的AAV载体进行HindIII和RsrII双酶切，同时PCR法获得bGHpolyA(模板来源于pCMV3-3-Flag)片段，上述双酶切的载体和PCR获得的bGHpolyA片段纯化后进行同源重组连接，大肠杆菌转化后挑选克隆测序验证序列。同时对测序正确的载体进行SmaI酶切验证ITR是否有缺失。验证完成后获得目标质粒Variant 10-pAAV(如SEQ ID NO.12所示)。
2、AAV病毒包装
采用三质粒共转染Expi293F细胞的方法包装AAV病毒，其中的三质粒分别是辅助包装质粒、AAV8rep-cap质粒、转基因质粒。
(1)质粒扩增及抽提
将步骤1构建好的Variant 10-pAAV载体、AAV8rep-cap质粒和pHelper辅助质粒需经过大量抽提，浓度大于1μg/μL，A260/280在1.8-2.0间用以包装病毒。
(2)病毒包装
无血清悬浮培养Expi293F细胞至细胞密度为1E+6cells/ml。将抽提的三质粒按摩尔比1:1:1的比例混合，再把质粒DNA按总质量与PEIpro转染试剂按1:2的质量比例进行混匀，室温孵育20分钟后缓慢加到细胞悬液(细胞与三质粒的比例为1mL：1μg)中混匀。放37℃，8％CO2摇床培养3天，收集细胞悬液。
(3)AAV病毒浓缩
1)将细胞悬液10,000g离心10min，所得离心上清转移到一个新离心管中，所得细胞沉淀用少量PBS溶液重悬后反复冻融法裂解细胞；冻融后细胞再10,000g离心10min，收集离心所得上清。
2)将两次离心收集的上清混合在一起，用0.45μm滤器过滤除杂质。
3)加入1/2体积的1M NaCl，10％PEG8000溶液，混合均匀，4度过夜。
4)12,000rpm离心2h，弃上清，病毒沉淀用适量的PBS溶液溶解，待完全溶解后用0.22μm滤器过滤除菌。
5)加入Benzonase核酸酶消化去除残留的质粒DNA(终浓度为50U/ml)。合上管盖，颠倒几次以充分混合。在37℃孵育30分钟；
6)用0.45μm针头滤器过滤，取滤出液，即为浓缩的AAV病毒。
(4)AAV的纯化
1)向病毒浓缩液中添加固体CsCl直到密度为1.41g/ml(折射率为1.372)；
2)将样品加入到超速离心管中，用预先配好的1.41g/ml CsCl溶液将离心管剩余空间填满；
3)在175,000g下离心24小时，以形成密度梯度。按顺序分步收集不同密度的样品，取样进行滴度测定。收集富集有AAV颗粒的组分。
4)重复上述过程一次。
5)将病毒装入100kDa的透析袋，4度透析脱盐过夜，透析缓冲液成分为 含0.001％Pluronic F68的PBS，pH7.2。收获的透析后样品即为纯化的AAV病毒AAV8-Afli Fc Variant10，可用于体外和体内分析测试。
6)纯化后的病毒保存于-80℃超低温冰箱。
本发明使用的AAV病毒均采用上述方法进行制备，根据需要选择具体的AAV载体、表达元件以及VEGF受体重组融合蛋白突变体或VEGF重组抗体即可。其中，将Aflibercept替换为Bevacizumab，并发生Fc片段的H310E突变后采用上述同样方法构建的质粒如SEQ ID NO.13所示，以此质粒再采用上述同样的方法构建的AAV病毒命名为AAV8-Bevacizumab Fc Variant10。
实施例8 AAV载体分子表达蛋白的生物学活性
候选载体分子表达产物的生物学活性验证，是基因疗法药学评价的重要考量点。本研究以实施例7制备的AAV8-Afli Fc Variant 10以及AAV8空载体分别以MOI为2E+5感染HEK293T细胞，72h后收集细胞上清。将6E+3HUVEC细胞接种于96孔板中(每孔50μL)，培养于含100ng/ml VEGF165的基础培养基中。4-6h后，加入50μL病毒感染的细胞上清，继续培养基72h。每孔加10μL CCK-8溶液，置于37℃孵育，3h后读取OD450nm。结果如图8所示，表明候选载体分子的表达产物对HUVEC细胞的增殖产生抑制作用，具体为表达产物能与VEGF结合，进而抑制VEGF对于HUVEC细胞的增殖作用。
实施例9 AAV载体分子不同给药方式的转导表达
