WO2023005906A1 - Novel adeno-associated virus serotype mediated angiogenesis inhibitor and application thereof - Google Patents

Abstract

A novel adeno-associated virus serotype, an adeno-associated virus serotype-mediated gene therapeutic drug, and in particular, an angiogenesis inhibitor. The adeno-associated virus serotype has good retinal tissue targeting, and can be used as a delivery carrier of a therapeutic gene for the treatment of eye-related diseases. The drug can be used for treating various retinal diseases and multiple cancers that use angiogenesis as a major pathological mechanism.

Description

Novel adeno-associated virus serotype-mediated angiogenesis inhibitor and its application technical field

The present disclosure relates to a new adeno-associated virus serotype and gene therapy drugs mediated by the adeno-associated virus serotype, especially an angiogenesis inhibitor.

Background technique

Pathological neovascularization occurs in a large number of retinal diseases and affects millions of people worldwide each year. For example, approximately 30 million people worldwide, especially those over the age of 60, suffer from age-related macular degeneration (AMD), which is the leading cause of blindness in the elderly (Klein R, Peto T et al, Am J Ophthalmol (2004 )137(3):486-95; Friedman DS, O'Colmain BJ et al., Arch Ophthalmol (2004)122(4):564-72; AI-Zamil WM et al., Clin Interv Aging (2017):28860733). AMD is characterized by progressive retinal damage caused by multiple factors. Among them, there is a severe form of AMD known as wet AMD, in which choroidal neovascularization (CNV) gradually expands throughout the retina, and due to the fragility of the vessel wall, its internal contents are prone to leakage, which further damages the structural integrity of the retina and hinder visual function. Similar neovascular problems are prevalent in diabetic retinopathy (DR) and retinopathy of prematurity (ROP), where neovascular bleeding and scarring occur in diabetics and newborns, respectively.

In addition, excessive neovascularization is induced during tumorigenesis to facilitate the supply of oxygen and nutrients to cancer cells. First, neovascularization can be induced from pre-existing vascular networks and infiltrate tumor tissue; second, tumor cells can recruit endothelial progenitor cells to form secondary vasculature; third, tumor cells accumulate around pre-existing blood vessels and Organize endothelial cells in a tubular fashion to generate new vasculature (Jain RK, Science (2005) 307(5706): 58-62; Carmeliet P et al, Nature (2011) 473(7347): 298-307; Majidpoor J et al, Cell Oncol. 2021).

The pathological conditions described above demonstrate the importance of anti-angiogenesis in the treatment of retinal diseases and cancers. Vascular endothelial growth factor (VEGF) is a key factor in initiating the growth of new blood vessels in these diseases. Anti-VEGF approaches have attracted considerable attention. Endostatin is a 20kD fragment produced from the C-terminus of collagen XVIII, and angiostatin is a Kringle domain-containing protein produced by the proteolytic cleavage of plasminogen. They all exhibit excellent anti-angiogenic activity and antagonize the biological effects of VEGF. In the past decade, these two angiogenesis inhibitors have been widely used in retinopathy and cancer therapy to inhibit neovascularization (O'Reilly MS et al., Cell (1994) 79(2): 315-28; O'Reilly MS et al., Cell (1997) 88(2): 277-85; Patent Nos. US 9707304 B2 and US 2004/0156828 A1). Therefore, in order to achieve a better therapeutic effect on retinal diseases and cancers, it is also expected to obtain endostatin coding sequences and angiostatin coding sequences with higher expression levels.

Adeno-associated virus (AAV, adeno-associated virus) has low pathogenicity and the ability to stably express proteins in various organs and tissues for a long time. These characteristics make AAV have obvious advantages in the field of gene therapy and are suitable for delivering therapeutic genes. However, wild-type AAV serotypes usually broadly infect multiple tissues/organs in mammals with broad tissue targeting, resulting in gene delivery to off-target tissues, exacerbating adverse reactions. The capsid proteins of AAV particles not only regulate AAV assembly during replication, but also facilitate virus interaction with receptors on the plasma membrane and entry into target cells. Studies have shown that the tissue tropism and cell transformation efficiency of AAV vectors are mainly determined by their capsids. In view of this, in order to achieve better therapeutic effects, it is desirable to properly modify the AAV capsid protein to obtain an organ (eg eye) specific AAV vector.

In addition, repeated administration of angiogenesis inhibitors is undesirable due to the inconvenience and pain caused by repeated injections to patients. Therefore, it is desirable to obtain a gene delivery system that can simultaneously deliver two or more angiogenesis inhibitors.

Contents of the invention

In order to solve the above technical problems, in a first aspect, the present disclosure provides an AAV capsid protein, wherein the amino acid sequence of the AAV capsid protein is the same as that of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14 The amino acid sequences shown are at least 50%, 60%, 70% or 80% identical.

In one embodiment, the amino acid sequence of the AAV capsid protein has at least 85%, 90%, 95%, 99%, or 100% identity.

In one embodiment, the AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14. In a preferred embodiment, the amino acid sequence of the AAV capsid protein is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14.

In a second aspect, the present disclosure provides a nucleic acid molecule encoding the AAV capsid protein according to the first aspect.

In one embodiment, the nucleotide sequence of the nucleic acid molecule is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13. In one embodiment, the nucleotide sequence of the nucleic acid molecule has at least 85%, 90%, 95%, 99% of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13 % or 100% identity.

In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13. In one embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13.

The modified AAV capsid protein of the present disclosure can be used to produce novel AAV vectors, so as to carry out related research on novel AAV vectors or be used for disease treatment. The new AAV vector packaging the AAV capsid protein has good retinal tissue targeting, has lower toxic side effects and better safety potential, and can be applied to the prevention, diagnosis and treatment of eye-related diseases.

Therefore, in a third aspect, the present disclosure provides an AAV vector comprising the AAV capsid protein according to the first aspect.

In one embodiment, the AAV vector further comprises an exogenous polynucleotide comprising a nucleotide sequence encoding a Therapeutic protein. In one embodiment, the therapeutic protein is an anti-angiogenic protein. In one embodiment, the therapeutic protein is endostatin and/or angiostatin.

In one embodiment, the exogenous polynucleotide comprises a nucleotide sequence encoding endostatin; preferably, the nucleotide sequence is shown in SEQ ID NO: 15 or SEQ ID NO: 17.

In one embodiment, the exogenous polynucleotide comprises a nucleotide sequence encoding angiostatin; preferably, the nucleotide sequence is as shown in SEQ ID NO: 16 or SEQ ID NO: 18.

In a fourth aspect, the present disclosure provides a nucleic acid molecule encoding endostatin, the nucleotide sequence of which has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17, Preferably at least 85%, 90%, 95%, 99% or 100% identity. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17. In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 15 or SEQ ID NO: 17.

In a fifth aspect, the present disclosure provides a nucleic acid molecule encoding angiostatin, the nucleotide sequence of which has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18, Preferably at least 85%, 90%, 95%, 99% or 100% identity. In one embodiment, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18. In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 16 or SEQ ID NO: 18.

