WO2023005906A1 - Nouvel inhibiteur de l'angiogenèse à médiation par le sérotype du virus adéno-associé et son application - Google Patents

Nouvel inhibiteur de l'angiogenèse à médiation par le sérotype du virus adéno-associé et son application Download PDF

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WO2023005906A1
WO2023005906A1 PCT/CN2022/107847 CN2022107847W WO2023005906A1 WO 2023005906 A1 WO2023005906 A1 WO 2023005906A1 CN 2022107847 W CN2022107847 W CN 2022107847W WO 2023005906 A1 WO2023005906 A1 WO 2023005906A1
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seq
nucleic acid
promoter
acid molecule
expression cassette
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PCT/CN2022/107847
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English (en)
Chinese (zh)
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赵锴
肖啸
陈晨
杜增民
蒋威
吴侠
郑静
薛华清
王慧
赵阳
王天翼
田善水
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上海信致医药科技有限公司
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Priority claimed from CN202110872927.4A external-priority patent/CN113563430B/zh
Priority claimed from CN202110874563.3A external-priority patent/CN113584043B/zh
Priority claimed from CN202110874960.0A external-priority patent/CN113564187A/zh
Priority claimed from CN202110874776.6A external-priority patent/CN113480615B/zh
Application filed by 上海信致医药科技有限公司 filed Critical 上海信致医药科技有限公司
Publication of WO2023005906A1 publication Critical patent/WO2023005906A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • 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.
  • AMD age-related macular degeneration
  • AMD choroidal neovascularization
  • CNV choroidal neovascularization
  • ROP retinopathy of prematurity
  • neovascularization is induced during tumorigenesis to facilitate the supply of oxygen and nutrients to cancer cells.
  • neovascularization can be induced from pre-existing vascular networks and infiltrate tumor tissue;
  • tumor cells can recruit endothelial progenitor cells to form secondary vasculature;
  • 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).
  • VEGF Vascular endothelial growth factor
  • Endostatin is a 20kD fragment produced from the C-terminus of collagen XVIII
  • 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.
  • 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.
  • 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.
  • angiogenesis inhibitors are 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.
  • 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.
  • the amino acid sequence of the AAV capsid protein has at least 85%, 90%, 95%, 99%, or 100% identity.
  • the AAV capsid protein comprises the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 14.
  • 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.
  • the present disclosure provides a nucleic acid molecule encoding the AAV capsid protein according to the first aspect.
  • 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.
  • 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.
  • the present disclosure provides an AAV vector comprising the AAV capsid protein according to the first aspect.
  • the AAV vector further comprises an exogenous polynucleotide comprising a nucleotide sequence encoding a Therapeutic protein.
  • the therapeutic protein is an anti-angiogenic protein.
  • the therapeutic protein is endostatin and/or angiostatin.
  • 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.
  • 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.
  • 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.
  • the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 15 or SEQ ID NO: 17.
  • the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 15 or SEQ ID NO: 17.
  • 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.
  • the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 18.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • a signal peptide such as an SP signal peptide, an ALB signal peptide, and a PLS signal peptide
  • 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.
  • the transgenic expression cassette includes the nucleic acid molecule according to the fourth aspect and the nucleic acid molecule according to the fifth aspect.
  • the transgene expression cassette further includes a linker sequence.
  • 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.
  • nucleotide sequence of the transgene expression cassette is shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.
  • the present disclosure provides a gene delivery system comprising: an AAV capsid protein and a transgene expression cassette.
  • 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.
  • 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.
  • the AAV capsid protein in the gene delivery system of the present disclosure is the AAV capsid protein according to the first aspect.
  • 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.
  • the transgene expression cassette in the gene delivery system of the present disclosure is the transgene expression cassette according to the sixth aspect.
  • the nucleotide sequence of the transgene expression cassette is shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.
  • 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.
  • endostatin and/or angiostatin anti-angiogenic proteins
  • 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.
  • 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.
  • the disease has angiogenesis as the main pathological mechanism or inducing factor.
  • the disease is an ocular disease such as a retinal disease or cancer.
  • the present disclosure provides a medicament comprising: the gene delivery system according to the seventh aspect; and an excipient.
  • 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.