8～10周龄SPF级C57BL/6J雄性小鼠，在实验室饲养3～5天。分组前，对动物进行一般眼科检查、眼底照相(FP)和眼底血管荧光造影(FFA)，筛选合格的动物用于试验。筛选合格的小鼠于给药前根据体重进行随机分组，分为4组，以实施例7制备的AAV病毒AAV8-Afli Fc Variant10为供试品，动物分组及给药处理见下表7：
表7小鼠分组及不同途径给药剂量表
注：本发明实施例使用的溶媒皆为含0.001％(W/V)Pluronic F68的PBS，pH7.2。
供试品在使用时根据剂量要求，必要时采用溶媒稀释。
每只小鼠双眼单次给药，给药方法：
动物在给药前需要进行麻醉。根据最近一次称量的动物体重，用于计算麻药量。动物麻醉至苏醒期间做好保温措施(例如加盖毛毯)。
连接注射针头和注射器(每组动物使用不同的注射针头和注射器)，吸取所需量的供试品/对照品溶液用于给药，给药在手术显微镜下进行。
视网膜下腔注射：盖玻片覆盖在角膜上，用卡波姆滴眼液排除其间空气，用一次性胰岛素针与巩膜成小角度行巩膜、脉络膜穿刺，形成通道；退出胰岛素针，更换显微注射器从通道口进入直至视网膜下进行注射。
玻璃体腔注射：盖玻片覆盖在角膜上，用卡波姆滴眼液排除其间空气，用一次性胰岛素针与巩膜成小角度行巩膜、脉络膜穿刺，形成通道；退出胰岛素针，更换显微注射器从通道口进入玻璃体腔进行注射。
脉络膜上腔注射：用一次性胰岛素针行巩膜穿刺，形成通道；退出胰岛素针，更换显微注射器从通道口进入脉络膜上腔进行注射。
给药结束后连续3天给予左氧氟沙星滴眼液和/或涂抹氧氟沙星眼膏，每天2-3次。
给药后第8天，采集动物双侧眼球，每只眼加入200μL含蛋白酶抑制剂的PBS，使用组织匀浆机进行匀浆，离心(5000g，5min，4℃)后取上清液，-60℃及以下暂存。
深孔酶标板包被VEGF165蛋白，洗涤后加入HRP偶联的羊抗人IgG(Fc特异)抗体，检测捕获于板上的阿柏西普；经最后一步洗涤后，加入TMB底物溶液，HRP催化底物发生显色反应；最后加终止液终止反应，溶液由蓝色变为黄色，颜色深浅与样品中阿柏西普蛋白浓度成正相关。用酶标仪检测显色反应后的溶液，计算眼球组织匀浆上清里阿柏西普蛋白量。
检测结果显示，3种不同给药，小鼠均有较强阿柏西普蛋白表达。

实施例10 AAV载体分子注射小鼠后长期表达试验
8～10周龄SPF级C57BL/6J雄性小鼠，在实验室饲养3～5天。分组前，对动物进行一般眼科检查、眼底照相(FP)和眼底血管荧光造影(FFA)，筛选合格的动物用于试验。筛选28只合格的小鼠于给药前根据体重进行随机分组，分为7组，以实施例7制备的AAV病毒AAV8-Afli Fc Variant10为供试品，动物分组及给药处理见下表8：
表8小鼠分组及给药剂量表二
每只小鼠双眼单次给药，采用实施例9相同的视网膜下腔注射给药方法及后处理方法，并在给药后不同时间点检测眼球中的阿柏西普蛋白表达量。阿柏西普蛋白量检测结果如表9，时间表达曲线如图9所示。
表9小鼠注射AAV后不同时间点蛋白表达量检测结果
从试验结果可以看出，候选载体分子在3E+8vg/eye剂量下，有长达12周的持续高表达，相比较文献报道，蛋白药物Eylea给药后只能维持4—8周时间。
实施例11 AAV载体分子小鼠剂量爬坡试验
采购8～10周龄SPF级C57BL/6J雄性小鼠，在实验室饲养3～5天。分组前， 对动物进行一般眼科检查、眼底照相(FP)、OCT和眼底血管荧光造影(FFA)，筛选合格的动物用于试验。筛选30只合格的小鼠于给药前根据体重进行随机分组，分为6组，以实施例7制备的AAV病毒AAV8-Afli Fc Variant10为供试品，动物分组及给药处理见表10(各组给药体积均为1μL/eye)：
表10小鼠注射不同剂量AAV后蛋白表达量检测结果
每只小鼠双眼单次给药，分别视网膜下腔注射溶媒和不同剂量AAV8-Afli Fc Variant10供试品，给药方法同实施例9。
一般眼科检查、眼底照相(FP)、OCT、FFA显示无明显眼部不良反应。从不同剂量蛋白表达结果看，蛋白表达体现明显的剂量依赖相关性。
实施例12候选载体分子激光诱导小鼠CNV模型-药效试验