The nucleic acid molecule encoding endostatin of the present disclosure comprises a codon-optimized human or murine endostatin-encoding nucleic acid sequence, which has the same Higher expression levels of endostatin. Likewise, the nucleic acid molecule encoding angiostatin of the present disclosure comprises a codon-optimized human or murine angiostatin encoding nucleic acid sequence, and an uncodon-optimized original human or murine angiostatin encoding nucleic acid sequence have a higher expression level of angiostatin than

In the sixth aspect, the present disclosure provides a transgene expression cassette, which includes: a promoter, the nucleic acid molecule according to the fourth aspect and/or the fifth aspect, and bGH polyA.

In one embodiment, the promoter is selected from: CB promoter, CAG promoter, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16, VP64 promoter, Tuj1 promoter, GFAP promoter, Vimentin promoter, RPE65 promoter, VMD2 promoter, synapsin promoter, VGAT promoter, DAT promoter, TH promoter and osteocalcin promoter; preferably, the promoter is a CB promoter.

In one embodiment, the transgene expression cassette further comprises: a signal peptide, such as an SP signal peptide, an ALB signal peptide, and a PLS signal peptide; and/or two ITRs located at both ends, each of which is independently a normal ITR or Shortened ITR peptide.

In one embodiment, the nucleic acid molecule according to the fourth aspect and/or the fifth aspect bears an oligopeptide tag, such as Flag, 6×His, 2×HA and Myc.

In a preferred embodiment, the transgenic expression cassette includes the nucleic acid molecule according to the fourth aspect and the nucleic acid molecule according to the fifth aspect.

In a preferred embodiment, the transgene expression cassette further includes a linker sequence. In a preferred embodiment, the connecting sequence is Furin protease sequence+connecting peptide+2A sequence, such as P2A, T2A or F2A. The use of such linking sequences allows for better separation of expression and secretion of two or more proteins such as endostatin and angiostatin.

In one embodiment, the nucleotide sequence of the transgene expression cassette is shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

In a seventh aspect, the present disclosure provides a gene delivery system comprising: an AAV capsid protein and a transgene expression cassette.

In one embodiment, the AAV capsid protein in the gene delivery system of the present disclosure is a natural AAV capsid protein or an artificially modified AAV capsid protein. In a preferred embodiment, the AAV in the gene delivery system of the present disclosure is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8 , AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh10.

In a preferred embodiment, the AAV capsid protein in the gene delivery system of the present disclosure is the AAV capsid protein according to the first aspect. For example, the amino acid sequence of the AAV capsid protein is shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14.

In a preferred embodiment, the transgene expression cassette in the gene delivery system of the present disclosure is the transgene expression cassette according to the sixth aspect. For example, the nucleotide sequence of the transgene expression cassette is shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

In a preferred embodiment, the gene delivery system of the present disclosure can express higher levels of anti-angiogenic proteins (endostatin and/or angiostatin), which can achieve better therapeutic effects on retinal diseases and cancers.

In a more preferred embodiment, the gene delivery system of the present disclosure can realize the simultaneous delivery of two or more angiogenesis inhibitors, reducing the number of administrations to patients.

In an eighth aspect, the present disclosure provides the application of the gene delivery system according to the seventh aspect in the preparation of a medicament for treating a disease. In one embodiment, the disease has angiogenesis as the main pathological mechanism or inducing factor. In a preferred embodiment, the disease is an ocular disease such as a retinal disease or cancer.

In a ninth aspect, the present disclosure provides a medicament comprising: the gene delivery system according to the seventh aspect; and an excipient.

In one embodiment, the drug is an angiogenesis inhibitor. In one embodiment, the drug is used to treat diseases whose main pathological mechanism or inducing factor is angiogenesis. In a preferred embodiment, the disease is an eye disease, such as age-related macular degeneration, diabetic retinopathy, and other retinal diseases caused by strong light. In one embodiment, the disease is cancer, such as lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, and glioblastoma cell tumor.

In a tenth aspect, the present disclosure provides a method of treating a retinal disease or cancer, comprising administering a therapeutically effective amount of the medicament according to the ninth aspect to a subject in need thereof.

In one embodiment, the drug is administered by systemic or local routes, such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, and intralesional. In a preferred embodiment, the drug is administered topically to the eye, for example by intravitreal injection, subretinal injection or suprachoroidal injection.

Description of drawings

Figure 1A shows images of GFP signals of AAV5, AAV8, AAV9, AAVH15, AAVXL32 and AAVT13 in mouse retina.

Figure 1B shows a 3D reconstruction of the GFP signal fluorescence image shown in Figure 1A. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium.

Figure 1C shows the relative GFP fluorescence signal intensity of different AAV serotypes relative to AAV5. n=4 mice/group, ***p<0.001.

Figure 2 shows retinal sections from mice transduced with AAV8, AAV9, AAVH15 and AAVT13. GFP, DAPI and cone cell marker (S-opsin)/RPE marker (RPE65) are shown. GFP signals reaching the photoreceptor layer are indicated by white arrows.

Figure 3A is a schematic representation of the B36, B110 and B111 expression cassettes.

Figure 3B shows the Western blot results of the expression and secretion of endostatin and angiostatin in HEK293 and Huh7 medium supernatants of B110 and B111 expression cassettes. A plasmid encoding GFP was used as a control.

Figure 3C shows the B110 expression cassette (comprising codon-optimized human endostatin and human angiostatin coding sequences) and the B111 expression cassette (comprising codon-optimized murine endostatin and murine angiostatin coding sequences) ) compared with the expression level of the original non-codon-optimized human or mouse endostatin coding nucleic acid sequence. Left panel: Western blot results plot. Right panel: Quantitative statistics of relative expression levels; n=3, ***p<0.001, t test.

Figure 3D shows the B110 expression cassette (comprising codon-optimized human endostatin and human angiostatin coding sequences) and the B111 expression cassette (comprising codon-optimized murine endostatin and murine angiostatin coding sequences ) compared with the expression level of the original non-codon-optimized human or mouse angiostatin coding nucleic acid sequence. Left panel: Western blot results plot. Right panel: Quantitative statistics of relative expression levels; n=3, ***p<0.001, t test.

Figure 4A shows that GFP-containing AAVH15 (hereinafter referred to as H15-GFP) transduces HUVEC at a multiplicity of infection (MOI) of 1×10 ⁵ , 1×10 ⁴ and 1×10 ³ vg/cell, 3 days after virus infection captured image. Upper panel: GFP fluorescence; lower panel: bright field. Scale bar: 100 μm.

Figure 4B shows quantification of the percentage of GFP-positive HUVEC cells.

Figure 4C shows the conditioned medium from HUVEC infected with H15-GFP, H15-B110 (AAVH15 packaging B110 expression cassette) and H15-B111 (AAVH15 packaging B111 expression cassette) at an MOI of 1×10 ⁵ vg/cell. Western blot analysis.

Figure 4D shows the captured images of H15-GFP, H15-B36 (AAVH15 packaging B36 expression cassette), H15-B110 and H15-B111 particles infected HUVEC at MOI of 1×10 ⁵ and 1×10 ⁴ vg/cell. Scale bar: 200 μm.

Figure 4E shows quantification of HUVEC tube formation (tube length per unit area ( ^mm2 )). n=5 cell wells. **p<0.01, ***p<0.001, t-test.

