阅读说明：本技术 用于治疗视网膜疾病和癌症的转基因表达盒 (Transgene expression cassette for treating retinal diseases and cancers ) 是由 赵锴 陈晨 杜增民 汤丁越 凌菲香 吴侠 王慧 蒋威 赵阳 王利群 郑静 肖啸 于 2021-07-30 设计创作，主要内容包括：本发明涉及涉及编码内皮抑素的核酸分子、包含该核酸分子的转基因表达盒以及包含该转基因表达盒的基因递送系统。本发明的转基因表达盒可用于治疗各种以新生血管为主要病理机制的视网膜疾病以及多种癌症。(The present invention relates to nucleic acid molecules encoding endostatin, transgenic expression cassettes comprising the nucleic acid molecules, and gene delivery systems comprising the transgenic expression cassettes. The transgene expression cassette of the invention can be used for treating various retina diseases taking new vessels as main pathological mechanisms and various cancers.)

1. A nucleic acid molecule encoding endostatin having a nucleotide sequence that is substantially identical to SEQ ID NO: 15 or SEQ ID NO: 17, preferably at least 85%, 90%, 95%, 99% or 100% identity.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises SEQ ID NO: 15 or SEQ ID NO: 17, preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 15 or SEQ ID NO: shown at 17.

3. A transgenic expression cassette comprising: a promoter, the nucleic acid molecule of claim 1 or 2, bGH polyA.

4. The transgenic expression cassette of claim 3, 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, the promoter is a CB promoter.

5. The transgenic expression cassette of claim 3 or 4, wherein the transgenic expression cassette further comprises: signal peptides, such as SP signal peptide, ALB signal peptide, and PLS signal peptide; and/or two ITRs at both ends, each independently a normal ITR or a shortened ITR peptide.

6. A transgenic expression cassette according to any one of claims 3 to 5, wherein the nucleic acid molecule carries an oligopeptide tag, such as Flag, 6 XHis, 2 XHA and Myc.

7. The transgenic expression cassette of any one of claims 3 to 6, wherein the nucleotide sequence of the transgenic expression cassette is as set forth in SEQ ID NO: shown at 7.

8. The transgenic expression cassette of any one of claims 3 to 7, wherein the transgenic expression cassette further comprises: a nucleotide sequence encoding one or more therapeutic proteins other than endostatin; preferably, the therapeutic protein is a protein having an anti-angiogenic effect; more preferably, the therapeutic protein is angiostatin.

9. The transgenic expression cassette of claim 8, wherein the transgenic expression cassette further comprises a linker sequence; preferably, the linker sequence is a Furin protease sequence + linker peptide +2A sequence, such as P2A, T2A, or F2A.

10. The transgenic expression cassette of claim 8 or 9, wherein the nucleotide sequence encoding angiostatin is as set forth in SEQ ID NO: 16 or SEQ ID NO: 18, respectively.

11. The transgenic expression cassette of any one of claims 8 to 10, wherein the nucleotide sequence of the transgenic expression cassette is as set forth in SEQ ID NO: 9 or SEQ ID NO: shown at 11.

12. A gene delivery system, comprising: the transgenic expression cassette and AAV capsid protein of any one of claims 3 to 11.

13. The gene delivery system of claim 12, wherein the AAV capsid protein is a native AAV capsid protein or an artificially engineered AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh 10.

14. The gene delivery system according to claim 12 or 13, wherein the amino acid sequence of the AAV capsid protein is as set forth in SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: as shown at 14.

15. Use of a gene delivery system according to any one of claims 12 to 14 in the manufacture of a medicament for the treatment of a disease in which neovasculature is the major pathological mechanism or causative factor.

16. A medicament, comprising: the nucleic acid molecule of claim 1 or 2, the transgenic expression cassette of any one of claims 3 to 11, or the gene delivery system of any one of claims 12 to 15, and an excipient; preferably, the medicament is used for treating diseases with new blood vessels as main pathological mechanisms or inducers; preferably, the disease is a retinal disease or cancer, such as age-related macular degeneration, diabetic retinopathy, and other retinal damage caused by intense light; lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, and glioblastoma.

17. The medicament according to claim 16, wherein the medicament is administered by systemic or local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration and intralesional administration, preferably topically to the eye, such as by intravitreal injection, subretinal injection or suprachoroidal injection.

Technical Field

The present disclosure relates to nucleic acid molecules encoding endostatin, transgenic expression cassettes comprising the nucleic acid molecules, and gene delivery systems comprising the transgenic expression cassettes.

Background

Pathological neovascularization occurs in a large number of retinal diseases and affects millions of people worldwide each year. For example, approximately 3000 million people worldwide, especially over the age of 60 years, have age-related macular degeneration (AMD), a 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 a variety of factors. Of these, one type of severe AMD, known as wet AMD, is characterized by Choroidal Neovascularization (CNV) that gradually spreads throughout the retina, the contents of which are prone to leakage due to the fragility of the vessel walls, further disrupting retinal structural integrity and impeding visual function. Similar neovascular problems are also prevalent in Diabetic Retinopathy (DR) and retinopathy of prematurity (ROP), i.e. neovascular bleeding and scarring occur in diabetic patients and neonates, respectively.

In addition, excessive neovascularization can be induced during tumorigenesis to facilitate the supply of oxygen and nutrients to cancer cells. First, neovascularization can be induced from a preexisting vascular network 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-.

The above pathological conditions indicate the importance of antiangiogenesis in the treatment of retinal diseases and cancer. Vascular Endothelial Growth Factor (VEGF) is a key factor in initiating the growth of new blood vessels in these diseases. anti-VEGF methods have attracted considerable attention. Endostatin is a 20kD fragment produced by the C-terminus of collagen XVIII, angiostatin is a Kringle domain-containing protein produced by 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 the treatment of retinopathies and cancers 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 9707304B 2 and US 2004/0156828A 1). Therefore, in order to achieve better therapeutic effects on retinal diseases and cancers, it is also desirable to obtain endostatin-encoding sequences and angiostatin-encoding sequences with higher expression levels.