采购8-10周龄SPF级C57BL/6J雄性小鼠，在实验室饲养3-5天。分组前，对动物进行一般眼科检查、眼底照相(FP)和眼底血管荧光造影(FFA)，筛选合格的动物用于试验。筛选30只合格的小鼠于给药前根据体重进行随机分为6组，分别以实施例7制备的AAV病毒AAV8-Afli Fc Variant10，以及蛋白药为供试品，动物分组及给药处理见表11(各组给药体积均为1μL/eye)：
表11小鼠分组及给药剂量表三

注：1)给药开始计为D1。
2)蛋白药在给药后可以很快起效，所以在造模后给药；AAV8-Afli Fc Variant10在给药后1-2周左右达到稳定的表达水平而且可以持续12周以上的长时间高表达，所以在造模前给药。
3)蛋白药作为治疗AMD的标准治疗方法，其给药途径是玻璃体腔注射。
每只小鼠双眼单次给药，分别视网膜下腔注射溶媒和不同剂量AAV8-Afli Fc Variant10供试品，给药方法同实施例9。其中第2组为蛋白对照组，玻璃体注射80μg/eye。
在D17进行双眼眼底激光诱导脉络膜新生血管模型，每只眼计划激光灼烧数量为4个。具体步骤是：
1)散瞳：动物双眼各滴0.5％复方托吡卡胺滴眼液1～2滴散瞳；
2)麻醉：动物肌肉注射50mg/kg，50mg/mL的舒泰50实施麻醉。
3)激光光凝：动物眼光凝前滴卡波姆滴眼液，放置眼底激光镜，看清眼底后距离视盘约1.5-2PD处围绕视乳头避开血管用532nm波长激光进行光凝(光斑直径约50μm)。
4)动物护理：激光光凝后动物双眼涂氧氟沙星眼膏，动物苏醒前放保温毯上维持体温，苏醒后放回笼中。
D31进行FFA(眼底血管荧光造影)检查，典型图谱如图10所示。FFA检查前，动物静脉注射给予荧光素钠注射液(10％，0.02mL/只)。
分析指标：对比眼底荧光血管造影早期(1.5min内)和晚期(大于3min)的图片，根据动物眼底有无荧光渗漏判断是否有脉络膜新生血管生成及渗漏，并对荧光渗漏程度进行评级，计算各级渗漏光斑的比率及光斑渗漏平均分。
光斑荧光渗漏评级标准为：0级(无荧光渗漏)，1级(轻度荧光渗漏，渗漏面积为激光光斑大小的1％～50％)，2级(中度荧光渗漏，渗漏面积为激光光斑大小的50％～100％)，3级(重度荧光渗漏，渗漏面积大于激光光斑大小)。
各级光斑比率(％)＝对应级别光斑总数÷4种光斑总数(即有效光斑数)×100％。
光斑渗漏平均分＝[(0级光斑数×0)+(1级光斑数×1)+(2级光斑数×2)+(3级光斑数×3)]÷4种光斑总数(即有效光斑数)，其中，有效光斑是指其附近无严重视网膜前、下出血且在荧光造影中能完整显示的光斑。
D31，溶媒对照、蛋白对照和1E+7、3E+7、1E+8、3E+8GC/眼的AAV8-Afli Fc Variant10组的3级渗漏光斑比率统计结果(表12)显示， 和AAV8-Afli Fc Variant各剂量组均显著低于溶媒对照组(p≤0.05)，AAV8-Fc varian各剂量组均显著低于蛋白对照组(p≤0.05)。
表12小鼠注射不同剂量AAV后激光诱导CNV模型产生的渗漏斑比率
光斑渗漏平均分见图11，D31时AAV8-Afli Fc Variant10组的平均分均低于溶媒对照和蛋白对照组。并且与D24相比，AAV8-Afli Fc Variant10各剂量组的光斑渗漏平均分均有所降低，而蛋白对照组的光斑渗漏平均分却有所升高，说明AAV8-Afli Fc Variant10各剂量组能够持续稳定表达，且相比蛋白药药效更加持久。
实施例13 AAV载体分子在非人灵长类(食蟹猴)激光模型动物上药效评价
非人灵长类动物与人的眼部结构类似，食蟹猴是生物药非临床研究常用的非啮齿类动物模型。采用激光光凝法建立食蟹猴脉络膜新生血管(Choroidal neovascularization,CNV)模型，考查实施例7制备的AAV病毒AAV8-Afli Fc Variant10单次视网膜下腔注射给药后对食蟹猴CNV的抑制作用。
9只已进行眼科常规检查确认无眼病的食蟹猴入组进行实验，以实施例7制备的AAV病毒AAV8-Afli Fc Variant10为供试品，分组及给药情况如表13所示：