Figure 4F shows the quantification of HUVEC tube formation (number of branch points per unit area ( ^mm2 )). n=5 cell wells. **p<0.01, ***p<0.001, t-test.

Figure 5A shows a mouse model of laser-induced neovascularization. C57BL/6 mice were injected intravitreously with 2×10 ¹⁰ vg/eye of AAV particles with H15 capsids and packaged B36, B110 or B111 expression cassettes (referred to as laser-B36, laser-B110, laser- B111). Fourteen days after virus injection, mice were treated with a laser-induced CNV model, and 12 days later, fluorescein angiography (FFA) and immunofluorescence (IF) were performed.

Figure 5B shows laser-induced neovascularization and scarring. Upper panel: Fluorescein angiography. Bottom: Laser-induced lesions. Scale bar: 1 mm.

Figure 5C shows fluorescent images of stained retinal sections showing activated retinal astrocytes and Müller cells (GFAP) and blood vessels (IB4). GFAP white arrows: CNV clusters; IB4 white arrows: GFAP-positive glial cell membranes associated with CNV clusters. Scale bar: 100 μm.

Figure 5D shows quantification of laser-induced CNV tuft area. *p<0.05, **p<0.01, ***p<0.001, n=5 eyes/group, one-way analysis of variance.

Figure 5E shows the quantification of the number of laser-induced CNV tufts. *p<0.05, **p<0.01, ***p<0.001, n=5 eyes/group, one-way analysis of variance.

Figure 5F shows laser-induced lesion size. *p<0.05, **p<0.01, ***p<0.001, n=5 eyes/group, one-way analysis of variance.

Figure 5G shows the % glial membrane coverage calculated by the ratio of the GFP-positive glial membrane area to the total visual field area. *p<0.05, **p<0.01, ***p<0.001, n=5 eyes/group, one-way analysis of variance.

Figure 6A shows a mouse model of laser-induced neovascularization. Intravitreal injection of 2×10 ⁹ vg/eye of AAV particles with AAVT13 as the capsid and packaged B36, B110 or B111 expression cassettes (referred to as Laser-B36, Laser-B110, Laser-B110, Laser-B111). Fourteen days after virus injection, mice were subjected to a laser-induced CNV model, and 12 days later, fluorescein angiography (FFA) was performed.

Figure 6B shows laser-induced neovascularization and scarring. Upper panel: Fluorescein angiography. Bottom: Laser-induced lesions. Scale bar: 1 mm.

Figure 6C shows the quantification of neovascularization. *p<0.05, **p<0.01, n=7 eyes/group.

Figure 6D shows quantification of laser-induced lesion area. *p<0.05, **p<0.01, n=7 eyes/group.

Figure 7A shows a flow chart of tumor transplantation in mice.

Figure 7B shows representative tumor images at day 21 after cell implantation and AAV treatment. The dotted circle marks the location of the tumor below the right forelimb.

Figure 7C shows the quantitative results of tumor size. Tumor volume was calculated by the formula V=0.5×L×W2. V: tumor volume; L: tumor length; W: tumor width. ***p<0.001, n=3 mice/group. Two-way ANOVA.

Figure 8 shows the nucleic acid sequence (SEQ ID NO: 1) encoding AAVH15 capsid protein.

Figure 9 shows the amino acid sequence of the AAVH15 capsid protein (SEQ ID NO: 2).

Figure 10 shows the nucleic acid sequence (SEQ ID NO:3) of encoding AAVT13 capsid protein.

Figure 11 shows the amino acid sequence of the AAVT13 capsid protein (SEQ ID NO: 4).

Figure 12 shows the nucleotide sequence (SEQ ID NO:5) of CB promoter.

Figure 13 shows the nucleotide sequence of bGH POLYA (SEQ ID NO: 6).

Figure 14 shows the nucleotide sequence (SEQ ID NO:7) of the B36 expression cassette.

Figure 15 shows the amino acid sequence (SEQ ID NO: 8) of the protein product of the B36 expression cassette.

Figure 16 shows the nucleotide sequence (SEQ ID NO:9) of the B110 expression cassette.

Figure 17 shows the amino acid sequence (SEQ ID NO: 10) of the protein product of the B110 expression cassette.

Figure 18 shows the nucleotide sequence (SEQ ID NO: 11) of B111 expression cassette.

Figure 19 shows the amino acid sequence (SEQ ID NO: 12) of the protein product of the B111 expression cassette.

Figure 20 shows the nucleic acid sequence (SEQ ID NO: 13) of encoding AAVXL32 capsid protein.

Figure 21 shows the amino acid sequence of the AAVXL32 capsid protein (SEQ ID NO: 14).

Figure 22 shows the codon-optimized human endostatin coding nucleic acid sequence (SEQ ID NO: 15).

Figure 23 shows the codon-optimized human angiostatin coding nucleic acid sequence (SEQ ID NO: 16).

Figure 24 shows the codon-optimized murine endostatin coding nucleic acid sequence (SEQ ID NO: 17).

Figure 25 shows the codon-optimized murine angiostatin coding nucleic acid sequence (SEQ ID NO: 18).

Figure 26 shows the original (not codon-optimized) human endostatin coding nucleic acid sequence (SEQ ID NO: 19).

Figure 27 shows the original (not codon-optimized) human angiostatin encoding nucleic acid sequence (SEQ ID NO: 20).

Figure 28 shows the original (not codon-optimized) murine endostatin encoding nucleic acid sequence (SEQ ID NO: 21).

Figure 29 shows the original (not codon-optimized) murine angiostatin encoding nucleic acid sequence (SEQ ID NO: 22).

Detailed ways

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Unless otherwise indicated, nucleic acid or polynucleotide sequences listed herein are in single-stranded form, oriented 5' to 3', left to right. Nucleotides and amino acids are presented herein using the format recommended by the IUPACIUB Biochemical Nomenclature Commission, using either the single-letter code or the three-letter code for the amino acids.

Unless otherwise stated, "polynucleotide" is synonymous with "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. A polynucleotide may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

As used herein, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including but not limited to").

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the medicaments of the present disclosure, and Humans and non-human animals (eg, rodents or other mammals) are included.

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other ape and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; experimental animals including rodents) animals such as mice, rats and guinea pigs; birds including domestic, wild and game birds such as chickens, turkeys and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

As used herein, the term "treating" includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) Ameliorating a disease, ie, causing regression of at least one of a condition, disease or disorder, or clinical or subclinical symptoms thereof.

As used herein, the term "therapeutically effective amount" refers to a dose that produces the therapeutic effect for which it is administered. For example, a therapeutically effective amount of a drug useful in the treatment of an ocular disease may be an amount capable of preventing or ameliorating one or more symptoms associated with the ocular disease.

As used herein, the term "amelioration" refers to an amelioration of a symptom associated with a disease, and may refer to an amelioration of at least one parameter that measures or quantifies the symptom.

As used herein, the term "preventing" a condition, disease or disorder includes preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a developing condition, disease or disorder in a subject, The subject may suffer from or be susceptible to the condition, disease or disorder but has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

As used herein, the term "topical administration" or "local route" refers to administration with a local effect.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process of delivering exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which involves the transfer of exogenous A source polynucleotide is introduced into a host cell.