Adeno-associated virus (AAV) has low pathogenicity and the ability to stably express proteins in various organ tissues for a long time, and these properties make AAV have significant advantages in the field of gene therapy and suitable for delivering therapeutic genes. However, wild-type AAV serotypes typically infect a broad spectrum of multiple tissues/organs in mammals, have extensive tissue targeting, resulting in gene delivery to off-target tissues, thereby exacerbating adverse reactions. The capsid proteins of AAV particles not only regulate AAV assembly during replication, but also facilitate viral interaction with receptors on the plasma membrane and entry into target cells. Studies have shown that tissue tropism and cellular transformation efficiency of AAV vectors are largely determined by their capsid. In view of this, in order to achieve better therapeutic effects, it is desirable to appropriately engineer AAV capsid proteins to obtain AAV vectors with organ (e.g., ocular) specificity.

In addition, multiple repeated administrations of the angiogenesis inhibitor are undesirable because repeated injections can cause inconvenience and pain to the patient. Therefore, it is desirable to have a gene delivery system that can deliver two or more angiogenesis inhibitors simultaneously.

Disclosure of Invention

To solve the above technical problem, in a first aspect, the present disclosure provides a nucleic acid molecule encoding endostatin, the nucleotide sequence of which is identical to 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 SEQ ID NO: 15 or SEQ ID NO: 17. In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 15 or SEQ ID NO: shown at 17.

The nucleic acid molecules encoding endostatin of the present disclosure comprise codon-optimized human or murine endostatin-encoding nucleic acid sequences that have higher endostatin expression levels than the original human or murine endostatin-encoding nucleic acid sequences that have not been codon-optimized.

In a second aspect, the present disclosure provides a nucleic acid molecule encoding angiostatin, whose nucleotide sequence is identical to 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 SEQ ID NO: 16 or SEQ ID NO: 18, or a nucleotide sequence shown in the specification. In a preferred embodiment, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 16 or SEQ ID NO: 18, respectively.

The disclosed angiostatin-encoding nucleic acid molecules comprise codon-optimized human-or murine-angiostatin-encoding nucleic acid sequences with higher angiostatin expression levels than the original human-or murine-angiostatin-encoding nucleic acid sequences that were not codon-optimized.

In a third aspect, the present disclosure provides a transgenic expression cassette comprising: a promoter, a nucleic acid molecule according to the first aspect, bGH polyA.

In one embodiment, 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, the promoter is a CB promoter.

In one embodiment, the transgenic expression cassette further comprises: signal peptides, such as SP signal peptide, ALB signal peptide, and PLS signal peptide; and/or two ITRs at both ends, each independently a normal ITR or a shortened ITR peptide.

In one embodiment, the nucleotide sequence encoding endostatin and/or the nucleotide sequence encoding angiostatin carry an oligopeptide tag, such as Flag, 6 × His, 2 × HA, and Myc.

In a preferred embodiment, the transgenic expression cassette further comprises: a nucleotide sequence encoding one or more therapeutic proteins other than endostatin; preferably, the therapeutic protein is a protein having an anti-angiogenic effect; more preferably, the therapeutic protein is angiostatin, for example angiostatin encoded by a nucleic acid molecule encoding angiostatin according to the second aspect.

In a preferred embodiment, the transgenic expression cassette further comprises a linker sequence. In a preferred embodiment, the linker sequence is a Furin protease sequence + linker peptide +2A sequence, such as P2A, T2A, or F2A. The use of such linker sequences can better separate the expression and secretion of two or more proteins (e.g., endostatin and angiostatin).

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

In a fourth aspect, the present disclosure provides a gene delivery system comprising: a transgenic expression cassette and an AAV capsid protein according to the third aspect.

In one embodiment, the AAV capsid protein described above is a native AAV capsid protein or an artificially engineered AAV capsid protein. In a preferred embodiment, the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrh 10.

In one embodiment, the amino acid sequence of the AAV capsid protein is as set forth in SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: as shown at 14.

The transgenic expression cassettes and gene delivery systems of the present disclosure can express higher levels of anti-angiogenic proteins (endostatin and/or angiostatin), and can achieve better therapeutic effects on retinal diseases and cancer. In addition, in the case where the transgene expression cassette includes a nucleotide sequence encoding endostatin and a nucleotide sequence encoding angiostatin, the gene delivery system of the present disclosure can achieve simultaneous delivery of two or more angiogenesis inhibitors.

In a fifth aspect, the present disclosure provides use of a transgenic expression cassette according to the third aspect or a gene delivery system according to the fourth aspect in the manufacture of a medicament for the treatment of a disease in which neovascularization is a major pathological mechanism or causative factor.

In a sixth aspect, the present disclosure provides a medicament comprising: a transgenic expression cassette according to the third aspect or a gene delivery system according to the fourth aspect; and an excipient. In one embodiment, the medicament is for treating a disease in which neovascularization is the major pathological mechanism or causative factor.

In one embodiment, the disease is a retinal disease or cancer, such as age-related macular degeneration, diabetic retinopathy, and other retinal damage caused by intense light; lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, and glioblastoma.

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

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

In an eighth aspect, the present disclosure provides an engineered AAV capsid protein, wherein the amino acid sequence of the AAV capsid protein is as set forth in SEQ ID NO: 2. SEQ ID NO: 4 or SEQ ID NO: as shown at 14.

In a ninth aspect, the present disclosure provides a nucleic acid molecule encoding an AAV capsid protein according to the eighth aspect. In one embodiment, the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID NO: 1. SEQ ID NO: 3 or SEQ ID NO: shown at 13.

