Gene therapy drug for treatment of age-related macular degeneration

Abstract

A gene therapy drug for treatment of AMD. An anti-VEGF fusion protein having a specific secretion signal peptide sequence, allowing for effective expression and secretion of anti-VEGF protein. And a modified AAV vector for increasing anti-VEGF protein retina transduction efficiency. The described fusion protein and the modified AAV vector can be used for treating ocular neovascular disease.

Description

Gene therapy drugs for the treatment of age-related macular degeneration Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a gene therapy drug for treating age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD) is a major public health problem that threatens visual health. Age is the primary risk factor for AMD. A comprehensive study from the Blue Mountains Eye Institute, Beaver Dam Eye Institute, and Rotterdam Research Center found that the prevalence of neovascular (wet) age-related macular degeneration (nAMD) was 0.17%, 0.54%, 2.52%, and 5.76% in people aged 55-64, 65-74, 75-84, and 85 years and older, respectively (Smith et al. 2001). In China, the prevalence of AMD in people aged 50 years and older is 15.5%. With the aging population and advances in fundus disease screening, the number of people with nAMD in China has increased from 1.71 million in 1990 to 3.81 million in 2015, and is projected to reach 8.84 million by 2050 (Clinical Pathway for Age-Related Macular Degeneration in China, 2013; Song et al. 2017).

[0003] Existing gene therapy technologies for nAMD mostly use adeno-associated virus (AAV) vectors carrying an anti-VEGF (vascular endothelial growth factor) expression cassette for intraocular injection. However, the combination of expression and other regulatory elements with the anti-VEGF molecule, as well as the combination of AAV vectors with different transduction efficiencies and cell tissue tropisms and injection methods, may affect the efficacy and safety of the therapeutic drug.

[0004] Currently approved anti-VEGF treatment regimens typically include an initial 3-month loading phase, followed by a maintenance phase. The maintenance phase includes fixed treatment regimens (monthly, bimonthly, or trimonthly), as-needed (PRN) regimens, and treatment-and-extend (T&E) regimens. These regimens require regular follow-up visits and multiple injections, placing high demands on patient compliance and placing a significant burden on both patients and healthcare providers (Mitchell et al. 2018; Roman et al. 2020).

[0005] Existing technologies often use the stronger broad-spectrum promoter CMV (see, for example, Preclinical Evaluation of ADVM-022, a Novel Gene Therapy Approach to Treating Wet Age-Related Macular Degeneration, doi: 10.1016 / j.ymthe.2018.11.003., hereinafter referred to as "ADVM-022"). However, in primate and clinical trials, the CMV promoter has the risk of being silenced and has certain toxicity to photoreceptors and retinal pigment epithelial cells in the eye.

[0006] At the same time, the serotypes disclosed in the prior art are generally AAV2, AAV8 and AAV2.7m8 (see, for example, ADVM-022, or Delivery of nVEGFi using AAV8 for the treatment of neovascular age-related macular degeneration, doi: 10.1016 / j.omtm.2022.01.002., hereinafter referred to as “nVEGFi”). Among them, wild-type AAV2 and AAV8 cannot efficiently penetrate the inner limiting membrane (ILM) of the retina to reach the main cell tissues (RPE and PR) of most types of nAMD lesions, so subretinal administration is generally adopted. Although this administration method can effectively deliver the target gene, it has high requirements for clinical surgery. When taking the route of intravitreal administration (clinical difficulty explanation and better patient compliance), these two wild-type AAV vectors will cause most of the rAAV and the expressed anti-VEGF molecules to remain in the vitreous fluid, which may affect the clinical treatment effect. The AAV2.7m8 disclosed in the ADVM-022 technology has better retinal penetration than wild-type AAV2 and can be used for intravitreal injection; however, there is still room for optimization in terms of transduction efficiency and cell tropism.

[0007] Therefore, there is a need in the art to develop a gene therapy drug that is easy to administer and can efficiently deliver and express the target gene for the treatment of age-related macular degeneration.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a gene therapy drug for the treatment of age-related macular degeneration that can efficiently deliver and express the target gene.

[0010] In the first aspect of the present invention, an anti-VEGF fusion protein is provided. The fusion protein has a structure of Formula I from N-terminus to C-terminus:

[0011] LVH-Fc (I)

[0012] In the formula, each “-” is independently a bond or a peptide linker;

[0013] L is a secretory signal peptide, which is derived from the Ig kappa chain;

[0014] V is an anti-VEGF protein, which includes the IgC domain 2 of VEGFR1 (VEGF recptor 1) and the IgC domain 3 of VEGFR2;

[0015] H is an optional hinge region;

[0016] Fc is an optional crystallizable fragment of an immunoglobulin.

[0017] In another preferred embodiment, the sequence of the secretory signal peptide is shown as SEQ ID NO:11.

[0018] In another preferred embodiment, the sequence of V is shown in SEQ ID NO: 3, positions 1-202.

[0019] In another preferred embodiment, the hinge region amino acid sequence is GPG.

[0020] In another preferred embodiment, the Fc is the Fc fragment of human IgG1.

[0021] In another preferred embodiment, the anti-VEGF fusion protein further includes an HA tag.

[0022] In a second aspect of the present invention, an expression cassette is provided, wherein the expression cassette comprises a nucleic acid molecule encoding the anti-VEGF fusion protein according to the first aspect of the present invention.

[0023] In another preferred embodiment, the expression cassette has a structure of Formula II from the 5'-3' end:

[0024] Z0-Z1-Z2-Z3-Z4-Z5 (II)

[0025] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0026] Z0 is none or 5′ITR sequence;

[0027] Z1 is the promoter;

[0028] Z2 is an intron;

[0029] Z3 is a nucleotide sequence encoding the anti-VEGF fusion protein according to the first aspect of the present invention;

[0030] Z4 is none or hGHpA (human growth hormone poly(A) tail) sequence;

[0031] Z5 is absent or has a 3' ITR sequence.

[0032] In another preferred embodiment, the nucleotide sequence of Z3 is as shown in SEQ ID NO: 4, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% to the nucleotide sequence shown in SEQ ID NO: 4.

[0033] In another preferred embodiment, the promoter includes a tissue-specific promoter.

[0034] In another preferred embodiment, the promoter is selected from the group consisting of UBC (ubiquitin C) promoter, CAG promoter, or a combination thereof.

[0035] In another preferred embodiment, the UBC promoter sequence is shown as SEQ ID NO:5.

[0036] In another preferred embodiment, the CAG promoter sequence is shown as SEQ ID NO: 6.

[0037] In another preferred embodiment, the intron is selected from the following group: UBC-CAG chimeric intron, CAG intron, UBC intron, or a combination thereof.

[0038] In another preferred example, the UBC-CAG chimeric intron sequence is shown as SEQ ID NO:7.

[0039] In another preferred embodiment, the UBC intron sequence is shown as SEQ ID NO:8.

[0040] In another preferred example, the CAG intron sequence is shown as SEQ ID NO: 9 or SEQ ID NO: 10.

[0041] In another preferred embodiment, the length of each nucleotide linker sequence is 0-30 nt, preferably 0-15 nt.

[0042] In another preferred embodiment, the nucleotide sequence of the expression cassette is as shown in SEQ ID NO: 2, or has ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.

[0043] In the third aspect of the present invention, a vector is provided, wherein the vector contains the expression cassette according to the second aspect of the present invention.

[0044] In another preferred embodiment, the vector further contains one or more additional promoters, enhancers, introns, transcription termination signals, polyadenylation sequences, replication origins, selectable markers, nucleic acid restriction sites and / or homologous recombination sites.

[0045] In another preferred embodiment, the selective marker includes a resistance marker and a fluorescent marker. Preferably, the selective marker is a kanamycin resistance sequence.

[0046] In another preferred embodiment, the vector includes a plasmid or a viral vector.

[0047] In another preferred embodiment, the plasmid is a shuttle plasmid.

[0048] In another preferred embodiment, the vector includes a DNA virus or a retroviral vector.

[0049] In another preferred embodiment, the vector is selected from the following group: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), or a combination thereof; preferably, the vector is an AAV vector.

[0050] In another preferred embodiment, the vector is used to express the anti-VEGF fusion protein as described in the first aspect of the present invention.

[0051] In a fourth aspect of the present invention, an adeno-associated virus (AAV) vector is provided, wherein the adeno-associated virus vector contains the expression cassette according to the second aspect of the present invention.

[0052] In another preferred embodiment, the serotype of the adeno-associated virus is selected from the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6 (Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAVclone 32 / 83, AAVShH10, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45, AAV2 or AAV5, or a combination thereof.