Herein, the term "gene delivery" refers to the introduction of exogenous polynucleotides into cells for gene delivery, including targeting, binding, uptake, transport, replicon integration and expression.

As used herein, the term "gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.

As used herein, the term "infection" refers to the process by which a virus or virus particle comprising a polynucleotide component delivers a polynucleotide into a cell and produces its RNA and protein products, and may also refer to the process of virus replication in a host cell .

Herein, the term "targeting" means that the virus preferentially enters some cells or tissues, and then further expresses the viral genome or the sequence carried by the recombinant transgene in the cells.

As used herein, the term "vector" refers to one or a series of macromolecules that encapsulate a polynucleotide, which facilitates the delivery of the polynucleotide to target cells in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotides to be delivered are sometimes referred to as "expression cassettes" or "transgene cassettes," which may include, but are not limited to, certain proteins or synthetic polypeptides (which can enhance, inhibit, impair, protect, trigger, or prevent certain biological and physiological functions), coding sequences of interest in vaccine development (e.g., polynucleotides expressing proteins, polypeptides or peptides suitable for eliciting an immune response in mammals), coding sequences of RNAi materials (e.g., shRNA, siRNA, antisense oligonucleotides) or alternative biomarkers.

As used herein, the term "oligopeptide" refers to a polymer of less than 20 amino acids linked by peptide bonds. The terms "polypeptide" and "protein" are used synonymously herein to refer to polymers consisting of 20 or more amino acids. These terms also encompass synthetic or artificial amino acid polymers.

Herein, the terms "expression cassette", "transgene cassette" and "transgene expression cassette" are used interchangeably to refer to a polynucleotide fragment encoding a specific protein, polypeptide or RNAi element, which can be cloned into a plasmid vector.

In some embodiments, a "cassette" can also be packaged into an AAV particle and used as the viral genome to deliver the transgene product into target cells. The "cassette" may also include other regulatory elements, such as specific promoters/enhancers, polyA, regulatory introns, etc., to enhance or attenuate the expression of the transgene product.

In one embodiment, the transgene cassette contains a number of regulatory elements to enable packaging of the transgene into the virus, such as a normal ITR of 145 bp, a shortened ITR of approximately 100 bp in length, in addition to the sequence encoding the protein product. In some embodiments, the transgenic cassette further comprises polynucleotide elements for controlling the expression of the protein product, such as an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), or a 2A signal (e.g., P2A, T2A, F2A), promoters and enhancers (e.g., CMV promoters with vertebrate β-actin, β-globin, or β-globin regulatory elements or other hybrid CMV promoters (termed CB and CAG promoters) , EF1 promoter, hypoxia response element, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter). Promoters and enhancers can be activated by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific time points. Furthermore, promoters and enhancers may be natural or artificial or chimeric sequences, ie prokaryotic or eukaryotic sequences.

In some preferred embodiments, the inducible regulatory element for gene expression may be a tissue or organ-specific promoter or enhancer, including but not limited to: promoters specific to various types of retinal cells, such as , ganglion cell-specific promoters (e.g., Tuj1 promoter), astrocyte- and Müller cell-specific promoters (e.g., GFAP or vimentin promoters), and retinal pigment epithelium-specific promoters (e.g., RPE65 or VMD2 promoters); specific promoters for various types of ocular neurons (e.g., synapsin, VGAT, DAT, TH promoters); and specific promoters for the osteoblast lineage (e.g., osteocalcin promoter), Liver, pancreas, spleen, and lung cancer cell-specific promoters.

As used herein, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and acts as a cis element to mediate viral replication, packaging and integration. ITRs herein include, but are not limited to, terminal repeats from AAV types 1-11 (avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). Furthermore, the AAV terminal repeats need not have native terminal repeats, so long as the terminal repeats are available for viral replication, packaging and integration.

As used herein, the term "cis-element" refers to a transgene cassette packaged in an AAV particle and expressed in target cells to produce a therapeutic protein product.

As used herein, the term "codon-optimized" refers to a polynucleotide sequence modified from its native form. Such modifications result in a difference of one or more base pairs, with or without a change in the corresponding amino acid sequence, which may enhance or inhibit gene expression and/or cellular response to the modified polynucleotide sequence.

Those skilled in the art know that the AAV capsid protein contains VP1, VP2 and VP3 proteins, and the VP2 and VP3 proteins undergo transcription and translation at the start codon inside the VP1 protein, that is, the VP1 sequence contains the VP2 and VP3 sequences. The present disclosure provides the amino acid sequence of the VP1 protein of the AAV capsid.

In one embodiment, the AAV capsid protein can be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). AAV capsid protein) and other engineered AAV capsid proteins (eg, engineered capsid proteins of types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). The genome sequences, ITR sequences, Rep and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, such as the GenBank database.

In one embodiment, the present disclosure provides therapeutic tools with anti-angiogenic effects that can be used to treat various diseases with associated pathological mechanisms, including but not limited to: neovascular retinopathy (eg, AMD, ROP, DR ) and eye damage caused by strong light or other causes. In addition, the therapeutic tools of the present disclosure can also treat various types of cancer in which angiogenesis promotes tumor growth and metastasis, such as lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer Carcinoma, leukemia, lymphoma, melanoma, and glioblastoma.

In one embodiment, the protein product of the therapeutic tool (e.g., a transgene expression cassette) includes an anti-angiogenic protein such as, but not limited to, Aflibercept, a recombinant VEGF soluble receptor (manufactured by Rengeron Pharmaceuticals, which inhibits neovascularization) , anti-VEGF antibodies (such as bevacizumab, ranibizumab, and blocizumab), other anti-angiogenic proteins or polypeptides (such as endostatin, angiostatin, platelet factor 4, pigment epithelium-derived factor), adult Fibroblast growth factor (FGF) inhibitor, metalloproteinase inhibitor BB94.

In one embodiment, the protein product of the therapeutic tool (e.g., a transgene expression cassette) also includes anti-tumor antibodies, such as anti-PD-1 antibodies (e.g., Nivolumab, Pembrolizumab, Cemiplimab) and PD-L1 antibodies (e.g., Avelumab, Atezolizumab), anti-CTLA-4 antibodies (eg Ipilimumab), anti-CGRP antibodies (eg Fremanezumab, Galcanezumab, Erenumab), anti-HER2 antibodies (eg Trastuzumab, Pertuzumab) and anti-EGFR antibodies (eg Cetuximab, Panitumumab, Necitumumab).

In some embodiments, AAV virions with AAVH15 capsid protein (SEQ ID NO: 2) exhibit more efficient retinal transduction efficiency than wild-type (WT) serotypes and are suitable for expressing anti-angiogenic proteins transmission of genes.

In some embodiments, compared with WT serotype, AAV virus particles with AAVT13 capsid protein (SEQ ID NO: 4) exhibit more efficient retinal transduction efficiency and are suitable for the delivery of genes expressing anti-angiogenic proteins .