The engineered AAV capsid proteins of the present disclosure described above can be used to produce novel AAV vectors, to develop relevant research for novel AAV vectors, or for disease treatment. The novel AAV vector for packaging the modified AAV capsid protein has good retina tissue targeting property, lower toxic and side effects and better safety potential, and can be applied to prevention, diagnosis and treatment of eye related diseases.

Accordingly, in a tenth aspect, the present disclosure provides an AAV vector comprising an AAV capsid protein according to the eighth 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 a protein having an anti-angiogenic effect. 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 as set forth in SEQ ID NO: 15 or SEQ ID NO: shown at 17.

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

Drawings

Fig. 1A shows images of GFP signal at mouse retina for AAV5, AAV8, AAV9, AAVH15, AAVXL32, and AAVT 13.

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

Fig. 1C shows the relative GFP fluorescence signal intensity of different AAV serotypes relative to AAV 5. n-4 mice/group, p < 0.001.

Figure 2 shows retinal sections of mice transduced with AAV8, AAV9, AAVH15, and AAVT 13. GFP, DAPI and cone markers (S-opsin)/RPE marker (RPE65) are shown. GFP signal reaching the photosensitive layer is indicated by the white arrow.

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

Fig. 3B shows a western blot result of expression and secretion of endostatin and angiostatin for B110 and B111 expression cassettes in HEK293 and Huh7 culture medium supernatants. A plasmid encoding GFP was used as a control.

Figure 3C shows the expression levels of 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 to the original non-codon-optimized human or murine endostatin coding nucleic acid sequence. Left panel: western blot results. Right panel: quantitative statistics of relative expression levels; n-3, p <0.001, t-test.

Figure 3D shows the expression levels of 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 to the original non-codon-optimized human or murine angiostatin coding nucleic acid sequences. Left panel: western blot results. Right panel: quantitative statistics of relative expression levels; n-3, p <0.001, t-test.

FIG. 4A shows AAVH15 containing GFP (hereinafter referred to as H15-GFP) at a multiplicity of infection (MOI) of 1X 10⁵、1×10⁴And 1X 10³Ratio vg/cell HUVEC was transduced, and images taken 3 days after viral infection. The upper diagram: GFP fluorescence; the following figures: bright field. Scale bar: 100 μm.

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

FIG. 4C shows the MOI from 1X 10⁵Western blot analysis of conditioned media of vg/cell HUVECs infected with H15-GFP, H15-B110(AAVH15 packaging B110 expression cassette) and H15-B111(AAVH15 packaging B111 expression cassette).

FIG. 4D shows particles of H15-GFP, H15-B36(AAVH15 packaging B36 expression cassette), H15-B110 and H15-B111 at 1X 10⁵And 1X 10⁴Images taken of the mog/cell MOI infected HUVEC. Scale bar: 200 μm.

FIG. 4E shows the quantification of HUVEC tube formation (per unit area)Volume (mm)²) Tube length of (1). n-5 wells. P<0.01，***p<0.001, t test.

FIG. 4F shows the quantification of HUVEC tube formation (per unit area (mm)²) The number of branch points). n-5 wells. P<0.01，***p<0.001, t test.

FIG. 5A shows a mouse model map of laser-induced neovascularization. Intravitreal injection of 2X 10 into C57BL/6 mice¹⁰vg/eye AAV particles having an H15 capsid and packaged with a B36, B110 or B111 expression cassette (referred to as laser-B36, laser-B110, laser-B111, respectively). Mice were treated with the laser-induced CNV model 14 days after virus injection, and after another 12 days, fluorescein angiography (FFA) and Immunofluorescence (IF) were performed.

Figure 5B shows laser-induced neovascularization and scarring. The upper diagram: fluorescein angiography. The following figures: laser induced lesions. Scale bar: 1 mm.

FIG. 5C shows a fluorescence image of retinal section staining showing activated retinal astrocytes and Muller cells (GFAP) and blood vessels (IB 4). GFAP white arrow: a CNV cluster; IB4 white arrow: GFAP positive glial cell membrane associated with CNV cluster. Scale bar: 100 μm.

Fig. 5D shows the quantification of the area of laser induced CNV tufts. P <0.05, p <0.01, p <0.001, n-5 eyes/group, one-way anova.

Fig. 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 anova.

Fig. 5F shows laser-induced lesion size. P <0.05, p <0.01, p <0.001, n-5 eyes/group, one-way anova.

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

FIG. 6A shows a mouse model map of laser-induced neovascularization. Intravitreal injection of 2X 10 into C57BL/6 mice⁹vg/eye of AAV particles encapsidated with AAVT13 and packaged with B36, B110 or B111 expression cassettes (referred to as laser-B36, laser-B110, laser-B111, respectively). Mice were laser-induced on the CNV model 14 days after virus injection and fluorescein angiography (FFA) was performed 12 more days later.

Figure 6B shows laser-induced neovascularization and scarring. The upper diagram: fluorescein angiography. The following figures: laser induced lesions. Scale bar: 1 mm.

Fig. 6C shows quantification of neovascularization. P <0.05, p <0.01, n-7 eyes/group.

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

Figure 7A shows a tumor implantation flowchart in mice.

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

Fig. 7C shows the quantification 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 analysis of variance.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

FIG. 22 shows a codon-optimized human endostatin-encoding nucleic acid sequence (SEQ ID NO: 15).

FIG. 23 shows a codon-optimized human angiostatin-encoding nucleic acid sequence (SEQ ID NO: 16).

FIG. 24 shows a codon-optimized murine endostatin-encoding nucleic acid sequence (SEQ ID NO: 17).

FIG. 25 shows a codon-optimized murine angiostatin-encoding nucleic acid sequence (SEQ ID NO: 18).

FIG. 26 shows the original (non-codon optimized) human endostatin-encoding nucleic acid sequence (SEQ ID NO: 19).

FIG. 27 shows the original (non-codon optimized) human angiostatin-encoding nucleic acid sequence (SEQ ID NO: 20).