[0053] In another preferred embodiment, the AAV vector is an AAV2 vector or an AAV9 vector.

[0054] In another preferred embodiment, the AAV vector is a modified AAV vector.

[0055] In another preferred embodiment, the capsid protein VP1 of the modified AAV vector has amino acids 584-598 replaced by LQRGNRQAATADVNT (SEQ ID NO: 17) relative to the wild-type AAV9 capsid protein VP1.

[0056] In another preferred embodiment, the modified AAV vector capsid protein contains an inserted functional peptide.

[0057] In another preferred embodiment, the inserted functional peptide has a function selected from the following group:

[0058] 1) Enhance the tissue-specific targeting of AAV capsid protein variants;

[0059] 2) altering the spatial structure of the receptor binding motif of the wild-type AAV capsid protein; or

[0060] 3) Weakening the binding activity of wild-type AAV capsid protein to galactose (Glu).

[0061] In another preferred embodiment, the functional peptide comprises the amino acid sequence shown as LALGDVTRPA (SEQ ID NO: 18).

[0062] In another preferred embodiment, the functional peptide is inserted between positions 586 and 587, between positions 587 and 588, or between positions 588 and 589 of the corresponding wild-type AAV capsid protein VP1 amino acid sequence.

[0063] In another preferred embodiment, the VP1 of the modified AAV vector capsid protein has the following mutation relative to the wild-type AAV9 capsid protein VP1: amino acids at positions 584-598 are replaced with the sequence shown as LQRGNLALGDVTRPARQAATADVNT (SEQ ID NO: 19).

[0064] In another preferred embodiment, the VP1 of the modified AAV vector capsid protein has the following mutations relative to the wild-type AAV capsid protein VP1:

[0065] a) Isoleucine I at position 240 is mutated to threonine T (I240T);

[0066] b) Valine V at position 708 was mutated to isoleucine I (V708I);

[0067] c) Tyrosine Y at position 444 was mutated to phenylalanine F (Y444F);

[0068] d) Any combination of a) to c) above.

[0069] In another preferred embodiment, the VP1 of the modified AAV vector capsid protein has the following mutations relative to the wild-type AAV capsid protein VP1: I240T, V708I.

[0070] In another preferred embodiment, the amino acid sequence of the modified AAV vector capsid protein is selected from the following group:

[0071] (i) the sequence shown in SEQ ID NO: 13 or 15;

[0072] (ii) a sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 13 or 15.

[0073] In another preferred embodiment, the AAV vector is used to treat eye diseases.

[0074] In another preferred embodiment, the modified AAV vector has a photoreceptor cell tropism that is increased by at least 1 order of magnitude, preferably 2 orders of magnitude, relative to the corresponding wild-type AAV vector.

[0075] In another preferred embodiment, the modified AAV vector has a higher penetration ability to the inner limiting membrane than the corresponding wild-type AAV vector.

[0076] In another preferred embodiment, the inner limiting membrane penetration of the modified AAV vector is increased by at least 15%, preferably by at least 25%, and more preferably by at least 35% relative to the corresponding wild-type AAV vector.

[0077] In the fifth aspect of the present invention, a host cell is provided, which contains the vector described in the third aspect of the present invention or the AAV vector described in the fourth aspect of the present invention, or an exogenous expression cassette as described in the second aspect of the present invention is integrated into its chromosome.

[0078] In another preferred embodiment, the host cell is a mammalian cell, and the mammal includes human and non-human mammals.

[0079] In another preferred embodiment, the host cell is selected from the group consisting of HEK cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as bipolar cells, horizontal cells), ganglion cells, or a combination thereof.

[0080] In another preferred embodiment, the host cell is selected from the group consisting of rod cells, cone cells, light-receiving bipolar cells, light-withdrawing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or a combination thereof.

[0081] In the sixth aspect of the present invention, a use of the vector described in the third aspect of the present invention or the adeno-associated virus vector described in the fourth aspect of the present invention is provided for preparing a preparation or composition for treating VEGF-related eye diseases.

[0082] In another preferred embodiment, the preparation or composition is also used to inhibit the expression of VEGF in the eye.

[0083] In another preferred embodiment, the preparation or composition is also used to reduce leakage caused by laser damage and / or inhibit angiogenesis.

[0084] In another preferred embodiment, the VEGF-related eye disease is selected from the group consisting of macular degeneration, diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, or a combination thereof.

[0085] In another preferred embodiment, the macular degeneration is age-related macular degeneration, more preferably wet age-related macular degeneration.

[0086] In the seventh aspect of the present invention, a pharmaceutical preparation is provided, which contains (a) the vector described in the third aspect of the present invention or the adeno-associated virus vector described in the fourth aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

[0087] In another preferred embodiment, the dosage form of the pharmaceutical preparation is selected from the following group: a lyophilized preparation, a liquid preparation, or a combination thereof.

[0088] In another preferred embodiment, the content of the carrier in the pharmaceutical preparation is 1×10 9 -1×10 16 viruses / ml, preferably 1×10 12 -1×10 13 viruses / ml.

[0089] In another preferred embodiment, the pharmaceutical preparation is used to treat VEGF-related eye diseases.

[0090] In another preferred embodiment, the VEGF-related eye disease is selected from the group consisting of macular degeneration, diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, or a combination thereof.

[0091] In another preferred embodiment, the macular degeneration is age-related macular degeneration, more preferably wet age-related macular degeneration.

[0092] In the eighth aspect of the present invention, a treatment method is provided, which comprises administering the vector described in the third aspect of the present invention or the adeno-associated virus vector described in the fourth aspect of the present invention to a subject in need.

[0093] In another preferred embodiment, the administration is selected from the group consisting of intravenous administration, intramuscular administration, subcutaneous administration, oral administration, mucosal contact, intraperitoneal administration, intralesional administration, or a combination thereof.

[0094] In another preferred embodiment, the adeno-associated viral vector is injected into the eye of a subject in need.

[0095] In another preferred embodiment, the injection includes subretinal injection, suprachoroidal injection, and vitreous cavity injection; preferably, vitreous cavity injection.

[0096] In another preferred embodiment, the subject in need includes humans and non-human mammals.

[0097] In another preferred embodiment, the treatment method is a method for treating VEGF-related eye diseases.

[0098] In another preferred embodiment, the method further comprises administering an immunomodulator to the subject.

[0099] In another preferred embodiment, in the method described above, 1×10 6 -1×10 16 The vector is administered at a dose of 1×10 VG / eye, preferably 1×10 7 -1×10 8 VG / eye.

[0100] In the ninth aspect of the present invention, a method for preparing the anti-VEGF fusion protein according to the first aspect of the present invention is provided, comprising culturing the host cell according to the fifth aspect of the present invention, thereby obtaining the anti-VEGF fusion protein.

[0101] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0103] Figure 1 shows the plasmid map of the shuttle gene GOI-D02 and the effects of regulatory elements on target gene expression.

[0104] FIG2 shows the effects of different secretion signal peptides on expression and secretion at the cellular level.

[0105] Figure 3 shows that the VEGF trap protein expressed and secreted by AAV-infected cells can effectively compete with VEGF antibodies for binding to VEGF165 protein and inhibit the signal transduction activity downstream of VEGFR1.

[0106] FIG4 shows the transduction activity of engineered AAV serotypes (RC-C14 and RC-C07V5 series) in mouse eyes and the comparison of tissue sections.

[0107] FIG5 shows that the modified capsid efficiently delivers and expresses VEGF trap protein in HEK293 cells in vitro.

[0108] Figure 6 shows that recombinant AAV injected into the mouse eye can long-term express VEGF trap protein molecules and significantly inhibit neovascularization leakage in the mouse choroidal neovascularization (CNV) model.

[0109] Figure 7 shows that intraocular injection of recombinant AAV into non-human primates (NHP) can long-term express VEGF trap protein molecules and significantly inhibit neovascularization leakage in the non-human primate CNV model. DETAILED DESCRIPTION

[0110] After extensive and in-depth research, the inventors have developed, for the first time, a gene therapy drug for the treatment of AMD. The therapeutic drug of the present invention comprises a gene expression cassette employing optimized expression regulatory elements and a specific secretion signal peptide sequence, capable of effectively expressing and secreting an anti-VEGF protein. The expression cassette of the present invention is compatible with common wild-type adeno-associated viruses that can be used for subretinal injection, as well as engineered AAV vectors that can be used for intravitreal and subretinal injection and effectively transduce the entire retinal layer. Consequently, the present invention is capable of expressing and secreting an anti-VEGF protein in multiple types of AMD lesion tissue, exerting a timely and effective therapeutic effect. This is the basis for the completion of the present invention.