  • the disease is an eye disease, such as age-related macular degeneration, diabetic retinopathy, and other retinal diseases caused by strong light.
  • 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.
  • 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.
  • the drug is administered by systemic or local routes, such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, and intralesional.
  • systemic or local routes such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal, and intralesional.
  • the drug is administered topically to the eye, for example by intravitreal injection, subretinal injection or suprachoroidal injection.
  • 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.
  • FIG. 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.
  • 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.
  • FIG. 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.
  • MOI multiplicity of infection
  • 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 ⁇ m.
  • 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).
  • B36 B110 or B111 expression cassettes
  • FFA fluorescein angiography
  • IF immunofluorescence
  • 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 ⁇ m.
  • 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.
  • FFA fluorescein angiography
  • Figure 6B shows laser-induced neovascularization and scarring.
  • Upper panel Fluorescein angiography.
  • Bottom Laser-induced lesions.
  • Scale bar 1 mm.
  • 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.
  • FIG. 7C shows the quantitative results of tumor size.
  • ***p ⁇ 0.001, n 3 mice/group.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.).
  • the subject is a mammal. In one embodiment, the subject is a human.
  • 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.
  • a therapeutically effective amount refers to a dose that produces the therapeutic effect for which it is administered.
  • 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.
  • 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.
  • 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.
  • topical administration or “local route” refers to administration with a local effect.
  • transduction refers 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.
  • gene delivery refers to the introduction of exogenous polynucleotides into cells for gene delivery, including targeting, binding, uptake, transport, replicon integration and expression.
  • gene expression refers to the process of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.
  • 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 .
  • 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.
  • 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.
  • 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.
  • expression cassettes or “transgene cassettes”
  • coding sequences of interest in vaccine development e.g., polynucleotides expressing proteins, polypeptides or peptides suitable for eliciting an immune response in mammals
  • RNAi materials e.g., shRNA, siRNA, antisense oligonucleotides
  • oligopeptide refers to a polymer of less than 20 amino acids linked by peptide bonds.
  • 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.
  • expression cassette refers to a polynucleotide fragment encoding a specific protein, polypeptide or RNAi element, which can be cloned into a plasmid vector.
  • 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.
  • 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.
  • 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 ⁇ -actin, ⁇ -globin, or ⁇ -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).
  • 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 enhancer
  • Promoters and enhancers can be activated by chemicals or hormones (such as doxycycline or tamoxifen) to ensure gene expression at specific time points.
  • promoters and enhancers may be natural or artificial or chimeric sequences, ie prokaryotic or eukaryotic sequences.
  • 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.
  • promoters specific to various types of retinal cells such as , ganglion cell-specific promoters (e.g., Tuj1 promoter), astrocyte- and Müller cell
  • ITR inverted terminal repeat
  • AAV inverted terminal repeat
  • AAV types 1-11 avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV.
  • AAV terminal repeats need not have native terminal repeats, so long as the terminal repeats are available for viral replication, packaging and integration.
  • trans-element refers to a transgene cassette packaged in an AAV particle and expressed in target cells to produce a therapeutic protein product.
  • cogniated 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • neovascular retinopathy eg, AMD, ROP, DR
  • 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.
  • the protein product of the therapeutic tool 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.
  • 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
  • the protein product of the therapeutic tool 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).
  • anti-PD-1 antibodies e.g., Nivolumab, Pembrolizumab, Cemiplimab
  • PD-L1 antibodies e.g., Avelumab, Atezolizumab
  • anti-CTLA-4 antibodies e.g Ipilimumab
  • AAV virions with AAVH15 capsid protein exhibit more efficient retinal transduction efficiency than wild-type (WT) serotypes and are suitable for expressing anti-angiogenic proteins transmission of genes.
  • AAV virus particles with AAVT13 capsid protein exhibit more efficient retinal transduction efficiency and are suitable for the delivery of genes expressing anti-angiogenic proteins .
  • AAV virions with the AAVXL32 capsid protein exhibit higher retinal transduction efficiency than wild-type (WT) serotypes and are suitable for expressing anti-angiogenic proteins transmission of genes.