FIG. 28 shows the original (non-codon optimized) murine endostatin-encoding nucleic acid sequence (SEQ ID NO: 21).

FIG. 29 shows the original (non-codon optimized) murine angiostatin-encoding nucleic acid sequence (SEQ ID NO: 22).

Detailed Description

Unless defined otherwise, 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, the nucleic acid or polynucleotide sequences set forth herein are in single stranded form in the orientation 5 'to 3', left to right. The nucleotides and amino acids provided herein are in the format suggested by the IUPACIUB Biochemical nomenclature Commission, and the single letter code or the three letter code is used for amino acids.

Unless otherwise indicated, "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. Polynucleotides may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism that has or is susceptible to a disorder that can be prevented or treated by administration of a medicament of the present disclosure, and include humans and non-human animals (e.g., rodents or other mammals).

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, pheasants, 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.

Herein, the term "treatment" 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 the disease, i.e., causing regression of at least one of the conditions, diseases or disorders or clinical or subclinical symptoms thereof.

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

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

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 condition, disease or disorder developing in a subject who may be suffering from or susceptible to the condition, disease or disorder but who has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

Herein, the term "topical administration" or "topical route" refers to an administration having a local effect.

As used herein, the terms "transduction," "transfection," and "transformation" refer to the process of delivering an exogenous nucleic acid into a host cell, followed by transcription and translation of the polynucleotide product, which includes the introduction of an exogenous polynucleotide into the host cell using a recombinant virus.

As used herein, the term "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell 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 by which a gene is transcribed, translated, and post-translationally modified to produce the RNA or protein product of the gene.

As used herein, the term "infection" refers to the process by which a virus or viral 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 replication of the virus in a host cell.

In this context, the term "targeted" means that the virus preferentially enters some cell or tissue and then further expresses the viral genome or a sequence carried by the recombinant transgene in the cell.

As used herein, the term "vector" refers to a macromolecule or series of macromolecules encapsulating a polynucleotide that facilitates delivery of the polynucleotide to a target cell in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as an "expression cassette" or "transgene cassette," may include, but is not limited to, the coding sequence of certain proteins or synthetic polypeptides (which may enhance, inhibit, impair, protect, trigger, or prevent certain biological and physiological functions), the coding sequence of interest in vaccine development (e.g., a polynucleotide that expresses a protein, polypeptide, or peptide suitable for eliciting an immune response in a mammal), the coding sequence of RNAi material (e.g., shRNA, siRNA, antisense oligonucleotides), or an optional biomarker.

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 more than 20 amino acids. These terms also encompass synthetic or artificial amino acid polymers.

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

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

In one embodiment, the transgene cassette comprises a number of regulatory elements in addition to the sequence encoding the protein product to allow for packaging of the transgene into the virus, e.g., a normal ITR of 145bp, a shortened ITR of approximately 100bp in length. In some embodiments, the transgene cassette further comprises polynucleotide elements for controlling expression of the protein product, such as origins of replication, polyadenylation signals, Internal Ribosome Entry Sites (IRES) or 2A signals (e.g., P2A, T2A, F2A), promoters and enhancers (e.g., CMV promoter with vertebrate β -actin, β -globin or β -globin regulatory elements or other hybrid CMV promoters (referred to as 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 (e.g., doxycycline or tamoxifen) to ensure gene expression at a particular point in time. Furthermore, promoters and enhancers may be natural or artificial or chimeric sequences, i.e., 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 for various types of retinal cells, e.g., ganglion cell-specific promoters (e.g., Tuj1 promoter), astrocyte and Muller cell-specific promoters (e.g., GFAP or vimentin promoters), and retinal pigment epithelium-specific promoters (e.g., RPE65 or VMD2 promoter); specific promoters for various types of ocular neurons (e.g., synaptophysin, VGAT, DAT, TH promoters); and promoters specific for osteoblast lineage (e.g. osteocalcin promoter), liver, pancreas, spleen and lung cancer cell specific promoters.

In this context, the term "Inverted Terminal Repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and serves as a cis element to mediate viral replication, packaging and integration. The 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 repeat need not have a native terminal repeat, so long as the terminal repeat is available for viral replication, packaging, and integration.

In this context, the term "cis-element" refers to a transgene cassette packaged in an AAV particle and expressed in a target cell to produce a protein product having a therapeutic effect.

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

As known to those skilled in the art, AAV capsid proteins contain VP1, VP2, and VP3 proteins, and VP2 and VP3 proteins undergo transcription and translation processes at the start codon inside the VP1 protein, i.e., the VP1 sequence comprises VP2 and VP3 sequences. The present disclosure provides the amino acid sequence of VP1 protein of AAV capsid.

In one embodiment, the AAV capsid protein may be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., capsid proteins of native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV) and other artificially engineered AAV capsid proteins (e.g., capsid proteins of artificially engineered AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV). The genomic 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 a variety of diseases with associated pathological mechanisms, including but not limited to: neovascular retinopathy (e.g., AMD, ROP, DR) and ocular damage from intense light or other causes. In addition, the therapeutic tools of the present disclosure can also treat various types of cancers that develop angiogenesis promoting tumor growth and metastasis, 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.

In one embodiment, the protein product of the therapeutic tool (e.g., a transgenic expression cassette) includes proteins with anti-angiogenic effects, such as, but not limited to, Aflibercept, recombinant VEGF soluble receptors (produced by renitron Pharmaceuticals, which inhibit neovascularization), anti-VEGF antibodies (e.g., bevacizumab, ranibizumab, and brevizumab), other anti-angiogenic proteins or polypeptides (e.g., endostatin, angiostatin, platelet factor 4, pigment epithelium derived factor), Fibroblast Growth Factor (FGF) inhibitors, metalloproteinase inhibitor BB 94.