表13食蟹猴分组及给药剂量
在D0单次视网膜膜下腔给药，使用碘伏消毒眼表，在眼科专用手术显微镜下用30G一次性注射针头在角巩膜缘内侧穿刺食蟹猴巩膜，用带有35G平针头的微量进样器沿穿刺口进入并绕过晶状体后到达玻璃体，避开主血管，然后逐渐进针至视网膜下腔，中心凹上方一个点缓慢推注。注射器拔出后即刻用带有碘伏的棉签压迫进针口5s。每只眼睛给药50μL，双眼单次给药。
在D56使用舒泰(5-10mg/kg,i.m.)麻醉动物，532nm激光在黄斑周边烧灼8～9个激光斑(光斑直径约50μm)，激光造模后立刻拍摄IR照片与OCT扫描确认造模是否到位。激光造模后第4周，动物经舒泰(5-10mg/kg,i.m.)麻醉后，拍摄IR照片并使用SD-OCT扫描检查激光斑愈合程度，拍摄早期及晚期荧光素眼底血管造影(FFA)图像，用于渗漏评分，评分细节如表14所示。
表14渗漏斑分级标准表
注：III级-IV级光斑为渗漏病灶。
给药后持续检测房水中目标蛋白含量，12周持续稳定表达，并还在持续监测中(而蛋白药仅能维持4-8周)。
激光造模后4周眼底血管造影(FFA)典型图谱如图12所示，AAV病毒AAV8-Afli Fc Variant10低剂量组(1E+9GC/eye)和高剂量组(1E+10GC/eye)相比溶媒组，在激光造模后的渗漏斑明显减少，均有显著性差异。
计算光斑面积和光斑评级后进行Two way Anova/Dunnett统计学评价，低剂量组和高剂量组相比对照组均有显著差异，统计结果见表15。
表15食蟹猴激光诱导CNV模型后渗漏斑统计

备注：显著性分析为实验组跟溶媒对照组相比显著性分析，P<0.05为有统计学差异，P<0.01为有显著统计学差异，P<0.001为有极其显著的统计学差异。
实施例14 AAV载体分子在非人灵长类(食蟹猴)DL-AAA模型动物上药效评价
与人类眼睛非常相似的物种(例如非人类灵长类动物(NHP))中的视网膜血管疾病模型的一个挑战是缺乏慢性血管渗漏和/或人类eAMD，DME和PDR的新血管反应特征。在NHP激光诱导的eAMD的CNV模型中，脉络膜脉管系统的短暂血管渗漏和新血管反应在激光破坏Bruch膜后持续6-8周，此后自发地消退。虽然抗VEGF处理加速了这种诱导病理学的消退，但在人类中，CNV的病理学在抗VEGF药物被清除后恢复并持续存在。因此，建立一个持续性和复发性血管渗漏和新生血管的模型，将大大有助于并加速对长效干预措施的评估，以解决病理性血管不稳定和新血管形成的多种临床表现。DL-α-氨基己二酸(DL-AAA)模型是一种已经在大鼠和兔中描述过的慢性渗漏模型，并且已经在这些物种中进行常规候选药物筛选。DL-AAA是一种选择性神经胶质细胞毒素，据报道可抑制谷氨酰胺合成酶的作用，损害更广泛的Müller细胞视网膜稳态功能而导致神经胶质功能障碍和死亡，从而导致血视网膜屏障破坏。猴和人类的视网膜脉管系统，视网膜分割和基底层边界，视网膜神经元和神经胶质细胞亚型的比例丰度以及黄斑的存在是同源的。一种新的DL-AAA引起的慢性血管渗漏NHP模型，慢性视网膜血管渗漏和新血管形成的临床前模型允许在疾病发展的多个阶段对短效和长效抗血管生成化合物进行疗效筛查。
选1只食蟹猴(动物编号P1)，双眼视网膜下分别注入50mmol/L DL-α-AAA (sigma-aldrich)30μL。每眼注药前1w和注药后6w，12w行眼底彩色照相、荧光素眼底血管造影、黄斑自发荧光、光学相干断层成像(optical coherence tomography，OCT)、多焦视网膜电图检查，确认食蟹猴持续性视网膜新生血管(PRNV)造模成功，6-8周后开始成模。
选用食蟹猴持续性视网膜新生血管(PRNV)模型动物1只，成模时间已超过半年：双眼PRNV模型组1只(动物编号P1)，右眼注射实施例7制备的病毒AAV8-Afli Fc Variant10(1E+10GC/eye/50μL)，左眼注射AAV稀释液(溶媒对照组，50μL/eye)。观察Baseline、注射后2周、6周、8周、10周、12周长时间眼科FFA检查变化情况，目前已观察到的结果如下所示：