In some embodiments, AAV virions with the AAVXL32 capsid protein (SEQ ID NO: 14) exhibit higher retinal transduction efficiency than wild-type (WT) serotypes and are suitable for expressing anti-angiogenic proteins transmission of genes.

In one embodiment, the transgene expression cassette encoding an angiogenesis inhibitor comprises a CB promoter sequence (SEQ ID NO: 5), a bGH polyadenylation (polyA) sequence (SEQ ID NO: 6) and codon-optimized human Source endostatin sequence (SEQ ID NO: 15), this codon-optimized human endostatin sequence has an N-terminal ALB signal peptide and an inserted intron within the coding sequence to enhance protein expression, thus forming B36 expression Cartridge (SEQ ID NO: 7). The B36 expression cassette is flanked by a normal ITR and a shortened ITR to enable packaging of the B36 expression cassette into AAV particles as a self-complementary AAV vector.

In one embodiment, the transgene expression cassette encoding an angiogenesis inhibitor comprises a CB promoter sequence (SEQ ID NO: 5), a bGH polyA sequence (SEQ ID NO: 6), a codon-optimized Human endostatin and codon-optimized human angiostatin sequences (SEQ ID NO: 15 and SEQ ID NO: 16), the coding sequences of these two proteins are passed through the Furin protease sequence (Lys-Arg-Lys-Arg- Arg)+connecting peptide (Ser-Gly-Ser-Gly)+F2A sequence connection, thus forming B110 expression cassette (SEQ ID NO: 9). The B110 expression cassette also contains two ITRs that allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.

In one embodiment, the transgene expression cassette encoding an angiogenesis inhibitor comprises a CB promoter sequence (SEQ ID NO: 5), a bGH polyA sequence (SEQ ID NO: 6), a codon-optimized Murine endostatin and codon-optimized mouse angiostatin sequences (SEQ ID NO: 17 and SEQ ID NO: 18), the coding sequences of these two proteins are passed through the Furin protease sequence (Lys-Arg-Lys-Arg- Arg)+connecting peptide (Ser-Gly-Ser-Gly)+P2A sequence connection, thus forming B111 expression cassette (SEQ ID NO: 11). The B111 expression cassette also contains two ITRs that allow packaging of the expression cassette into AAV virions as single-chain AAV vectors.

In some embodiments, anti-angiogenic AAV particles are produced by triple-plasmid (plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid) transfection of HEK293 cells.

In one embodiment, to generate therapeutically functional AAV particles, three-plasmid transfection of HEK293 cells is performed as follows: plasmid 1: cis-element plasmid with ITR (e.g., B36, B110, and B111 expression cassettes); plasmid 2 : AAV Rep/Cap plasmid with coding sequences for capsid proteins (e.g., AAVH15, AAVT13, and AAVXL32 capsid proteins); Plasmid 3: a helper plasmid with adenoviral components that facilitates replication, assembly, and packaging of AAV virions. In one embodiment, AAV particles produced by HEK293 cells are purified by affinity chromatography and iodixanol density gradient ultracentrifugation (Xiao X et al., J Virol (1998) 72(3):2224-32).

Those skilled in the art can use known standard methods to produce recombinant and synthetic polypeptides or proteins thereof, design nucleic acid sequences, produce transformed cells, construct recombinant AAV mutants, transform capsid proteins, package and express AAV Rep and/or Cap sequences vector, and transiently or stably transfect packaging cells. These techniques are known to those skilled in the art. See, eg, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, (Cold Spring Harbor, NY, 1989).

In some embodiments, the gene delivery systems of the present disclosure are used to aid in cell transplantation therapy. Specifically, AAV particles with transgenes can be used to transduce various types of cells in vitro to generate stable cell lines expressing protein products, which can then be introduced in vivo for therapeutic purposes. Types of cells include, but are not limited to, endothelial cells, myoblasts, fibroblasts, astrocytes, Müller cells, oligodendrocytes, microglia, rods and cones, neurons, hematopoietic Stem cells, monocytes, granulocytes, lymphocytes, osteoclasts and macrophages.

In one embodiment, the cells used for transplantation are autologous cells of the subject, which allow for in vitro culture. The principles and techniques of introducing or transplanting cells into a subject are known to those skilled in the art.

In one embodiment, AAV particles are harvested from the culture medium and lysates of HEK293 cells. Purification methods such as affinity chromatography, ion exchange chromatography, cesium chloride and iodixanol gradient ultracentrifugation. Chemicals or reagents related to AAV production and purification include, but are not limited to: Chemicals or reagents used in cell culture (e.g., components of cell culture media including bovine, equine, goat, chicken or other vertebrate serum, glutamine Amides, glucose, sucrose, sodium pyruvate, phenol red; antibiotics such as penicillin, kanamycin, streptomycin, tetracycline); chemicals or reagents for cell lysis, polynucleotide precipitation, or ultracentrifugation (such as Triton X-100, NP-40, sodium deoxycholate, sodium lauryl sulfate, domiphene bromide, sodium lauryl salicylate, sodium chloride, magnesium chloride, calcium chloride, barium chloride, nitric acid Salt, potassium chloride, ammonium chloride, ammonium persulfate, ammonium sulfate, PEG-20, PEG-40, PEG-400, PEG-2000, PEG-6000, PEG-8000, PEG-20000, Tris-HCl, Tris - acetate, manganese chloride, phosphate, bicarbonate, cesium chloride, methanol, ethanol, glycerol, iodixanol, isopropanol, butanol, benzoin enzyme, DNase I, RNase); affinity column Materials (e.g., AAVX affinity resins, heparan sulfate proteoglycan and mucin resins, other materials related to AAV-specific antibodies); acids, bases, and organics (e.g., hydrochloric acid, sulfuric acid) contained in ion-exchange chromatography materials and wash buffers , acetic acid, formic acid, nitric acid, urea, acetone, chloroform, acetonitrile, trifluoroacetic acid, sodium hydroxide, potassium hydroxide, barium hydroxide, ammonium hydroxide, Tris base or other organic amines, poloxamer 188, Tween 20, Tween 40, Tween 80, guanidine hydrochloride).

In one embodiment, the protein product encoded by the transgene cassette is linked to an oligopeptide tag (eg, Flag, 6xHis, 2xHA, Myc), which facilitates the purification of the protein product. Those of skill in the art understand the techniques and procedures involved in protein purification. Briefly, transgenic plasmids were transfected into eukaryotic cells (eg, HEK293 and CHO cells), and then the target protein was collected by affinity chromatography. For example, Flag-M2 resin beads are often used to specifically attract Flag-tagged proteins, which are then eluted with 3×Flag soluble oligopeptides. It is also possible to use nickel nitrilotriacetate (Ni-NTA) columns for reversible binding followed by exclusive purification of 6×His-tagged proteins.

In one embodiment, the AAV vectors of the present disclosure can be loaded with exogenous polynucleotides for gene delivery into target cells. Thus, the AAV vectors of the present disclosure can be used to deliver nucleic acids to cells in vitro or in vivo.

In one embodiment, the exogenous polynucleotide delivered by the AAV vector encodes a polypeptide that acts as a reporter (ie, a reporter protein). A reporter protein is used to indicate cells successfully infected by AAV. These reporter proteins include, but are not limited to, green fluorescent protein (GFP), β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.