In one embodiment, the protein product of the therapeutic tool (e.g., the transgene expression cassette) further comprises an antibody having anti-tumor properties, such as an anti-PD-1 antibody (e.g., Nivolumab, Pembrolizumab, cemipimab) and PD-L1 antibody (e.g., Avelumab, Atezolizumab), an anti-CTLA-4 antibody (e.g., Ipilimumab), an anti-CGRP antibody (e.g., Fremanezumab, galvaezumab, erelumab), an anti-HER 2 antibody (e.g., Trastuzumab, Pertuzumab), and an anti-EGFR antibody (e.g., Cetuximab, Panitumumab, necumab).

In some embodiments, AAV viral particles having an AAVH15 capsid protein (SEQ ID NO: 2) exhibit more efficient retinal transduction efficiency as compared to the Wild Type (WT) serotype, suitable for delivery of genes expressing anti-angiogenic proteins.

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

In some embodiments, AAV viral particles having an AAVXL32 capsid protein (SEQ ID NO: 14) exhibit more efficient retinal transduction efficiency as compared to the Wild Type (WT) serotype, suitable for delivery of genes expressing anti-angiogenic proteins.

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

In one embodiment, the transgene expression cassette encoding the angiogenesis inhibitor comprises a CB promoter sequence (SEQ ID NO: 5), a bGH polyA sequence (SEQ ID NO: 6), a codon optimized human endostatin with an N-terminal SP signal peptide, and a codon optimized human angiostatin sequence (SEQ ID NO: 15 and SEQ ID NO: 16), the coding sequences of which are linked by a Furin protease sequence (Lys-Arg-Lys-Arg-Arg) + linker peptide (Ser-Gly-Ser-Gly) + F2A sequence, thus forming the B110 expression cassette (SEQ ID NO: 9). The B110 expression cassette also comprises two ITRs, which allow the expression cassette to be packaged into AAV viral particles as a single-stranded 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 murine endostatin with an N-terminal PLS signal peptide, and a codon optimized murine angiostatin sequence (SEQ ID NO: 17 and SEQ ID NO: 18), the coding sequences of which are linked by a Furin protease sequence (Lys-Arg-Lys-Arg-Arg) + linker peptide (Ser-Gly-Ser-Gly) + P2A sequence, thus forming the B111 expression cassette (SEQ ID NO: 11). The B111 expression cassette also comprises two ITRs that allow the expression cassette to be packaged into AAV viral particles as a single-stranded AAV vector.

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

In one embodiment, to produce AAV particles with therapeutic function, three plasmid transfection of HEK293 cells is performed as follows: plasmid 1: cis-element plasmids with ITRs (e.g., B36, B110, and B111 expression cassettes); plasmid 2: an AAV Rep/Cap plasmid having capsid protein (e.g., AAVH15, AAVT13, and AAVXL32 capsid protein) coding sequences; plasmid 3: a helper plasmid having an adenoviral component, which is capable of facilitating 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).

One skilled in the art can use standard methods known to produce recombinant and synthetic polypeptides or proteins thereof, design nucleic acid sequences, produce transformed cells, construct recombinant AAV mutants, engineer capsid proteins, package vectors expressing AAV Rep and/or Cap sequences, and transiently or stably transfect packaging cells. These techniques are known to those skilled in the art. See, e.g., MOLECULAR CLONING (MOLECULAR CLONING): a laboratory Manual (ALABORATORY MANUAL), second edition, (Cold spring harbor, N.Y., 1989).

In some embodiments, the gene delivery system of the present disclosure is used in adjuvant cell transplantation therapy. In particular, 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, muller cells, oligodendrocytes, microglia, rod and cone cells, neurons, hematopoietic stem cells, monocytes, granulocytes, lymphocytes, osteoclasts, and macrophages.

In one embodiment, the cells for transplantation are autologous cells of the subject, which allow for in vitro culture. The principles and techniques for 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 lysate of HEK293 cells. Purification methods such as affinity chromatography, ion exchange chromatography, cesium chloride and iodixanol gradient ultracentrifugation. Chemicals or reagents involved in AAV production and purification include, but are not limited to: chemicals or reagents for cell culture (e.g., components of cell culture media including bovine, equine, caprine, chicken or other vertebrate serum, glutamine, glucose, sucrose, sodium pyruvate, phenol red; antibiotics such as penicillin, kanamycin, streptomycin, tetracycline); chemicals or reagents for cell lysis, polynucleotide precipitation or ultracentrifugation (e.g., Triton X-100, NP-40, sodium deoxycholate, sodium dodecyl sulfate, domiphen bromide, sodium dodecyl salicylate, sodium chloride, magnesium chloride, calcium chloride, barium chloride, nitrate, potassium 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, DNase I, RNase); affinity column materials (e.g., AAVX affinity resin, heparin sulfate proteoglycan and mucin resin, other materials related to AAV-specific antibodies); acids, bases and organics contained in the ion exchange chromatography material and wash buffer (e.g., hydrochloric acid, sulfuric acid, 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 (e.g., Flag, 6 × His, 2 × HA, Myc), which aids in the purification of the protein product. Those skilled in the art understand the techniques and procedures associated with protein purification. Briefly, transgenic plasmids are transfected into eukaryotic cells (e.g., HEK293 and CHO cells) and the target protein is then collected by affinity chromatography. For example, Flag-M2 resin beads are typically used to specifically attract Flag-tagged proteins, which are then eluted with 3 xflag soluble oligopeptides. It is also possible to use a nickel nitrilotriacetate (Ni-NTA) column for reversible binding and then to purify specifically the 6 XHis-tagged protein.

In one embodiment, an AAV vector of the present disclosure may be loaded with an exogenous polynucleotide for delivery of the gene into a target cell. 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 (i.e., a reporter protein). The reporter protein is used to indicate cells successfully infected with 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, which native protein is codon-optimized or not codon-optimized.