[0111] The gene expression cassettes and secretory peptides screened in this invention demonstrated effective molecular expression and secretion. Furthermore, the modified AAV capsids of this product demonstrated high transduction efficiency both in vitro and in vivo. In vivo, intravitreal administration significantly enhanced transduction activity in all layers of retinal tissue in animal eyes.

[0112] Compared to other related gene therapy drugs in clinical and preclinical studies, the drug molecule of the present invention can more efficiently express and secrete anti-VEGF protein molecules at the same dosage level. Furthermore, the modified high-efficiency targeting vector also allows for the delivery of the target gene via intravitreal injection. Overall, the drug molecule of the present invention has a better dose-response relationship (lower therapeutically effective dose) than known AMD gene therapy drugs, as well as long-term effective clinical results.

[0113] the term

[0114] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0115] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0116] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0117] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), ie, has a reasonable benefit / risk ratio.

[0118] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is tolerated by humans and / or animals. Those skilled in the art will appreciate that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and combination therapy with other drugs.

[0119] Anti-VEGF fusion protein

[0120] As used in the present invention, the terms "fusion protein of the present invention", "polypeptide of the present invention", "anti-VEGF fusion protein", and "VEGF trap fusion protein" are used interchangeably and all refer to the fusion protein described in the first aspect of the present invention.

[0121] The fusion protein of the present invention has the structure of formula I from N-terminus to C-terminus:

[0122] LVH-Fc (I)

[0123] In the formula, each “-” is independently a bond or a peptide linker;

[0124] L is a secretory signal peptide, which is derived from the Ig kappa chain;

[0125] V is an anti-VEGF protein, wherein the anti-VEGF protein comprises the IgC domain 2 of VEGFR1 and the IgC domain 3 of VEGFR2;

[0126] H is an optional hinge region;

[0127] Fc is an optional crystallizable fragment of an immunoglobulin.

[0128] The fusion protein of the present invention has a secretory signal peptide (or secretory peptide or signal peptide) derived from the Ig kappa immunoglobulin light chain, which can achieve better extracellular secretion effect compared with the secretory peptide derived from VEGFR1 used in the prior art.

[0129] In some embodiments, the fusion protein of the present invention may contain an immunoglobulin crystallizable fragment (Ig fragment), which may be derived from Ig1, Ig4, or other immunoglobulins. The Ig fragment and the anti-VEGF protein sequence may be connected by a hinge region, such as a GPG hinge region.

[0130] In some embodiments, the fusion protein of the present invention may further contain other tags to facilitate purification or expression, such as an HA tag.

[0131] In a preferred embodiment, the amino acid sequence of the fusion protein of the present invention is as shown in SEQ ID NO: 3, or has ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0132] Expression cassette

[0133] The present invention provides an expression cassette for expressing an anti-VEGF fusion protein, which comprises a nucleic acid sequence encoding the anti-VEGF fusion protein of the present invention. The structure of the expression cassette is as described in the second aspect of the present invention.

[0134] In one embodiment, the nucleotide sequence encoding the fusion protein of the present invention is as shown in SEQ ID NO:4, or has a sequence identity of ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% to the nucleotide sequence shown in SEQ ID NO:4.

[0135] In the expression cassette of the present invention, the promoter, introns, and other regulatory elements can be modified and optimized so that the expression amount and effect of the active ingredient can be further optimized under the same vector dosage, thereby achieving long-term and effective expression of anti-VEGF molecules in the eye.

[0136] In one embodiment, the expression cassette of the present invention comprises a UBC promoter of a ubiquitin gene. In one embodiment, the expression cassette of the present invention comprises a UBC-CAG chimeric intron.

[0137] In one embodiment, the nucleotide sequence of the expression cassette of the present invention is as shown in SEQ ID NO: 2, or has ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.

[0138] The expression cassette of the present invention can be a nucleic acid in the form of DNA or RNA. In another preferred embodiment, the nucleotide is DNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The AAV viral vector of the present invention expresses an anti-VEGF fusion protein.

[0139] The nucleic acid sequence can be DNA, RNA, cDNA, or PNA. The nucleic acid sequence can be genomic, recombinant, or synthetic. The nucleic acid sequence can be isolated or purified. The nucleic acid sequence can be single-stranded or double-stranded. Preferably, the nucleic acid sequence will encode an anti-VEGF fusion protein as described herein. The nucleic acid sequence can be derived by cloning, for example, using standard molecular cloning techniques including restriction enzyme digestion, ligation, and gel electrophoresis, such as those described in Sambrook et al. (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press). The nucleic acid sequence can be isolated, for example, using PCR techniques. Isolation means separating the nucleic acid sequence from any impurities and from other nucleic acid sequences and / or proteins that are naturally found associated with the nucleic acid sequence in its source. Preferably, it will also be free of cellular material, culture medium, or other chemicals from the purification / production process. The nucleic acid sequence can be synthetic, for example, produced by direct chemical synthesis. The nucleic acid sequence can be provided as naked nucleic acid or can be provided in complex with a protein or lipid.

[0140] The full-length nucleotide sequence of the present invention or its fragment can usually be obtained by PCR amplification, recombinant method or artificial synthesis method. For PCR amplification, primers can be designed based on the disclosed relevant nucleotide sequence, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify and obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified each time are spliced ​​together in the correct order. At present, the DNA sequence encoding the polypeptide of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0141] The present invention also relates to vectors comprising the nucleic acid of the present invention, and host cells produced by genetic engineering using the vector or polypeptide encoding sequence of the present invention. The above nucleic acid, vector or host cell may be isolated.

[0142] As used herein, "isolated" means a substance that has been separated from its original environment (in the case of a naturally occurring substance, the original environment is the natural environment). For example, nucleic acids and polypeptides in their natural state within living cells are not isolated and purified. However, the same nucleic acid or polypeptide is isolated and purified if it is separated from other substances with which it is naturally present.

[0143] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0144] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0145] Methods using PCR technology to amplify DNA / RNA are preferably used to obtain the genes of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. Amplified DNA / RNA fragments can be separated and purified using conventional methods, such as by gel electrophoresis.

[0146] The present invention also relates to vectors comprising the nucleic acid of the present invention, host cells produced by genetic engineering using the vector or protein coding sequence of the present invention, and methods for expressing anti-VEGF fusion proteins using the host cells by recombinant technology.

[0147] The nucleic acid sequences of the present invention can be used to obtain host cells (e.g., mammalian cells) expressing the anti-VEGF fusion protein of the present invention through conventional recombinant DNA techniques. Generally, the process comprises the steps of: transducing the expression cassette of the second aspect of the present invention, the vector of the third aspect of the present invention, or the adeno-associated virus vector of the fourth aspect of the present invention into the host cells.

[0148] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a polypeptide of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0149] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0150] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the polypeptide.

[0151] The host cell can be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell (including human and non-human mammals). Representative examples include animal cells such as CHO, NS0, COS7, or 293 cells. In a preferred embodiment of the present invention, 293T cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as bipolar cells), and neural cells are selected as host cells. In another preferred embodiment, the host cell is selected from the following group: rod cells, cone cells, light-receiving bipolar cells, light-removing bipolar cells, horizontal cells, ganglion cells, amacrine cells, or a combination thereof.

[0152] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0153] The transformants obtained can be cultured using conventional methods to express the protein encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0154] The polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0155] carrier

[0156] The present invention also provides an expression vector for expressing the anti-VEGF fusion protein, which contains the anti-VEGF fusion protein coding sequence of the present invention.

[0157] Given the sequence information, a skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.

[0158] Preferably, the nucleic acid sequence encoding the anti-VEGF fusion protein is provided as a vector, preferably an expression vector. Preferably, it can be provided as a gene therapy vector, preferably suitable for transduction and expression in retinal target cells. The vector can be viral or non-viral (e.g., a plasmid).

[0159] In one embodiment, the vector of the present invention is a shuttle plasmid vector, preferably, its full-length DNA sequence is as shown in SEQ ID NO: 1, or has ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0160] Viral vectors include those derived from adenovirus, adeno-associated virus (AAV), including mutated forms, retroviruses, lentiviruses, herpes viruses, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxviruses, and SV40. Preferably, the viral vector is replication defective, although it is envisioned that it may be replication deficient, capable of replication, or conditionally replicating. Viral vectors can typically remain in an extrachromosomal state without integrating into the genome of target retinal cells. Preferred viral vectors for introducing nucleic acid sequences encoding anti-VEGF fusion proteins into retinal target cells are AAV vectors, such as self-complementary adeno-associated viruses (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotype 1 to AAV serotype 13) or modified versions of any of these serotypes.