如图13所示(图中*p<0.05,**p<0.01,***p<0.001表示具有显著统计学差异)，PRNV造模给药组(右眼)荧光渗漏2周相比基线已有明显下降，后续每2周至第12周继续下降，持续下降效果明显。PRNV造模猴溶媒组(左眼)荧光渗漏2周至第12周跟基线相比没有明显变化。
AAV8-Afli Fc Variant10供试品持续缓解时间已超出CNV激光造模缓解时间，体现该模型在观察AAV长期疗效上的优势。统计分析发现，无论渗漏面积还是渗漏强度供试品相比溶媒组均有显著性差异，说明了AAV8-Afli Fc Variant10腺相关病毒在PRNV模型非人灵长类动物有明显的疗效。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。

Claims (24)

  1. 一种人源IgG1的Fc片段突变体，其特征在于，所述突变体具有以下突变中的至少一种：
    T250A，L251A，M252L，I253A/D/P，S254A，T256A，L309A，H310L/V/A/D/E/Q，Q311A，L314A，M428L/I，H433L/V/A，N434L/V/A，H435L/V/A，Y436A；
    其中的编号由EU索引指示。
  2. 如权利要求1所述的一种人源IgG1的Fc片段突变体，其特征在于，具有如下单点突变中的一种：H310A、H310E或H435A；或者具有如下双突变：H310A/H435A；
    氨基酸序列如SEQ ID NO.1、2、3或4所示。
  3. 一种包含权利要求1所述Fc片段突变体的VEGF受体重组融合蛋白，其特征在于，所述VEGF受体重组融合蛋白由VEGFR-1的结构域2，VEGFR-2的结构域3，以及权利要求1所述的Fc片段突变体融合而成；
    所述VEGFR-1的结构域2如SEQ ID NO.5所示；
    所述VEGFR-2的结构域3如SEQ ID NO.6所示。
  4. 如权利要求3所述的VEGF受体重组融合蛋白，其特征在于，氨基酸序列如SEQ ID NO.7、8、9或10所示。
  5. 一种包含权利要求1所述Fc片段突变体的抗VEGF重组抗体，其特征在于，是在抗VEGF抗体的基础上将其中的Fc片段替换为权利要求1所述Fc片段突变体所得。
  6. 如权利要求5所述的抗VEGF重组抗体，其特征在于，所述抗VEGF重组抗体是将贝伐单抗Fc片段进行H310E突变获得，具有SEQ ID NO.11所示的氨基酸序列。
  7. 一种表达权利要求3所述VEGF受体重组融合蛋白或权利要求5所述抗VEGF重组抗体的AAV病毒载体表达盒，其特征在于，所述表达盒从5’-3’端包含如下式I所述的结构：
    ITR-E1-E2-E3-E4-ITR   式(I)，其中：
    ITR为反向末端重复序列；
    E1为启动子；
    E2为信号肽；
    E3为所述VEGF受体重组融合蛋白或抗VEGF重组抗体的核苷酸序列；
    E4为Poly A序列。
  8. 如权利要求7所述的载体表达盒，其特征在于，所述ITR来自于AAV1，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8或AAV9；
    所述启动子优选CMV、CBA、EF1a、SV40、PGK1、Ubc、CAG、TEF1、U6或H1启动子；
    所述信号肽来源包括但不限于Human OSM、Gaussia luc、或者Albumin(HSA)；
    所述Poly A序列选自bGH PolyA、SV40 PolyA或hGH PolyA。
  9. 如权利要求7所述的载体表达盒，其特征在于，所述表达盒还包括调控元件，所述表达调控元件包括但不限于以下功能的调控元件：(1)用于调控目的蛋白的表达的元件；(2)用于表达miRNA和siRNA序列的调控元件；(3)内含子；(4)将目的蛋白定位表达到细胞核、细胞质或各类细胞器，以及分泌到细胞外的定位序列；(5)Kozak序列；(6)增强子；(7)WPRE。