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

In one embodiment, the AAV vector or transgene expression cassette or gene delivery system of the present disclosure is formulated into a pharmaceutical formulation (e.g., injection, tablet, capsule, powder, eye drop) for administration to a human or other mammal. The pharmaceutical preparation may further comprise other ingredients, such as pharmaceutical excipients, water-soluble or organic solvents (e.g. water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (e.g. sodium chloride, potassium chloride, phosphate, acetate, bicarbonate, Tris-HCl and Tris-acetate), dissolution retarding agents (e.g. paraffin), surfactants, antimicrobial agents, liposomes, lipid complexes, immunosuppressants (e.g. cortisone, prednisone, cyclosporine), microspheres of non-steroidal anti-inflammatory drugs (NSAIDs, e.g. aspirin, ibuprofen, paracetamol), rigid matrices, semi-solid carriers, nanospheres or nanoparticles. In addition, the pharmaceutical formulations can be delivered in single or multiple doses by inhalation, systemic or local (e.g., intravenous, subcutaneous, intraocular, intravitreal, subretinal, suprachoroidal, parenteral, intramuscular, intracerebroventricular, oral, intraperitoneal, and intrathecal) administration.

In one embodiment, the present disclosure provides a medicament comprising an AAV vector or transgene expression cassette or gene delivery system of the present disclosure and an excipient. The medicaments of the present disclosure may be used to transduce cells in vitro or mammals (e.g., rodents, primates, and humans) in vivo, thereby treating various diseases, such as ocular diseases.

Herein, the ocular disease is selected from: inherited dystrophies of the retina, glaucoma, glaucomatous neuropathy, age-related macular degeneration, refractive error, dry eye, inherited dystrophies of ocular inflammation, uveitis, orbital inflammation, cataracts, allergic conjunctivitis, diabetic retinopathy, macular edema, corneal edema, keratoconus, proliferative vitreoretinopathy (fibrosis), periretinal fibrosis, central serous chorioretinopathy, vitreoretinopathy, vitreous macular traction, and vitreous hemorrhage. In one embodiment, the ocular disease involves a deterioration 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 of the patient receiving the treatment.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1: retinal affinities for AAVH15, AAVXL32, and AAVT13

The AAVT13 capsid (SEQ ID NO: 4) was constructed by replacing the N-terminal region of VP1, VP2 (amino acids 1-203) of AAV8 with the N-terminal region of AAV5 (amino acids 1-192) (with point mutation site G257R) and joining to the AAV5 capsid protein sequence. A DNA shuffling experiment was performed to construct the AAVH15 capsid protein (SEQ ID NO: 2) and AAVXL32 capsid protein (SEQ ID NO: 14).

The retinal affinities of the modified AAV serotypes (AAVH15, AAVXL32, and AAVT13) were studied by intravitreal injection of AAV particles capable of expressing GFP protein in C57BL/6 mice with WT serotypes AAV5, 8, and 9 as controls. At 2X 10⁹vg/eye dose, each AAV serotype carrying the GFP gene was injected intravitreally into C57BL/6 mice, and images of GFP signal taken 3 weeks after injection are shown in figure 1A. At 2X 10⁹Vg/eye dose, C57BL/6 mice were injected intravitreally with AAV particles bearing the GFP gene. At 3 weeks post-injection, retinal cross sections were removed for immunofluorescent staining and the results are shown in figure 2.

The results showed that the retinal GFP fluorescence levels were significantly higher in the AAVH15 and AAVT13 groups than in the AAV5, 8, 9 groups (fig. 1A), about 10-12 times higher (fig. 1C, p <0.001, n 4 eyes/group). The retinal GFP fluorescence levels of the AAVXL32 group were also significantly higher than those of AAV5, 8, 9 groups (fig. 1A), by a factor of about 5 (fig. 1C, p <0.001, n 4 eyes/group). It can be seen that AAVH15, AAVXL32, and AAVT13 have significantly increased retinal affinity and transduction efficiency compared to wild-type AAV.

Furthermore, despite intravitreal administration, the improved serotypes AAVH15 and AAVT13 were able to diffuse into the photosensitive layer and even the retinal pigment epithelium layer (RPE) (fig. 1B and fig. 2), significantly better than AAV5, 8, 9. Thus, serotypes AAVH15 and AAVT13 have greater affinity for the retina and are able to deliver genes to each layer of the retina.

Example 2: expression of codon optimized human and mouse endostatin coding nucleic acid sequence

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

HEK293 cells were transfected with B110 and B111 plasmids and then assayed for endostatin expression in cell lysates by Western blotting. Non-codon optimized endostatin of human and murine origin was constructed into plasmids as controls (original human, original murine). The endostatin protein expression of each group relative to the original human-derived group was quantitatively counted.

As shown in fig. 3C, the endostatin protein expression levels of the B110 and B111 expression cassettes were significantly higher than the control group that was not codon optimized. Quantitative statistics of the relative expression levels of the proteins showed that the endostatin protein expression levels of the B110 and B111 expression cassettes were 4-6 fold elevated compared to the non-codon optimized sequences (fig. 3C, right panel). The above results indicate that the codon optimized human and murine endostatin encoding nucleic acid sequences of the present disclosure have significant advantages in expression compared to the original natural protein coding sequence.

Example 3: expression of codon optimized human and mouse angiostatin coding nucleic acid sequences

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

HEK293 cells were transfected with B110 and B111 plasmids and then assayed for angiostatin expression in cell lysates by Western blotting. Human and murine angiostatin without codon optimization were constructed into plasmids as controls (original human, original murine). The angiostatin protein expression of each group relative to the original human-derived group was quantitatively counted.

As shown in fig. 3D, the angiostatin protein expression levels of the B110 and B111 expression cassettes were significantly higher than the control group that was not codon optimized. Quantitative statistics of the relative expression levels of the proteins showed that the angiostatin protein expression levels of the B110 and B111 expression cassettes were increased by about 4-fold compared to the non-codon optimized sequences (fig. 3D, right panel). The above results indicate that the codon optimized human and murine angiostatin coding nucleic acid sequences of the present disclosure have significant advantages in expression compared to the original natural protein coding sequences.