  • 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.
  • 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.
  • 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.
  • 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.
  • triple-plasmid plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid
  • 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.
  • 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).
  • the gene delivery systems of the present disclosure are used to aid in cell transplantation therapy.
  • 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.
  • 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.
  • 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
  • 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.
  • an oligopeptide tag eg, Flag, 6xHis, 2xHA, Myc
  • transgenic plasmids were transfected into eukaryotic cells (eg, HEK293 and CHO cells), and then the target protein was collected by affinity chromatography.
  • Flag-M2 resin beads are often used to specifically attract Flag-tagged proteins, which are then eluted with 3 ⁇ Flag soluble oligopeptides.
  • Ni-NTA nickel nitrilotriacetate
  • the AAV vectors of the present disclosure can be loaded with exogenous polynucleotides for gene delivery into target cells.
  • the AAV vectors of the present disclosure can be used to deliver nucleic acids to cells in vitro or in vivo.
  • 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.
  • reporter proteins include, but are not limited to, green fluorescent protein (GFP), ⁇ -galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.
  • 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.
  • the exogenous polynucleotide delivered by the AAV vector to the target cell encodes a synthetic polypeptide.
  • 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 Paraffin
  • Surfactants e.g, cortisone, prednisone, cyclosporine
  • Immuno Inhibitors eg, cortisone, prednisone, cyclosporine
  • nonsteroidal anti-inflammatory drugs e.g, aspirin, ibuprofen, acetaminophen
  • microspheres rigid matrices, semisolid carriers, Nanospheres or nanoparticles.
  • compositions can be administered by inhalation, systemic or topical (e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal)
  • 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.
  • 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.
  • 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.
  • the ocular disease involves degeneration of eye and/or visual function.
  • treating an ocular disorder refers to improving visual acuity, contrast vision, color vision, and visual field in the treated patient.
  • 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).
  • 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.
  • AAV particles carrying GFP gene were injected into the vitreous of C57BL/6 mice.
  • the retinal cross-sections were taken out for immunofluorescence staining, and the results are shown in Figure 2.
  • AAVH15, AAVXL32, and AAVT13 had significantly increased retinal affinity and transduction efficiency compared with wild-type AAV.
  • 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.
  • RPE retinal pigment epithelium
  • 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.
  • 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.
  • Example 4 Novel transgenic expression cassette expressing endostatin and angiostatin
  • 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.
  • single-chain AAV transgene expression cassettes B110 and B111 were designed to simultaneously express endostatin and angiostatin and simultaneously release them out of cells.
  • 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.
  • 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.
  • flag-tagged proteins can be purified using commercial Flag M2 magnetic beads (Sigma-Aldrich, Cat. No. M8823).
  • 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
  • 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.
  • 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
  • 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).
  • B36 B110 or B111 expression cassettes
  • 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.
  • FFA fluorescein angiography
  • IF immunofluorescence
  • neovascularization and retinal gliosis can be attenuated by treatment with AAV with H15 capsid and packaged B36, B110 or B111 expression cassettes.
  • 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.
  • FFA fluorescein angiography
  • IF immunofluorescence
  • Example 7 Inhibitory effect of AAV-mediated expression of endostatin and angiostatin on tumor growth
  • Hepa1-6 murine liver cancer cells were subcutaneously injected into CByJ.
  • AAVH15 of B36 and B111 transgene cassettes H15-B36 and H15-B111.
  • 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 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.

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Abstract

L'invention concerne un nouveau sérotype du virus adéno-associé, un médicament thérapeutique génique à médiation par le sérotype du virus adéno-associé, et en particulier, un inhibiteur d'angiogenèse. Le sérotype du virus adéno-associé a un bon ciblage de tissu rétinien, et peut être utilisé comme vecteur d'administration d'un gène thérapeutique pour le traitement de maladies de l'œil. Le médicament peut être utilisé pour traiter diverses maladies rétiniennes et de multiples cancers qui utilisent l'angiogenèse en tant que mécanisme pathologique majeur.
PCT/CN2022/107847 2021-07-30 2022-07-26 Nouvel inhibiteur de l'angiogenèse à médiation par le sérotype du virus adéno-associé et son application WO2023005906A1 (fr)

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