  10. 如权利要求7所述的载体表达盒，其特征在于，所述表达盒还包括标签元件，所述标签元件包括但不限于FLAG，HA，MYC，荧光蛋白，荧光素酶，SUMO蛋白，泛素蛋白，GST等。
  11. 如权利要求7所述的载体表达盒，其特征在于，是将VEGF受体重组融合蛋白或抗VEGF重组抗体在AAV病毒载体上进行表达；
    所述AAV病毒载体包括但不限于pAAV-CMV、pX601、pX551、pAAV-MCS质粒。
  12. 如权利要求7所述的载体表达盒，其特征在于，所述表达VEGF受体重组融合蛋白的AAV病毒载体表达盒具有SEQ ID NO.12所示的序列；
    所述表达抗VEGF重组抗体的AAV病毒载体表达盒具有SEQ ID NO.13所示的序列。
  13. 一种腺相关病毒包装载体系统，其特征在于，所述包装载体系统包括：权利要求7所述的表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，携带AAV rep、cap基因的载体以及辅助病毒载体，上述载体通过包装成为AAV病毒。
  14. 如权利要求13所述的一种腺相关病毒包装载体系统，其特征在于，所述携带AAV rep、cap基因的载体包括但不限于：AAV1，AAV2，AAV5，AAV8， AAV9，AAV-R100，AAV-NN，AAV-GL，AAV8-Y447F，AAV8-Y733F，AAV8-Y447F/Y733F，AAV-DJ或AAV7m8载体；
    所述辅助病毒载体为pHelper质粒。
  15. 一种腺相关病毒包装方法，其特征在于，将权利要求13所述包装载体系统转入宿主细胞中进行病毒包装；
    所述宿主细胞是能够进行病毒复制且稳定遗传的细胞系，包括但不限于Hela-S3，HEK-293，HEK-293T，HEK-293FT，Expi293F，A549或Sf9等细胞。
  16. 一种由权利要求15所述的腺相关病毒包装方法包装制备的腺相关病毒。
  17. 如权利要求16所述的腺相关病毒，其特征在于，是将SEQ ID NO.12所示的表达VEGF受体重组融合蛋白的AAV病毒载体表达盒、AAV8 Rep-Cap质粒、pHelper质粒转入宿主细胞Expi293F细胞中进行病毒包装获得的。
  18. 如权利要求16所述的腺相关病毒，其特征在于，是将SEQ ID NO.13所示的表达抗VEGF重组抗体的AAV病毒载体表达盒、AAV8 Rep-Cap质粒、pHelper质粒转入宿主细胞Expi293F中进行病毒包装获得的。
  19. 包含权利要求7所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或权利要求16所述腺相关病毒的制剂或配方或药物。
  20. 如权利要求19所述的制剂或配方或药物，其特征在于，可以是任何剂型，包括但不限于注射剂型和软膏剂型。
  21. 如权利要求19所述的制剂或配方或药物，其特征在于，所述制剂或配方或药物中，上述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或腺相关病毒为唯一活性成分。
  22. 权利要求7所述所述表达VEGF受体重组融合蛋白或抗VEGF重组抗体的AAV病毒载体表达盒，或权利要求16所述腺相关病毒在制备治疗新生血管相关眼底疾病的制剂或配方或药物中的应用。
  23. 如权利要求22所述的应用，其特征在于，所述新生血管相关眼底疾病包括年龄相关黄斑变性、湿性黄斑病变、糖尿病视网膜病变。
  24. 如权利要求22所述的应用，其特征在于，所述制剂或配方或药物的给药方法为单侧眼给药或双侧眼给药；
    给药方式为玻璃体注射、视网膜下腔注射、脉络膜上腔注射等注射方式；
    终身单次给药或多次给药，给药总剂量为1×108-1×1013个病毒基因组/眼。
PCT/CN2023/102771 2022-06-27 2023-06-27 一种治疗新生血管相关眼底疾病的aav药物 WO2024002076A1 (zh)

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