In one embodiment, the exogenous polynucleotide delivered to the target cell by the AAV vector encodes a native protein for therapeutic use, either codon-optimized or non-codon-optimized.

In one embodiment, the exogenous polynucleotide delivered by the AAV vector to the target cell encodes a synthetic polypeptide.

In one embodiment, the AAV vector or transgene expression cassette or gene delivery system of the present disclosure is made into pharmaceutical preparations (eg, injections, tablets, capsules, powders, eye drops) and administered to humans or other mammals. The pharmaceutical preparation also contains other ingredients such as pharmaceutical excipients, water-soluble or organic solvents (such as water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (such as sodium chloride, chloride Potassium, Phosphate, Acetate, Bicarbonate, Tris-HCl and Tris-Acetate), Delaying Dissolving Agents (e.g. Paraffin), Surfactants, Antimicrobials, Liposomes, Lipoplexes, Immuno Inhibitors (eg, cortisone, prednisone, cyclosporine), nonsteroidal anti-inflammatory drugs (NSAIDs, eg, aspirin, ibuprofen, acetaminophen) microspheres, rigid matrices, semisolid carriers, Nanospheres or nanoparticles. In addition, pharmaceutical formulations can be administered by inhalation, systemic or topical (e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal) The drug is delivered in single or multiple doses.

In one embodiment, the present disclosure provides a medicament comprising the AAV vector or transgene expression cassette or gene delivery system of the present disclosure and excipients. The medicaments of the present disclosure can be used to transduce cells in vitro or mammals (such as rodents, primates and humans) in vivo to treat various diseases, such as eye diseases.

Herein, the eye disease is selected from the group consisting of: hereditary dystrophies of the retina, glaucoma, glaucomatous neuropathy, age-related macular degeneration, refractive error, dry eye, hereditary dystrophies of ocular inflammation, ocular inflammation, Uveitis, orbital inflammation, cataract, allergic conjunctivitis, diabetic retinopathy, macular edema, corneal edema, keratoconus, proliferative vitreoretinopathy (fibrosis), periretinal fibrosis, central serous chorioretinopathy, Vitreoretinopathy, vitreomacular traction, and vitreous hemorrhage. In one embodiment, the ocular disease involves degeneration of eye and/or visual function.

In one embodiment, treating an ocular disorder refers to improving visual acuity, contrast vision, color vision, and visual field in the treated patient.

The present disclosure will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are only for illustrating the present disclosure and are not intended to limit the scope of the present disclosure. The experimental methods without specific conditions indicated in the examples were operated according to conventional conditions known in the art, or according to the conditions suggested by the manufacturer.

Example

Example 1: Retinal affinity of AAVH15, AAVXL32 and AAVT13

By replacing the N-terminal region of VP1, VP2 (amino acid 1-203) of AAV8 with the N-terminal region of AAV5 (amino acid 1-192) (with point mutation site G257R) and connecting to the AAV5 capsid protein sequence, construct AAVT13 capsid (SEQ ID NO: 4). Perform DNA shuffling experiments to construct AAVH15 capsid protein (SEQ ID NO: 2) and AAVXL32 capsid protein (SEQ ID NO: 14).

Using WT serotypes AAV5, 8, and 9 as controls, AAV particles expressing GFP protein were injected into the vitreous of C57BL/6 mice to study the retinal affinity of the improved AAV serotypes (AAVH15, AAVXL32, and AAVT13). At a dose of 2×10 ⁹ vg/eye, each AAV serotype carrying GFP gene was injected intravitreally into C57BL/6 mice. The GFP signal images taken 3 weeks after injection are shown in Figure 1A. At a dose of 2×10 ⁹ vg/eye, AAV particles carrying GFP gene were injected into the vitreous of C57BL/6 mice. Three weeks after the injection, the retinal cross-sections were taken out for immunofluorescence staining, and the results are shown in Figure 2.

The results showed that the retinal GFP fluorescence levels in AAVH15 and AAVT13 groups were significantly higher than those in AAV5, 8, and 9 groups (Fig. 1A), about 10-12 times higher (Fig. 1C, ***p<0.001, n=4 eyes/group) . The retinal GFP fluorescence level in the AAVXL32 group was also significantly higher than that in the AAV5, 8, and 9 groups (Fig. 1A), about 5 times higher (Fig. 1C, ***p<0.001, n=4 eyes/group). Thus, AAVH15, AAVXL32, and AAVT13 had significantly increased retinal affinity and transduction efficiency compared with wild-type AAV.

Furthermore, despite intravitreal administration, the modified serotypes AAVH15 and AAVT13 were able to diffuse into the photoreceptor layer and even the retinal pigment epithelium (RPE) (Fig. 1B and Fig. 2), significantly better than AAV5, 8, and 9. It can be seen that serotypes AAVH15 and AAVT13 have stronger retinal affinity and can deliver genes to retinal layers.

Example 2: Expression of codon-optimized human and murine endostatin-encoding nucleic acid sequences

In this example, the inventors compared B110 and B111 containing codon-optimized endostatin-encoding nucleic acid sequences with plasmids constructed from non-codon-optimized endostatin-encoding nucleic acid sequences (also carrying Flag and HA tags) protein expression ability.

HEK293 cells were transfected with B110 and B111 plasmids, and the expression of endostatin in cell lysates was detected by Western blot. Human and mouse endostatin without codon optimization were constructed into plasmids as control groups (original human source, original mouse source). The endostatin protein expression of each group relative to the original human group was quantitatively counted.

As shown in Figure 3C, the endostatin protein expression levels of B110 and B111 expression cassettes were significantly higher than those of the control group without codon optimization. Quantitative statistical results of relative protein expression levels showed that the endostatin protein expression levels of the B110 and B111 expression cassettes were 4-6 times higher than those of sequences without codon optimization (Fig. 3C, right panel). The above results show that the codon-optimized human and mouse endostatin coding nucleic acid sequences of the present disclosure have significant advantages in expression compared with the original natural protein coding sequences.

Example 3: Expression of codon-optimized human and mouse angiostatin-encoding nucleic acid sequences

In this example, the inventors compared B110 and B111 containing codon-optimized angiostatin-encoding nucleic acid sequences with plasmids constructed from non-codon-optimized angiostatin-encoding nucleic acid sequences (also carrying Flag and HA tags) protein expression ability.

HEK293 cells were transfected with B110 and B111 plasmids, and the expression of angiostatin in cell lysates was detected by Western blot. Angiostatin of human and mouse origin without codon optimization was constructed into the plasmid as a control group (original human origin, original mouse origin). Quantitative statistics of angiostatin protein expression in each group relative to the original human group.

As shown in Figure 3D, the angiostatin protein expression levels of B110 and B111 expression cassettes were significantly higher than that of the control group without codon optimization. Quantitative statistical results of relative protein expression levels showed that the expression levels of angiostatin proteins in the B110 and B111 expression cassettes were about 4 times higher than those of sequences without codon optimization (Fig. 3D, right panel). The above results show that the codon-optimized human and mouse angiostatin coding nucleic acid sequences of the present disclosure have significant advantages in expression compared with the original natural protein coding sequences.