Example 4: novel transgenic expression cassette for expressing endostatin and angiostatin

To make the anti-angiogenic polypeptide more stably expressed, self-complementary and single-stranded AAV were used. As shown in fig. 3A, a B36 transgene cassette was constructed by adding an ALB signal peptide to the N-terminus of a codon-optimized human endostatin sequence that enhances endostatin protein expression and secretion and inserting an intron inside. 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 cassette can be packaged into AAV particles as a self-complementing AAV vector.

In addition, single-chain AAV transgene expression cassettes B110 and B111 were designed which simultaneously express endostatin and angiostatin and release them out of the cells simultaneously. As shown in fig. 3A, the B110 cartridge includes: two normal ITR sequences, the CB promoter (SEQ ID NO: 5), the codon-optimized human endostatin sequence (SEQ ID NO: 15), the codon-optimized human angiostatin sequence (SEQ ID NO: 16), and the bGH polyA tail (SEQ ID NO: 6). SP signal peptides are used to control the secretion of endostatin and angiostatin outside the cell. The coding sequences of the two proteins are linked by a linker comprising the sequence Furin protease (krkrkrr) + linker (SGSG) + F2A to better separate endostatin and angiostatin expression during translation. In addition, endostatin and angiostatin carry Flag tags and 2 × HA tags, so that endostatin and angiostatin can be prepared and purified based on tag-dependent affinity chromatography. For example, purification of Flag-tagged protein can be performed using commercial Flag M2 magnetic beads (Sigma-Aldrich, cat # M8823).

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

B110 and B111 plasmids were transfected into HEK293 cells and Huh7 cells. After 48 hours, the expression levels of endostatin and angiostatin proteins in the medium were examined by western blotting. Results as shown in fig. 3B, both B110 and B111 expression cassettes achieved significant endostatin and angiostatin protein expression. It can be seen that endostatin and angiostatin are successfully expressed and secreted by transfecting B110 and B111 plasmids, and the B110 and B111 expression cassettes realize the simultaneous stable expression of the two proteins.

Example 5: inhibition of HUVEC tubulogenesis by novel AAV-mediated anti-neovascular protein delivery

The production process of anti-angiogenic AAV particles was started by three plasmid transfections of B36, B110, B111 cis-element plasmid, AAVH15 capsid plasmid and adenovirus helper plasmid, and 0.2-5mg/ml PEI, and terminated with 10-35g/L CDM4 solution of one quarter of the cell culture medium volume. HEK293 cells were lysed 40-80 hours after transfection and the polynucleotides were removed. Centrifugation was carried out at 4000-15000rpm 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 volume for use.

Transduction efficiency of AAVH15 in HUVEC cells was tested. Based on the expression of GFP, the MOI was 1X 10⁵In the case of vg/cell, approximately 87% of HUVEC cells were infected with AAVHH 15; at an MOI of 1X 10⁴In the case of vg/cell, the cell transduction ratio of AAV decreased to 45.8% (fig. 4A and fig. 4B). When the MOI is reduced to 1 × 10³At vg/cell, approximately 10.9% of HUVEC cells showed a clear GFP fluorescence signal.

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

AAVH15 carrying B36, B110 and B111 expression cassettes (designated H15-B36, H15-B110 and H15-B111, respectively) at MOI of 1X 10⁵And 1X 10⁴vg/cell infected HUVEC cells. Cells were harvested and transferred to Matrigel-plated 24-well plates for pre-incubation for 45 min for tube formation, and images were taken after 6 hours, with the results shown in fig. 4D. Analysis per unit area (mm) by Image J²) Tube length and number of branches. The results showed that both the tube length and branch point number of H15-B36, H15-B110, and H15-B111 infected cells were significantly reduced compared to the control group (H15-GFP) (FIG. 4E and FIG. 4F). The above results indicate that H15-B36, H15-B110 and H15-B111 viral vectors can inhibit HUVEC tube formation.

Example 6: AAV-mediated expression of endostatin and angiostatin inhibits retinal neovascularization and neurogenesis Proliferation of glial cells

2 x 10 to¹⁰vg/eye of AAV particles with H15 capsid packaged with B36, B110 or B111 expression cassettes (referred to as laser-B36, laser-B110, laser-B111, respectively) were injected intravitreally into C57BL/6 mice. As shown in fig. 5A, mice were subjected to a laser-induced CNV model 14 days after virus injection, and then to fluorescein angiography (FFA) and Immunofluorescence (IF) after another 12 days.

The results show that the size of the laser-induced CNV clusters was reduced by about 75% (shown in fig. 5B and 5D) and the number of CNV clusters was also reduced (fig. 5B and 5E) after AAVH 15-mediated endostatin and angiostatin treatment. At the same time, AAVH 15-mediated neovascularization accelerated recovery from laser-induced lesions. After 12 days of laser-induced retinal damage, lesion volume was reduced by about 50-75% in the different treated groups compared to the untreated control group (laser model) (fig. 5B and 5F). To further investigate the cellular mechanisms, the retina was co-stained with the vascular marker IB4 and the retinal glial cell marker GFAP. In healthy retinas, there were astrocytes or muller cells around the vessels, showing a clear network (fig. 5C). Following laser-induced injury, activated retinal glia, including astrocytes and muller cells, accumulate to form glial scar membranes with a close affinity for CNV clusters, whereas AAVH 15-mediated release of endostatin and angiostatin significantly improved retinal performance. As shown in fig. 5G, the area covered by the glial cell membrane dropped sharply from 29.9% to around 12% (p <0.001), indicating that proliferation of glial cells was inhibited. It can thus be seen that treatment by AAV (which has an H15 capsid and packaged with a B36, B110 or B111 expression cassette) can reduce neovascularization and retinal gliosis.