[0161] The viral vector can be modified to delete any non-essential sequence. For example, in AAV, the virus can be modified to delete all or part of the IX gene, Ela and / or Elb gene. For wild-type AAV, without the presence of a helper virus such as adenovirus, replication is very inefficient. For recombinant adeno-associated viruses, preferably, the replication gene and capsid gene are provided in trans (in the pRep / Cap plasmid), and only the 2ITR of the AAV genome is retained and packaged into the virion, while the required adenoviral genes are provided by adenovirus or another plasmid. Similar modifications can also be made to lentiviral vectors.

[0162] Viral vectors have the ability to enter cells. However, non-viral vectors such as plasmids can be complexed with agents to facilitate the uptake of viral vectors by target cells. Such agents include polycationic agents. Alternatively, delivery systems such as liposome-based delivery systems can be used. The carriers used in the present invention are preferably suitable for use in vivo or in vitro, and are preferably suitable for use in humans.

[0163] The vector will preferably contain one or more regulatory sequences to direct expression of the nucleic acid sequence in the retinal target cells. Regulatory sequences may include promoters, introns, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence. The vector may also include a selective marker, for example, to determine expression of the vector in a growth system (e.g., bacterial cells) or in the retinal target cells.

[0164] "Operably linked" means that a nucleic acid sequence is functionally related to the sequence to which it is operably linked so that they are linked in a manner that allows them to affect the expression or function of each other. For example, a nucleic acid sequence operably linked to a promoter will have an expression pattern affected by the promoter.

[0165] The promoter mediates the expression of the nucleic acid sequence to which it is connected. The promoter can be constitutive or inducible. The promoter can direct ubiquitous expression in inner retinal cells, or neuron-specific expression. In the latter case, the promoter can direct cell type-specific expression, such as to optic ganglion cells. Suitable promoters will be known to those skilled in the art. For example, a suitable promoter can be selected from the group consisting of: L7, thy-1, recovery protein, calbindin, human CMV, GAD-67, chicken beta-actin, hSyn, Grm6, Grm6 enhancer SV40 fusion protein.

[0166] An example of a suitable promoter is the human UBC promoter, which is derived from the upstream expression regulatory sequence (-1225-6) of the human ubiquitin protein C gene (GENE ID: 7316).

[0167] Many expression vectors can be used to express anti-VEGF fusion protein in mammalian cells (preferably human, more preferably human optic nerve cells or photoreceptor cells). In the present invention, adeno-associated virus is preferably used as an expression vector.

[0168] The present invention also provides a method for constructing a recombinant adeno-associated virus vector containing an anti-VEGF fusion protein coding sequence. The method can quickly and easily construct a recombinant adeno-associated virus vector carrying an anti-VEGF fusion protein coding sequence and package it to obtain a complex defective adeno-associated virus vector.

[0169] The present invention also provides a host cell for expressing an anti-VEGF fusion protein. Preferably, the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell), which increases the expression level of the anti-VEGF fusion protein.

[0170] AAV vectors

[0171] The gene therapy vector of the present invention is a viral expression vector. In a preferred embodiment, the viral expression vector is an adeno-associated virus (AAV) vector, such as an AAV vector selected from the group consisting of serotypes AAV1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or chimeric AAVs derived therefrom, such as AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6 (Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2 (Y->F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, AAVr3.45, AAV2 or AAV5, which are better suited for efficient transduction in the tissue of interest. Upon transfection, AAV induces only a mild immune response (if any) in the host. In a preferred embodiment of the present invention, the gene therapy vector is an AAV serotype 2 or 9 vector. In a further preferred embodiment, the gene therapy vector is an AAV 9 vector.

[0172] Recombinant viral vectors can be produced according to standard techniques. For example, recombinant adeno-associated viral vectors can be propagated in human 293 cells (which provide trans-acting E1A and E1B properties) to achieve 10 7 ~10 13 The titer of the virus vector can be within the range of 100 virus particles / mL. Before in vivo application, the viral vector can be desalted by gel filtration method (such as agarose column) and purified by subsequent filtration. Purification reduces potential harmful effects in the main body of the drug delivery vehicle. The administered virus is substantially free of wild-type virus and replicable virus. The purity of the virus can be demonstrated by suitable methods, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining.

[0173] A suitable dose of AAV for human use is approximately 1 × 10 6 -1×10 14 Virus particles range, for example 1×10 7 -1×10 12 within the range of a virus particle.

[0174] The gene therapy vector can be administered by intraocular injection, either into the subretinal space or into the vitreous cavity.

[0175] AAV vector modification

[0176] The present invention also provides an AAV vector for efficiently delivering a target gene in the eye, which has a modified capsid protein.

[0177] Specifically, the present invention is based on rational design, inserting short amino acid peptides and mutating specific sites in the AAV capsid protein, and obtaining an AAV vector that can efficiently deliver target genes in the eye.

[0178] 1) AAV2.7m8 variant

[0179] The main receptor for the capsid of serotype AAV2 to enter cells is heparan sulfate glycoprotein (HSPG), and it can also enter cells with the help of auxiliary receptors such as integrin, FGFR, HGFR, LamR, and AAVR.

[0180] Based on rational design, the capsid protein VP1 of AAV2.7m8 (RC-C01) was mutated to generate RC-C02 (two-point mutation I240T-V708I) and RC-C03 (three-point mutation I240T-V708I-Y444F). RC-C02 virus packaging stability was improved, and in vitro transduction efficiency was further enhanced compared to the AAV2.7m8 serotype.

[0181] Further computer simulation of tissue-targeted short peptides was used to screen out serotypes such as RC-C14. Compared with the RC-C02 capsid, RC-C14 has 10 amino acids inserted after position 587 of VP1 (587-LALGDVTRPA). Analysis of the three-dimensional spatial structure of the RC-C14 capsid revealed that the integration of the 10 peptides in the variable region of VP1VIII introduced characteristic targeting short peptides on the surface of the AAV capsid protein, which changed the original spatial structure of receptor binding and caused the capsid to produce a switch that specifically binds to potential new receptors, while weakening the binding activity of rAAV2 to HSPG. Therefore, RC-C14 can efficiently enter photoreceptors and melanocyte epithelial cells through multiple receptors (rather than relying on the heparin recognition receptor HSPG as the main binding receptor), significantly improving the transduction efficiency in in vitro cell lines and in vivo retinal tissues.

[0182] Compared to wild-type AAV2 VP1 capsid amino acid sequences, the RC-C14 serotype contains the following mutations: a 587-LALGDVTRPA-588 insertion and an I240T-V708I point mutation. RC-C14 exhibits the strongest transduction activity in ARPE19 and 293T cells in vitro. RC-C14 is described in detail in patent application number CN202310084749.8.

[0183] 2) AAV9 variants

[0184] Wild-type AAV9 uses galactose as its primary receptor. Based on rational design, the present invention mutates the AAV9 capsid protein at specific amino acid positions and chimerizes tissue-targeting peptides on the capsid surface (VR variable region), resulting in a series of capsid variants represented by RC-C07V5. For example, the RC-C07V5 serotype is derived by replacing 15 functional amino acids (HQSAQAQAQTGWVQN, SEQ ID NO: 20) in the VRVIII variable region (584-598aa) of the AAV9 capsid VP1 with 25 specific amino acids (LQRGNLALGDVTRPARQAATADVNT). The mutation in the VRIII region weakens the AAV9 capsid's ability to bind to the cell surface galactosidase receptor and, at the same time, enables the RC-C07V5 capsid to more efficiently penetrate the inner limiting membrane, making it more suitable for intravitreal administration. Compared with the parent, the tropism of RC-C07V5 for photoreceptor cells is increased by 1-2 orders of magnitude, which is superior to the currently known AAV serotypes (including but not limited to AAV1-13 serotypes). Whether it is administered subretinal or intravitreal, RC-C07V5 can be distributed throughout the retina, while stably and continuously expressing exogenous supplementary proteins. The stability of the modified virus is consistent with the existing serotype. The modified serotype (RC-C07V5) can enter cells through multiple receptors (including heparin recognition receptor HSPG main receptor, Galactose auxiliary receptor), significantly increasing the penetration of the static barrier (ILM) and reaching the outer layer of the retina. Compared with the parent serotype, the transduction efficiency is significantly improved.