Example 4: Novel transgenic expression cassette expressing endostatin and angiostatin

For more stable expression of anti-angiogenic polypeptides, self-complementary and single-chain AAVs were used. As shown in Figure 3A, the B36 transgene cassette was constructed by adding the ALB signal peptide to the N-terminal of the codon-optimized human endostatin sequence and inserting an intron inside it. sequence enhances the expression and secretion of the endostatin protein. The CB promoter is used to promote protein expression, and the bGH polyA sequence is used to stop mRNA transcription. The B36 transgene cassette is flanked by a normal ITR and a shortened ITR, so that the expression cassette can be packaged into AAV particles as a self-complementary AAV vector.

In addition, single-chain AAV transgene expression cassettes B110 and B111 were designed to simultaneously express endostatin and angiostatin and simultaneously release them out of cells. As shown in Figure 3A, the B110 cassette includes: two normal ITR sequences, a CB promoter (SEQ ID NO: 5), a codon-optimized human endostatin sequence (SEQ ID NO: 15), a codon-optimized Human angiostatin sequence (SEQ ID NO: 16) and bGH polyA tail (SEQ ID NO: 6). The SP signal peptide is used to control the extracellular secretion of endostatin and angiostatin. The coding sequences of the two proteins were joined by a linker containing the Furin protease sequence (KRKRR)+linker (SGSG)+F2A sequence to better separate the expression of endostatin and angiostatin during translation. In addition, endostatin and angiostatin have Flag tags and 2×HA tags, so endostatin and angiostatin can be prepared and purified based on tag-dependent affinity chromatography. For example, flag-tagged proteins can be purified using commercial Flag M2 magnetic beads (Sigma-Aldrich, Cat. No. M8823).

Similarly, the B111 expression cassette was constructed in a manner similar to B110, except that codon-optimized murine endostatin (SEQ ID NO: 17) and codon-optimized murine angiostatin (SEQ ID NO: 18) were used. ), and to better separate the expression and secretion of the two proteins, F2A was replaced by P2A.

B110 and B111 plasmids were transfected into HEK293 cells and Huh7 cells. After 48 hours, the expression levels of endostatin protein and angiostatin protein in the medium were detected by Western blot. Results As shown in FIG. 3B , both B110 and B111 expression cassettes achieved significant expression of endostatin protein and angiostatin protein. It can be seen that endostatin and angiostatin were successfully expressed and secreted by transfecting B110 and B111 plasmids, and the B110 and B111 expression cassettes realized the simultaneous stable expression of these two proteins.

Example 5: Inhibitory effect of novel AAV-mediated delivery of anti-angiogenic proteins on HUVEC tube formation

Start the anti-angiogenic AAV particle production process by three-plasmid transfection of B36, B110, B111 cis-element plasmid, AAVH15 capsid plasmid, and adenovirus helper plasmid with 0.2-5 mg/ml PEI and use cell culture medium volume quartered One of the 10-35g/L CDM4 solution was terminated. HEK293 cells were lysed 40-80 hours after transfection and polynucleotides were removed. Centrifuge at 4000-15000 rpm for 15-45 minutes. The supernatant with AAV particles was loaded on an AAVX affinity column and then eluted. The eluted AAV particles were subjected to iodixanol gradient ultracentrifugation and concentrated to a certain volume for use.

The transduction efficiency of AAVH15 in HUVEC cells was tested. According to the expression of GFP protein, about 87% of HUVEC cells were infected by AAVHH15 when the MOI was 1×10 ⁵ vg/cell ^; The conductance ratio dropped to 45.8% (Figure 4A and Figure 4B). When the MOI was reduced to 1×10 ³ vg/cell, about 10.9% of HUVEC cells showed obvious GFP fluorescence signal.

As shown in Figure 4C, AAVH15-transduced HUVEC cells successfully released endostatin and angiostatin into the conditioned medium.

HUVEC cells were infected with AAVH15 carrying B36, B110 and B111 expression cassettes (referred to as H15-B36, H15-B110 and H15-B111, respectively) at MOIs of 1×10 ⁵ and 1×10 ⁴ vg/cell. Cells were harvested and transferred to Matrigel-coated 24-well plates and pre-incubated for 45 minutes to form tubes. Images were taken 6 hours later, and the results are shown in Figure 4D. Tube length and number of branch points per unit area (mm ² ) were analyzed by Image J. The results showed that compared with the control group (H15-GFP), the tube length and the number of branch points of H15-B36, H15-B110 and H15-B111 infected cells were significantly reduced (Fig. 4E and Fig. 4F). The above results indicated that H15-B36, H15-B110 and H15-B111 viral vectors could inhibit HUVEC tube formation.

Example 6: AAV-mediated expression of endostatin and angiostatin inhibits retinal neovascularization and glia Plastid cell hyperplasia

C57BL/6 mice were injected intravitreally with 2×10 ¹⁰ vg/eye of AAV particles with H15 capsids and packaged B36, B110 or B111 expression cassettes (referred to as Laser-B36, Laser-B110, Laser-B110, Laser-B111). As shown in Figure 5A, mice were subjected to a laser-induced CNV model 14 days after virus injection, followed by fluorescein angiography (FFA) and immunofluorescence (IF) an additional 12 days later.

The results showed that after AAVH15-mediated endostatin and angiostatin treatment, the size of laser-induced CNV clusters was reduced by about 75% (shown in Figure 5B and Figure 5D), and the number of CNV clusters was also reduced (Fig. 5B and 5E). Meanwhile, AAVH15-mediated inhibition of neovascularization accelerated the recovery of laser-induced lesions. After 12 days of laser-induced retinal damage, the lesion volume in the different treatment groups was reduced by about 50-75% compared with the untreated control group (laser model) (Fig. 5B and Fig. 5F). To further investigate cellular mechanisms, retinas were co-stained with the vascular marker IB4 and the retinal glial cell marker GFAP. In healthy retinas, blood vessels were surrounded by astrocytes or Müller cells, showing a clear network (Fig. 5C). After laser-induced injury, activated retinal glia, including astrocytes and Müller cells, accumulate to form glial scar membranes with tight affinity to CNV clusters, whereas AAVH15-mediated endostatin and vascular The release of statins significantly improved retinal conditions. As shown in Figure 5G, the area covered by the glial cell membrane decreased sharply from 29.9% to about 12% (***p<0.001), indicating that the proliferation of glial cells was inhibited. Thus, neovascularization and retinal gliosis can be attenuated by treatment with AAV with H15 capsid and packaged B36, B110 or B111 expression cassettes.

In addition, the therapeutic effect of low-dose AAV particles was also investigated. 2×10 ⁹ vg/eye of AAV particles (with AAVT13 capsid and packaging B36, B110 and B111 transgene expression cassettes) were injected intravitreally into C57BL/6 mice. As shown in Figure 6A, mice were subjected to a laser-induced CNV model 14 days after virus injection, followed by fluorescein angiography (FFA) and immunofluorescence (IF) an additional 12 days later.