[0185] In one embodiment of the present invention, the nucleic acid sequence of the present invention is contained in a modified AAV vector (e.g., RC-C14 or RC-C07V5 serotype), thereby further improving the expression and distribution of anti-VEGF protein in the eye. The modified AAV vector of the present invention has significantly enhanced tropism for the entire layer of retinal cells (especially PR and RPE) compared with existing serotypes, while ensuring efficient inner limiting membrane shuttling ability. Compared with existing serotypes, the tropism for retinal pigment epithelial cells and photoreceptor cells is further improved. Whether it is administered subretinaly or into the vitreous cavity, the modified AAV vector can be distributed throughout the entire layer of the retina, stably and continuously express exogenous supplementary proteins, and the stability of the virus is better than that of existing known serotypes.

[0186] In a preferred embodiment, the sequence of the capsid protein of the adeno-associated virus vector carrying the anti-VEGF fusion protein coding sequence of the present invention is shown as SEQ ID NO: 13 or 15.

[0187] Formulations and compositions

[0188] The present invention provides a preparation or composition, which contains (a) the vector described in the third aspect of the present invention or the adeno-associated virus vector described in the fourth aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.

[0189] In another preferred embodiment, the pharmaceutical preparation is used to treat an eye disease, preferably, the pharmaceutical preparation is used to treat a VEGF-related eye disease, preferably, to treat macular degeneration and / or diabetic retinopathy, more preferably, to treat wet age-related macular degeneration.

[0190] To facilitate clinical application, the pharmaceutical composition of the present invention can be contained in an injection device (such as an injection needle), which can contain a single dose of the pharmaceutical composition. The injection device can be contained in a medicine box for convenient storage and use. During transportation, the small container containing the drug suspension needs to be placed on dry ice. It should be stored in a freezer at -80°C.

[0191] The medicine box or test kit of the present invention may also include instructions for use to facilitate those skilled in the art to use it in the correct manner.

[0192] The "active ingredient" in the pharmaceutical composition of the present invention refers to the vector of the present invention, such as a viral vector (including an adeno-associated viral vector). The "active ingredient", preparation and / or composition of the present invention can be used to treat eye diseases. "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier or excipient" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0193] The composition can be a liquid or solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0194] In the present invention, the vector can be administered by a method selected from the group consisting of intravenous, intramuscular, subcutaneous, oral, mucosal contact, intraperitoneal, and intralesional, but is not limited thereto.

[0195] In the present invention, the vector can be administered to the eye by injection. For example, the vector can be administered to the eye by subretinal injection, suprachoroidal injection, or intravitreal injection. In any mode of administration, the vector is preferably provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0196] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0197] The composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0198] The nucleic acid or fusion nucleic acid encoding the anti-VEGF fusion protein provided by the present invention can be used to produce the anti-VEGF fusion protein in vitro or in vivo, and the preparation containing the anti-VEGF fusion protein can be used to prepare drugs for treating eye diseases.

[0199] Treatment

[0200] The present invention provides a method for treating VEGF-mediated neovascular eye diseases, the method comprising introducing a vector comprising a nucleic acid sequence encoding an anti-VEGF fusion protein into the eye. The method may comprise administering a vector comprising a nucleic acid of the present invention subretinal, suprachoroidal, or intravitreal.

[0201] The present invention provides a nucleic acid vector for use in a method for treating neovascular eye diseases by providing photoreceptor function to cells. The nucleic acid vector comprises a nucleic acid sequence encoding an anti-VEGF fusion protein. The composition of the present invention can be administered alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0202] As used herein, treating a disease means administering a nucleic acid or vector as described herein to improve or alleviate one or more symptoms of the disease, including reducing vascular leakage, reducing angiogenesis, and the like.

[0203] The methods of the present invention comprise introducing a nucleic acid sequence encoding an anti-VEGF fusion protein into the vitreous cavity of an eye. Preferably, the method comprises contacting a cell with a vector (preferably a virus, more preferably an adeno-associated virus) comprising the nucleic acid sequence encoding the anti-VEGF fusion protein. Preferably, the cell is a retinal cell, preferably a cone cell, a rod cell, a light-receiving bipolar cell, a light-withdrawing bipolar cell, a horizontal cell, a ganglion cell, and / or an amacrine cell.

[0204] When the nucleic acid sequence and one or more enzymes are provided in multiple (two or more) doses, these doses may be separated by a suitable time interval, for example 30 seconds to several hours or one or more days.

[0205] Each dose may contain an effective amount of nucleic acid sequence or viral vector. The effective dose of nucleic acid sequence or viral vector may be 1×10 6 -1×10 16 The range of viruses, for example, 1×10 7 -1×10 12 range.

[0206] The main advantages of the present invention include:

[0207] (1) The anti-VEGF fusion protein of the present invention optimizes the extracellular secretion signal peptide of the VEGF trap protein. Compared with the fusion protein in CN112342228A, it can achieve a higher expression and secretion level of the VEGF trap molecule at the same dose of AAV product.

[0208] (2) The anti-VEGF gene expression cassette of the present invention achieves more efficient target gene expression than existing drugs by optimizing element selection, especially the combination of promoter and intron, and is expected to exert better and longer-term therapeutic effects.

[0209] (3) The AAV2 / GOI-D02 gene therapy drug combination of the present application is used for a single subretinal injection. Compared with the existing nVEGFi drug, the therapeutically effective dose in the animal CNV model is lower (40 times) and can more effectively inhibit neovascular leakage.

[0210] (4) The nucleic acid of the present application can be expressed using modified AAV vectors of the AAV2.RC-C14 (CN202310084749.8) and AAV9.RC-C07V5 series. Compared with wild-type AAV2 and AAV9, the modified AAV vector can more effectively penetrate the inner and outer retinal membranes and transduce the entire retina, including the photoreceptor layer (PR) and retinal pigment epithelium (RPE). Compared with the existing AAV2.7m8, the modified AAV vector has better whole-retinal infection and RPE transduction in primates, and is more suitable for delivering shuttle genes to multiple layers of the retina by intravitreal injection (IVT) for diseases, including different types of AMD. Compared with the existing drug ADVM-022, the RC-C14 / GOI-D02 gene therapy drug has a better dose-target gene expression relationship (~4 times).

[0211] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0212] Example 1 Effects of Shuttle Gene Plasmid Map and Regulatory Elements on Target Gene Expression

[0213] First, multiple GOI plasmids with different combinations of promoters and introns were constructed. The VEGF trap coding sequence shown in SEQ ID NO:4 was used, in which the percentage of CpG sequences, human codons, etc. were optimized. The Ig kappa chain secretion signal peptide (SEQ ID NO:11) was used. This coding sequence was constructed downstream of the CAG promoter (SEQ ID NO:6) and the ubiquitin gene UBC promoter (SEQ ID NO:5), and combined with the CAG intron (SEQ ID NO:9, 10), or the UBC intron (SEQ ID NO:8), or the UBC-CAG chimeric intron (SEQ ID NO:7) to form GOI plasmids with different combinations. HEK293 suspension cells were transfected with three plasmids using different designs of GOI plasmids, wild-type AAV2 capsid plasmids, and helper plasmids for AAV virus packaging and purification (two-step purification by AAV affinity chromatography and anion chromatography). The purified virus was used for infection and expression detection of HEK293T cells.

[0214] 293T cells were seeded into 24-well plates at 4E5 cells / well and cultured as usual. The next day, the amount of AAV required per well was calculated based on the number of cells in the plate and at different MOIs (3000 Vg / cell). The desired amount of AAV was added to the cells for infection. Culture was continued for 72 hours, and the cell and supernatant suspension was collected. VEGF trap expression was detected by native SDS-PAGE and western blot using HRP-conjugated goat anti-human IgG (H+L).

[0215] The Western blot results in Figure 1 show that different promoter and intron combinations exhibited different VEGF trap expression levels. Although the longer CAG intron enhanced expression under the CAG promoter (CAGp) and was slightly higher than the UBC promoter / UBC intron combination, the combination of the UBC-CAG chimeric intron and the UBC promoter (UBCp) exhibited even higher VEGF trap expression levels. Grayscale quantification revealed that the UBCp-UBC / CAG chimeric intron combination produced 22% higher VEGF trap protein expression than the CAGp-long CAG intron and 35% higher than the UBCp-UBC intron.

[0216] The left side of Figure 1 shows a map of the shuttle gene plasmid GOI-D02 (SEQ ID NO: 1), in which the single-stranded DNA sequence (SEQ ID NO: 2) packaged in the AAV plasmid capsid includes a 5'-ITR (inverted terminal repeat), a UBC (ubiquitin C) promoter (SEQ ID NO: 5), a UBC-CAG chimeric intron (SEQ ID NO: 7), an Ig kappa chain secretion signal peptide (SEQ ID NO: 11), a VEGF trap coding sequence (SEQ ID NO: 4), an hGH poly (A) human growth hormone polyadenylation signal, and a 3'-ITR element sequence.

[0217] Example 2 Effects of different secretion signal peptides on expression and secretion at the cellular level

[0218] To further compare the effects of different signal peptides on VEGF trap protein expression and secretion, the coding sequences of different signal peptides were constructed into the above-mentioned plasmids, and three plasmid packaging and purification were performed (using AAV2 capsid). Using an experimental method similar to that of Example 1, the purified virus was infected with HEK293T cells at different MOIs (0, 300, 1000, 3000, and 10,000 VG / cell). The cells were cultured for 72 hours, and the cells and supernatant were collected. The expression and intracellular distribution of VEGF trap were detected by native SDS-PAGE and western blot (using HRP horseradish peroxidase-conjugated goat anti-human IgG (H+L)).

[0219] Figure 2 WB (semi-quantitative estimation) shows that signal peptides from Ig kappa chain (SEQ ID NO: 11) and VEGFR1 (SEQ ID NO: 12, see CN112342228A) can help express and secrete VEGF trap protein, but the expression effect of Ig kappa chain and VEGFR1 signal peptides is better. The total expression of VEGF trap in cells transduced with Ig kappa chain secretory peptide (SEQ ID NO: 11) is approximately 1.86 times that of cells carrying VEGFR1 secretory peptide molecules (the expression multiples corresponding to 300, 1000, 3000, and 10000 VG / cell in Figure 2 are 2.78, 2.29, 1.47, and 1.28 times, respectively). According to the MOI amount and expression level calculation, when expressing a considerable amount of VEGF trap protein, the amount of virus carrying Ig kappa chain signal peptide required is only 1 / 3 of that of virus carrying VEGFR1 signal peptide, which greatly improves expression efficiency and reduces the amount of virus required. At the same time, the ratios of extracellular secretion and intracellular secretion of VEGF trap protein expressed in expression vectors carrying the Ig kappa chain signal peptide and the VEGR1 signal peptide were 2.47 and 2.08, respectively, suggesting that the Ig kappa chain secretory peptide can promote the extracellular secretion of VEGF trap. The extracellular secretion of the biologically active VEGF trap produced by the Ig kappa chain secretory peptide was 1.96 times that of the VEGFR1 secretory peptide (in Figure 2, the ratios of 300, 1000, 3000, and 10000 VG / cell were 2.50, 2.30, 1.48, and 1.15 times, respectively).

[0220] The results of this example suggest that the signal peptide derived from Ig kappa chain has a greater advantage in the expression and secretion of VEGF trap.

[0221] Example 3 VEGF trap protein expressed and secreted by AAV-infected cells can effectively compete with VEGF antibodies for binding to VEGF165 protein and inhibit the signal transduction activity downstream of VEGFR1

[0222] Experimental methods:

[0223] HEK293T cells were infected with the recombinant virus prepared in Example 1 (MOI: 5000 VG / cell). After 72 hours, the supernatant samples were collected and the VEGF trap protein in the supernatant was concentrated using an Amicon Ultra centrifugal filter. The concentration of the expressed VEGF trap protein was quantified by Western blotting.

[0224] VEGF165 (the most common VEGFa isoform) competition binding ELISA assay: Free VEGF165 levels were measured using an ELISA kit to assess the amount of VEGF165 bound to the VEGF trap protein, thereby reflecting the activity of secreted VEGF trap protein. Aflibercept was used as a control. The assay was performed according to the manual of the Human VEGF165 ELISA Kit (Sinobiological, KIT11066), using 2 ng / ml VEGF165 protein. The results are shown in Figure 3A.

[0225] The competitive activity of VEGF165 was assessed using H_VEGF Reporter 293 cells (Jiman Biotechnology, GM-C09057). VEGF165 binds to the surface VEGFR receptors on H_VEGF Reporter 293 cells, promoting luciferase expression in the cells. When VEGF165 is bound by the VEGF trap protein expressed and secreted by recombinant AAV-infected cells, luciferase expression in H_VEGF Reporter 293 cells decreases. Therefore, measuring luciferase expression in cells can reflect the activity of the VEGF trap protein. Aflibercept was used as a control.

[0226] Experimental results:

[0227] Figure 3 shows the competitive binding of AAV2 / GOI-D02-expressed VEGF trap protein to VEGFA (Figure 3A) and its inhibitory effect on downstream cellular bioactivity (Figure 3B). Compared with the control aflibercept, the VEGF trap protein expressed and secreted after recombinant molecule transduction into cells showed superior in vitro binding and bioactivity. The average IC values ​​of the VEGF trap protein expressed by AAV2 / GOI-D02 and aflibercept in the in vitro competitive binding ELISA assay for VEGF165 were 0. 50 The average IC values ​​in the H_VEGF Reporter 293 cell reporter gene assay were 9.6 and 33.6 pM, respectively. 50 The concentrations of VEGF trap encoded by GOI-D02 were 242.95 and 496.0 pM, respectively. This indicates that the VEGF trap encoded by GOI-D02 has excellent VEGFA binding and inhibits its interaction with VEGFR and downstream cell signaling. This superior biological activity may be due to the additional GPG linker peptide in the protein encoded by GOI-D02, which further improves the stability of the protein molecule.

[0228] Example 4 Comparison of transduction activity and tissue distribution of modified AAV serotypes (RC-C14 and RC-C07V5 and series) in mouse eyes

[0229] Autofluorescence (AF) detection method:

[0230] In vivo autofluorescence (AF) detection steps: 6-8 week old C57 mice (provided by Jicui Bio) were divided into 6 groups of 2 mice per group, and the drug was administered into the vitreous cavity of both eyes; AF of 4 eyes in each group was examined. Drugs were added to the ocular surface of both eyes to dilate the pupils, and the mice were anesthetized with a 60 mg / kg anesthetic dose of Zotai mixture. Topical anesthetics were added to both eyes, and gel was applied to the ocular surface to wear corneal contact lenses. The HRA control panel of the inspection equipment was put into IR mode, and the fundus of the mouse was focused until the image was clear. Then, it was switched to FA mode, the SENS value was adjusted to 100, the focus was adjusted until the retinal blood vessels could be clearly seen, the SENS value was lowered to 60, and the photo was taken to ensure that the exposure intensity of the photo was within a reasonable range.

[0231] Existing literature records that under the intravitreal administration method, the transduction efficiency of the AAV2.7m8 serotype virus in mouse retinal tissue is significantly improved compared to AAV2. In this example, RC-C14 (AAV2.7m8 variant serotype, whose viral capsid VP1 sequence is shown in SEQ ID NO: 13) and AAV2 serotype are used as controls to detect the transduction activity of the RC-C07 variant serotype. The RC-C07 serotype is a modified form of AAV9. The results of Example 2 show that the in vitro transduction activity of wild-type AAV2 is low, which suggests that the parent strain has limited ability to transduce retinal tissue via the vitreous injection route, while the rationally designed variant serotype has significantly improved transduction activity in photoreceptor cells. In this example, the transduction activity of the variant capsid is verified through in vivo experiments.

[0232] Figures 4A-4B show a comparison of the viral distribution and transduction activity of four variant capsids of the RC-C07 series and two existing serotype vectors in retinal tissue under the IVT vitreous injection method. Six serotypes (AAV2, RC-C14, RC-C07V1, RC-C07V2, RC-C07V5 and RC-C07V7, of which RC-C07V1, RC-C07V2, and RC-C07V7 structures and sequences are shown in Chinese patent application 202311491510.9) were administered intravitreally, and the viral dose per eye was E9vg. The transduction activity of the rAAV-EGFP virus in retinal tissue was evaluated after 6 weeks of administration. In vivo autofluorescence photography (BAF) was performed at three time points: 2 weeks, 4 weeks, and 6 weeks of administration, and the fluorescence intensity of the left and right fundus of each group of mice was detected.

[0233] Figures 4A-4B show that after two weeks of IVT administration, spontaneous green fluorescence signals were detected in the fundus of mice corresponding to all four variant serotypes. The fluorescence signal in the AAV2-administered group was the weakest, while that in the RC-C14-administered group was slightly stronger than that of the other serotypes, although there was variability between individuals. After six weeks of administration, the fluorescence signals of the C07V1, C07V2, and C07V7 variants continued to increase with prolonged administration, while no significant increase was observed in the control group (RC-C14).

[0234] The statistical results of fluorescence area (Figures 4C-4D) and average fluorescence intensity (Figures 4E-4F) further showed that after 6 weeks of intravitreal administration, the fundus fluorescence signal of mice in the RC-C07V2 and RC-C07V7 administration groups was significantly enhanced compared with 4 weeks, while the fundus fluorescence intensity and fluorescence area of ​​mice in the RC-C07V5 administration group were comparable to those of the experimental control (RC-C14).