The results showed that, as shown in Figure 6B to Figure 6D, smaller CNV clusters and laser-induced lesions were observed in the B36 and B111-treated groups compared to the untreated control group (laser model) (*p<0.05, **p<0.01, n=7 eyes/group), indicating that AAVT13-mediated expression of endostatin and angiostatin at low doses ameliorated neovascularization and lesion-induced scarring.

Example 7: Inhibitory effect of AAV-mediated expression of endostatin and angiostatin on tumor growth

In order to study the effect of AAV gene delivery system on cancer therapy, Hepa1-6 murine liver cancer cells were subcutaneously injected into CByJ. AAVH15 of B36 and B111 transgene cassettes (H15-B36 and H15-B111). As shown in Figure 7A, Hepa1-6 cells (ATCC CRL-1830) grown in culture dishes were digested with 0.05% trypsin, centrifuged at 800 rpm for 5 minutes, and then resuspended in PBS for mouse injection. A mixture of 2×10 ⁶ Hepa1-6 cells and 1×10 ¹¹ vg of AAVH15 (packaging B36 and B111 expression cassettes, respectively) was injected subcutaneously into CByJ.Cg-Foxn1nu/J mice (Jackson Laboratories Stock No. 000711). Tumor size was measured at 7, 14 and 21 days after implantation. Mice injected with Hepa1-6 cells and AAV encoding GFP (AAVH15) were used as controls.

As observed in Figure 7B and Figure 7C, at day 14 after cancer cell implantation and H15-B36 and H15-B111 treatment, the tumor volumes of the B36 and B111 treated groups had been significantly reduced compared with the control mice. Small. On the 21st day after treatment, the mean tumor volumes of the B36 and B111 treatment groups were 211.3 and 75.9 mm ² , respectively, which were less than one-tenth of the control group (the mean tumor volume was 2240 mm ² ). The above results indicated that AAVH15-mediated expression of endostatin and angiostatin had a significant inhibitory effect on tumor growth.

Although the present disclosure has been illustrated and described with reference to certain preferred embodiments of the present disclosure, those skilled in the art should understand that the above content is a further detailed description of the present disclosure in conjunction with specific embodiments, and cannot be deemed The specific implementation of the present disclosure is limited only by these descriptions. Those skilled in the art may make various changes in form and details, including several simple deduction or substitutions, without departing from the spirit and scope of the present disclosure.

Claims (27)

1. AAV capsid protein, wherein, the amino acid sequence of the AAV capsid protein has at least 50%, 60%, 70% or 80% identity; preferably, the amino acid sequence of the AAV capsid protein has at least 85%, 90%, 95% with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14 %, 99% or 100% identity.
2. The AAV capsid protein according to claim 1, wherein the AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14.
3. The AAV capsid protein according to claim 1, wherein the amino acid sequence of the AAV capsid protein is as shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14.
4. A nucleic acid molecule encoding the AAV capsid protein of any one of claims 1 to 3.
5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence of the nucleic acid molecule has at least 80% of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13 Preferably, the nucleotide sequence of the nucleic acid molecule has at least 85%, 90%, 95% of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13 %, 99% or 100% identity.
6. The nucleic acid molecule according to claim 4, wherein said nucleic acid molecule comprises a nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13; preferably, said nucleic acid molecule The nucleotide sequence is shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 13.
7. A nucleic acid molecule encoding endostatin whose nucleotide sequence has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17, preferably at least 85%, 90%, 95% %, 99% or 100% identity.
8. The nucleic acid molecule encoding endostatin according to claim 7, wherein said nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17.
9. The nucleic acid molecule encoding endostatin according to claim 7, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 15 or SEQ ID NO: 17.
10. A nucleic acid molecule encoding angiostatin whose nucleotide sequence has at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18, preferably at least 85%, 90%, 95% %, 99% or 100% identity.
11. The nucleic acid molecule encoding angiostatin according to claim 10, wherein said nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18.
12. The nucleic acid molecule encoding angiostatin according to claim 10, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 16 or SEQ ID NO: 18.
13. A transgenic expression cassette comprising: a promoter, the nucleic acid molecule encoding endostatin described in any one of claims 7 to 9 and/or the nucleic acid molecule encoding angiostatin described in any one of claims 10 to 12 Molecule, bGH polyA.
14. The transgenic expression cassette according to claim 13, wherein the promoter is selected from the group consisting of: CB promoter, CAG promoter, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16, VP64 promoter , Tuj1 promoter, GFAP promoter, vimentin promoter, RPE65 promoter, VMD2 promoter, synapsin promoter, VGAT promoter, DAT promoter, TH promoter and osteocalcin promoter; preferably, all The above-mentioned promoter is the CB promoter.
15. The transgene expression cassette according to claim 13 or 14, wherein the transgene expression cassette further comprises: a signal peptide, such as an SP signal peptide, an ALB signal peptide and a PLS signal peptide; and/or two ITRs located at both ends, the The two ITRs are each independently a normal ITR or a shortened ITR peptide.
16. The transgenic expression cassette according to any one of claims 13 to 15, wherein the nucleic acid molecule encoding endostatin and/or the nucleic acid molecule encoding angiostatin has an oligopeptide tag, such as Flag, 6 ×His, 2×HA and Myc.
17. The transgenic expression cassette according to any one of claims 13 to 16, wherein the transgenic expression cassette comprises the nucleic acid molecule encoding endostatin according to any one of claims 7 to 9 and any one of claims 10 to 12 The nucleic acid molecule encoding angiostatin according to any one of the above.
18. The transgene expression cassette according to claim 17, wherein the transgene expression cassette further comprises a connecting sequence; preferably, the connecting sequence is a Furin protease sequence+connecting peptide+2A sequence, such as P2A, T2A or F2A.
19. The transgenic expression cassette according to any one of claims 13 to 18, wherein the nucleotide sequence of the transgenic expression cassette is as shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.
20. A gene delivery system, comprising: the AAV capsid protein according to any one of claims 1 to 3 and the transgene expression cassette according to any one of claims 13 to 19.
21. Application of the AAV capsid protein according to any one of claims 1 to 3 and/or the transgene expression cassette according to any one of claims 13 to 19 in the preparation of medicines for treating diseases.
22. According to the application of claim 21, the disease takes new blood vessels as the main pathological mechanism or inducing factor; preferably, the disease is an eye disease, such as age-related macular degeneration, diabetic retinopathy, and other diseases caused by strong light Retinal diseases such as retinal damage; preferably, the disease is cancer, such as lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, Tumors and glioblastomas.
23. medicines, which contain:

    The AAV capsid protein according to any one of claims 1 to 3 and/or the transgenic expression cassette according to any one of claims 13 to 19; and

    Optional excipients.
24. The medicine according to claim 23, wherein the medicine is an angiogenesis inhibitor.
25. A method for treating retinal diseases or cancers, comprising administering a therapeutically effective amount of the drug of claim 23 or 24 to a subject in need.
26. The method according to claim 25, wherein the retinal diseases include senile macular degeneration, diabetic retinopathy, and retinal damage caused by strong light; the cancers include lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, Kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, and glioblastoma.
27. The method according to claim 25 or 26, wherein the drug is administered to the subject in need by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, topical contact, Intraperitoneal and intralesional administration, preferably topically to the eye, for example by intravitreal, subretinal or suprachoroidal injection.

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