[0235] Further, after 6 weeks of administration, the eyeballs of the mice were sampled, and frozen sections of the retinal tissue were subjected to immunofluorescence staining with RPE65 antibodies or Opsin antibodies (purchased from Abcam) (red marks the RPE cell layer, or photoreceptor cells; DAPI marks the cell nucleus; green fluorescence represents the autofluorescence after rAAV infection of the cells). From the immunofluorescence images of the mouse retinal tissue, it can be seen that with the IVT administration method (Figures 4G-4H), except for the control group (AAV2), the other four variant serotypes (RC-C07V1, RC-C07V2, RC-C07V5, RC-C07V7) can all efficiently infect the inner retinal tissues (ganglion cell layer, nerve fiber layer, inner nuclear layer). The tissue distribution of the variant serotypes can be clearly observed from the complete retinal image spliced ​​by the fully automatic scanning camera device. Among them, the green fluorescence signal expressed by RC-C07V5 (whose viral capsid VP1 sequence is shown in SEQ ID NO: 15) is the most widely distributed in the inner retinal tissues and has the strongest fluorescence brightness. Further comparative analysis of the fluorescence of the outer layer of the retina revealed that the two variants, RC-C07V2 and RC-C07V5, can fully transduce the outer plexiform layer, outer nuclear layer, and part of the inner segments of the photoreceptor layer (PR) of the retina. This further indicates that after intravitreal injection, RC-C07V2 and RC-C07V5 can be widely distributed throughout the retinal tissue, and the fluorescence brightness is significantly enhanced compared with the control group AAV2. The penetration of the inner limiting membrane (ILM) is higher than that of the existing serotype (AAV2.7m8).

[0236] Example 5: Efficient delivery and secretion of VEGF trap protein by modified capsid in HEK293 cells in vitro

[0237] Similar to Example 2, the RC-C14 / GOI-D02 recombinant AAV virus was prepared using a three-plasmid transfection method and infected HEK293T cells in vitro. After 72 hours, cells and supernatants were harvested for Western blotting, using GAPDH as an internal reference.

[0238] The results in Figure 5 show that recombinant AAV packaged with capsid RC-C14 can also efficiently infect HEK293T cells, express and secrete VEGF trap protein, and protein secretion is not affected by AAV capsid modification. The ratio of extracellular to intracellular protein amounts is approximately 2.1-3.1 (semi-quantitative Western blotting).

[0239] Example 6: Intraocular injection of recombinant AAV in mice can long-term express VEGF trap protein molecules and significantly inhibit neovascularization leakage in the mouse CNV model

[0240] Establishment of CNV model in mice by subretinal injection and fundus laser photocoagulation

[0241] Wild-type C57BL / 6J mice (6-8 months old) were divided into four groups, including three AAV2 / GOI-D02 dose groups (1E8, 1E7, and 1E6 VG / eye), a vehicle control group, and a non-treated control group (NC). General anesthesia was performed by intraperitoneal injection of anesthetic agent, 60 mg / kg 50 +8mg / kg xylazine hydrochloride, diluted to the desired concentration with 0.9% sodium chloride injection. Mydriasis was achieved with 0.5% compound tropicamide eye drops (Medo-Li), local anesthesia was achieved with 0.4% oxybuprocaine hydrochloride eye drops (Benoxi), and ofloxacin eye ointment (Dicloro) was applied to the ocular surface. Under a surgical microscope, a disposable needle was used to puncture the sclera at the inner side of the limbus. A microsyringe with a 36G flat needle was then inserted through the puncture, bypassing the lens and reaching the vitreous. The needle was then gradually advanced to the subretinal space, and the injection was slowly pushed to complete subretinal drug delivery. After the injection, the mouse was placed in a warm environment for recovery and returned to its cage after awakening.

[0242] The day of laser modeling was designated as day 0. Mice were anesthetized using the above method. The parameters of the ophthalmic laser therapy device (laser photocoagulation device) were adjusted to a laser energy of 160mw, an excitation time of 0.1s, and a laser spot diameter of 50μm. Gently press the mouse eyes with a coverslip to fully expose the visual axis, adjust the position of the laser photocoagulation device until a clear fundus and laser point can be seen, and emit the laser for modeling. Each mouse's eyes were photocoagulated at 3 or 4 points according to the actual situation. After the modeling is completed, the mice were placed in a warm environment for recovery and put back in the cage after waking up. The mice were continuously raised for 6 months before laser modeling.

[0243] Fundus fluorescein angiography (FFA)

[0244] Fundus fluorescein angiography (FFA) of mice was performed on days 3, 5, 10, and 13 after laser modeling. Fluorescein sodium stock solution was diluted 10-fold for later use. General anesthesia was administered via intraperitoneal injection of anesthetics, with 0.5% tropicamide eye drops (Medo-Rel) for mydriasis and 0.4% oxybuprocaine hydrochloride eye drops (Benoxi) for local anesthesia. After the mice were fully anesthetized, contact lenses were placed on the front operating table of the confocal laser synchronized angiography (HRA) system. Each mouse was subcutaneously injected with 0.1 mL of 10% sodium fluorescein dilution. The confocal laser synchronized angiography (HRA) system was adjusted to FA mode and the timing was started. Fundus fluorescence imaging was completed within 5-15 minutes after sodium fluorescein injection. The goal was to capture all laser-induced CNV lesions.

[0245] CNV lesion analysis

[0246] The results of fundus fluorescein angiography of mice were adjusted for brightness using ImageJ software. Under the premise of ensuring that the parameters of the inspection instrument remain unchanged, a picture with moderate brightness of the fundus artery and vein was selected from the original picture as the reference brightness, and the brightness of the remaining pictures was adjusted accordingly using ImageJ, ultimately achieving a similar brightness of the central retinal blood vessels in all pictures. According to the CNV lesion grading standard (Table 1), the levels of each lesion were distinguished and recorded. The chi-square test of Graphad's contingency tables was used to test whether there was a significant difference in the ratio of the number of different CNV lesions at a certain time point between the groups. Because the capillary leakage of grade 3-4 CNV lesions is too high, adjacent grade 3-4 lesions sometimes merge. For fused CNV lesions, the number of laser-induced spots is still counted.

[0247] Table 1 CNV lesion grading standards

[0248] result

[0249] To investigate the long-term effects of the drug described in this invention in animals, VEGF trap protein (Figure 6A) and RNA (Figure 6B) expression in the entire eyeball was measured periodically over the six months following drug administration (2, 4, 5, and 6 months after administration). Results were normalized using beta-actin expression as an internal reference. No protein or RNA was detected in the eyes of mice injected with the vehicle formulation or those receiving no drug (NC). However, all three dose groups demonstrated significant dose-dependent, stable expression, with VEGF trap protein levels increasing over time.

[0250] In addition, the therapeutic effect of AAV2 / GOI-D02 on laser-induced CNV neovascularization and leakage following intraocular injection was observed in middle-aged and elderly mice (12-14 months of age). Figure 6C shows that during the observation period after laser photocoagulation-induced CNV in wild-type C57BL / 6J mice, lesions and leakage became more severe (NC and vehicle (buffer) groups). The two groups of mice injected with 1E8 and 1E7 VG / eye AAV2 / GOI-D02 showed clear efficacy, as evidenced by a significant reduction or even disappearance of high-grade lesions on days 5, 10, and 13. The ultra-low-dose group (1E6 VG / eye) did not show any clear efficacy.

[0251] This result is consistent with the observed expression of the target gene. More importantly, it demonstrates that AAV2 / GOI-D02 can effectively express VEGF trap protein when administered subretinaally, inhibiting neovascularization leakage in the laser-induced CNV mouse model; and that 1E7 VG / eye is the minimum effective dose for this model.

[0252] Comparison with existing drugs

[0253] Compared with existing CNV gene therapy drugs, the effective dose required for the AAV2 / GOI-D02 molecule in the present invention is very low, reflecting the advantages of the gene therapy drug of the present invention.

[0254] Structurally, the VEGF trap protein of the present invention comprises the IgC domain 2 of VEGFR1, the IgC domain 3 of VEGFR2, and the GPG linker / Fc-hinge region. It differs from the prior art CN112342228A in that the C-terminus of the present invention is an IgG1 Fc CH2 domain, while CN112342228A utilizes an IgG4 Fc CH2 domain with a C-terminal HA tag. The HA tag is not required for VEGF trap activity; the sequences and functions of IgG1 and IgG4 are similar. Another difference is that the present invention utilizes a secretory signal peptide derived from the light chain of an Ig kappa chain immunoglobulin, while CN112342228A utilizes a signal peptide derived from VEGFR1.

[0255] Compared with aflibercept (used in ADVM-022 technology), the VEGF trap secretory protein of the present invention does not have the three amino acids "SDT" at the N-terminus, but has a short GPG linker added before the Fc-Hinge hinge sequence.

[0256] Figure 6D shows the results of a similar experimental method for testing the efficacy of the AAV2 molecule in CN112342228A in a mouse CNV model. The results showed that doses of 1E8 and 1E9 VG / eye effectively inhibited laser-induced neovascularization in mice, while a low dose of 1E7 VG / ml showed little improvement. Using the same administration route, the minimum effective dose of the drug molecule in CN112342228A in the mouse CNV model was approximately 1E8 VG / eye, 10 times that of the AAV2 / GOI-D02 of the present invention.

[0257] Another existing anti-VEGF molecule is nVEGFi, which has comparable activity to Aflibercept in binding and inhibiting VEGF. Both (nVEGFi & Aflibercept) use a gene expression cassette containing the CB7 (chicken beta actin) promoter and AAV8 as a delivery system for subretinal administration in C57BL / 6J mice and treatment experiments in the CNV model. Both showed a similar minimum effective dose of 4E8 VG / eye. At doses of 4E6 and 4E7 Vg / eye, AAV8 / CB7-nVEGFi showed no improvement in CNV neovascularization leakage, just like the PBS buffer group. Compared to existing drug molecules AAV8 / nVEGFi and AAV8 / Aflibercept, the minimum effective dose of the present invention's AAV2 / GOI-D02 is 40-fold lower, offering greater advantages in clinical effectiveness, reduced side effects, and lower production costs.

[0258] Example 7: Intraocular injection of recombinant AAV into NHP eyes can long-term express VEGF trap protein molecules and significantly inhibit neovascularization leakage in non-human primate CNV models.

[0259] Choroidal neovascularization (CNV) models were established in rhesus monkeys using laser photocoagulation. The inhibitory effects of a single subretinal administration of AAV2 / GOI-D02 (Group IV) and a single intravitreal injection of RC-C14 / GOI-D02 (Group III) on CNV were investigated. A vehicle control group (Group I) and a single intravitreal injection of AAV2 / GOI-D02 (Group II) served as controls. Drug administration was performed 4 weeks before laser modeling, with a 50 μl injection volume and a viral dose of 2.5E11 VG / eye. Three weeks after administration, a CNV model was induced using fundus laser photocoagulation. General clinical observations were performed daily throughout the study, along with periodic ophthalmological examinations and fundus photography (FP). The animals were tested with FFA at 5, 6, 7, and 8 weeks after administration. CNV was graded using a 4-grade method. The effectiveness was evaluated based on the fluorescence leakage area of ​​grade 4 (severe) leakage spots and the thickness of subretinal high reflective signal material (SHRM).

[0260] Experimental results showed that the vehicle control group showed no improvement in neovascular leakage after laser modeling, with worsening and fusion of some lesions observed in fundus photography (FP) and FFA images. However, significant improvements were observed 5-8 weeks after a single subretinal administration of AAV2 / GOI-D02 (Group IV, Figure 7A) and a single intravitreal injection of RC-C14 / GOI-D02 (Group III, Figure 7B), manifested by a reduction in lesions and leakage area. Statistical results are shown in Figure 7C, where a represents a comparison with the vehicle control group, p ≤ 0.05; b represents a comparison with a single intravitreal administration of AAV2 / GOI-D02, p ≤ 0.05. These imaging results were consistent with the results of VEGF trap protein detection (Figure 7D). The vehicle control group was negative for VEGF trap protein in both the vitreous humor and the retinal / choroidal complex. No expression product was detected in the retinal / choroidal complexes after intravitreal injection of AAV2 / GOI-D02; the average concentration of protein expression product in the vitreous humor was 37.63 ng / mL. In the intravitreal injection of RC-C14 / GOI-D02 group, the concentrations of protein expression product in the retinal / choroidal complexes and vitreous humor were 197.41 and 1727.82 ng / mL, respectively. In the subretinal injection of AAV2 / GOI-D02 group, the concentrations in the retinal / choroidal complexes and vitreous humor were 2308.02 and 380.72 ng / mL, respectively.

[0261] As expected, wild-type AAV2 capsids are suitable for treating subretinal neovascularization but are not well suited for intravitreal injection. However, the novel RC-C14 capsid, derived from AAV2-directed engineering, is well-suited for intravitreal injection, efficiently expressing VEGF trap, even reaching the retinal / choroidal complex. These results demonstrate that the GOI-D02 gene expression cassette can be used effectively with different capsids and injection methods, achieving long-term, high-efficiency expression and secretion of VEGF trap protein, effectively inhibiting neovascularization leakage.

[0262] Comparison with existing drugs

[0263] In experiments with the existing drug ADVM-022, ADVM-022 (AAV2.7m8 / CMVp-Aflibercept) was injected into the subretinal space of non-human primates at a dose of 2E12 Vg / eye. Three months later, approximately 2-4 μg / ml of Aflibercept was detected in the vitreous fluid of most monkeys. In Group III of this example, a single intravitreal injection of RC-C14 / GOI-D02 at 2.5E11 Vg / eye (1 / 8 the dose of ADVM-022) was administered. However, the average amount of VEGF trap protein detected in the vitreous fluid was approximately 1.7 μg / ml, only slightly lower than the expression level of ADVM-022. The RC-C14 / GOI-D02 combination of the present invention exhibits a dose-effect advantage of at least 3-4 times that of ADVM-022.

[0264] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0265] sequence

Claims

1. An anti-VEGF fusion protein, characterized in that: The fusion protein has a structure of formula I from N-terminus to C-terminus: LVH-Fc (I) In the formula, each "-" is independently a bond or a connecting peptide; L is a secretion signal peptide, and the secretion signal peptide is derived from the Ig kappa chain; V is an anti-VEGF protein, wherein the anti-VEGF protein comprises IgC domain 2 of VEGFR1 and IgC domain 3 of VEGFR2; H is an optional hinge region; Fc is an optional crystallizable fragment of an immunoglobulin.

2. The anti-VEGF fusion protein according to claim 1, characterized in that The sequence of the secretion signal peptide is shown in SEQ ID NO:

11.

3. The anti-VEGF fusion protein according to claim 1 or 2, characterized in that The sequence of V is shown in SEQ ID NO:3, positions 1-202.

4. An expression cassette, characterized in that The expression cassette comprises a nucleic acid molecule encoding the anti-VEGF fusion protein of claim 1.

5. The expression cassette according to claim 4, characterized in that The expression cassette has a structure of formula II from 5' to 3' end: Z0-Z1-Z2-Z3-Z4-Z5 (II) In the formula, each "-" is independently a bond or a nucleotide linking sequence; Z0 is none or 5′ITR sequence; Z1 is the promoter; Z2 is an intron; Z3 is a nucleotide sequence encoding the anti-VEGF fusion protein according to claim 1; Z4 is none or hGHpA sequence; Z5 is none or 3' ITR sequence.

6. The expression cassette according to claim 5, characterized in that The promoter is selected from the group consisting of a UBC promoter, a CAG promoter, or a combination thereof.

7. The expression cassette according to claim 5, characterized in that The intron is selected from the group consisting of a UBC-CAG chimeric intron, a CAG intron, a UBC intron, or a combination thereof.

8. The expression cassette of claim 4, wherein The nucleotide sequence of the expression cassette is as shown in SEQ ID NO:2, or has ≥85%, ≥90%, ≥95%, ≥97%, ≥98% or ≥99% sequence identity with the nucleotide sequence shown in SEQ ID NO:

2.

9. A carrier, characterized in that The vector contains the expression cassette according to claim 4.

10. An AAV vector, characterized in that The adeno-associated virus vector contains the expression cassette of claim 4.

11. A host cell, comprising the vector according to claim 9 or the AAV vector according to claim 10, or an exogenous expression cassette according to claim 4 integrated into its chromosome.

12. Use of the vector according to claim 9 or the AAV vector according to claim 10 for preparing a preparation or composition for treating VEGF-related eye diseases.

13. The use according to claim 12, characterized in that The VEGF-related eye disease is selected from the group consisting of macular degeneration, diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, or a combination thereof.

14. A pharmaceutical preparation comprising (a) the vector according to claim 9 or the adeno-associated virus vector according to claim 10, and (b) a pharmaceutically acceptable carrier or excipient.

15. A method for preparing the anti-VEGF fusion protein according to claim 1, comprising culturing the host cell according to claim 11, thereby obtaining the anti-VEGF fusion protein.

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