In addition, the therapeutic effect of low dose AAV particles was investigated. Intravitreal injection of 2X 10 into C57BL/6 mice⁹vg/ocular AAV particle (with AAVT13 capsid and packaging B36, B110 and B111 transgene expression cassettes). As shown in fig. 6A, mice were subjected to a laser-induced CNV model 14 days after virus injection, and then subjected to fluorescein angiography (FFA) and Immunofluorescence (IF) after another 12 days.

The results show that as shown in fig. 6B to 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 at low doses the expression of endostatin and angiostatin mediated by AAVT13 improved neovascularization and lesion-induced scarring.

Example 7: inhibition of tumor growth by AAV-mediated endostatin and angiostatin expression

To investigate the effect of AAV gene delivery systems on cancer therapy, Hepa1-6 murine hepatoma cells, as well as AAVH15(H15-B36 and H15-B111) with B36 and B111 transgene cassettes were injected subcutaneously into mice with systemic T cells mutated at Foxn1 gene and partially B cell deficient cbyj. cg-Foxn1 nu/J. As shown in FIG. 7A, Hepa1-6 cells (ATCC CRL-1830) grown in a culture dish were digested with 0.05% trypsin, centrifuged at 800rpm for 5 minutes, and then resuspended in PBS for mouse injection. Cg-Foxn1nu/J mice (Jackson laboratory stock number 000711) were injected subcutaneously with 2X 10⁶Hepa1-6 cells and 1X 10¹¹vg of AAVH15 (packaging B36 and B111 expression cassettes, respectively). Tumor sizes were measured 7, 14 and 21 days after implantation. Mice injected with Hepa1-6 cells and AAV encoding GFP (AAVH15) served as controls.

As observed in fig. 7B and 7C, at day 14 after cancer cell implantation and treatment with H15-B36 and H15-B111, tumor volumes in the B36 and B111 treated groups had been significantly reduced compared to control mice. On day 21 post-treatment, mean tumor volumes in B36 and B111 treatment groups were 211.3 and 75.9mm, respectively²Smaller than the control group (mean tumor volume 2240 mm)²) One tenth of the total. The results show that the AAVH 15-mediated endostatin and angiostatin expression has obvious inhibition effect on tumor growth.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Sequence listing

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accaccagca cccgaacatg ggccctgccc acctacaaca accacctcta caagcaaatc 780

tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840

tgggggtatt ttgatttcaa cagattccac tgccactttt caccacgtga ctggcagcga 900

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acggttcaag tcttctcgga ctcggagtac cagttgccgt acgtcctcgg ctctgcgcac 1080

cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140

acgctcaaca atggcagcca ggcagtggga cggtcatcct tttactgcct ggaatatttc 1200

ccatcgcaga tgctgagaac gggcaacaac tttaccttca gctacacctt cgaggacgtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccagtacc tgtattacct gaacagaact caaaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtctccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttatcggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aactttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacaaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaaaga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

attacagacg aagaggaaat caaagccact aaccccgtgg ccaccgaaag atttgggacc 1740

gtggcagtca atctccagag cagcagcaca gaccctgcga ccggagatgt gcatgttatg 1800

ggagccttac ctggaatggt gtggcaagat agagacgtgt acctgcaggg tcccatttgg 1860

gccaaaattc ctcacacaga tggacacttt cacccgtctc ctcttatggg cggctttgga 1920

ctcaagaacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcagagtttt cggctacaaa gtttgcttca ttcatcaccc agtattccac aggacaagtg 2040

agcgtggaga ttgaatggga gctgcagaaa gaaaacagca agcgctggaa tcccgaagtg 2100

cagtacacat ccaattatgc aaaatctgcc aacgttgatt ttactgtgga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctca cccgtcccct gtaa 2214

<210> 2

<211> 737

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAVH15 amino acid sequence

<400> 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn

435 440 445

Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe

450 455 460

Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp

485 490 495

Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu

500 505 510

Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His

515 520 525

Lys Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe

530 535 540

Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met

545 550 555 560

Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu

565 570 575

Arg Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro

580 585 590

Ala Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys Asn Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly

705 710 715 720

Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro

725 730 735

Leu

<210> 3

<211> 2205

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAVT13 coding sequence

<400> 3

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctgg agcacgacaa ggcctacgac 240

cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480

ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540

gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga 600

cctaatacaa tgtctgcggg aggtggcggc ccattgggcg acaataacca aggtgccgat 660

ggagtgggca atgcctcggg agattggcat tgcgattcca cgtggatggg ggacagagtc 720

gtcaccaagt ccacccgaac ctgggtgctg cccagctaca acaaccacca gtaccgagag 780

atcaaaagcg gctccgtcga cagaagcaac gccaacgcct actttggata cagcaccccc 840

tgggggtact ttgactttaa ccgcttccac agccactgga gcccccgaga ctggcaaaga 900

ctcatcaaca actactgggg cttcagaccc cggtccctca gagtcaaaat cttcaacatt 960

caagtcaaag aggtcacggt gcaggactcc accaccacca tcgccaacaa cctcacctcc 1020

accgtccaag tgtttacgga cgacgactac cagctgccct acgtcgtcgg caacgggacc 1080

gagggatgcc tgccggcctt ccctccgcag gtctttacgc tgccgcagta cggttacgcg 1140

acgctgaacc gcgacaacac agaaaatccc accgagagga gcagcttctt ctgcctagag 1200

tactttccca gcaagatgct gagaacgggc aacaactttg agtttaccta caactttgag 1260

gaggtgccct tccactccag cttcgctccc agtcagaacc tcttcaagct ggccaacccg 1320

ctggtggacc agtacttgta ccgcttcgtg agcacaaata acactggcgg agtccagttc 1380

aacaagaacc tggccgggag atacgccaac acctacaaaa actggttccc ggggcccacg 1440

ggccgaaccc agggctggaa cctgggctcc ggggtcaacc gcgccagtgt cagcgccttc 1500

gccacgacca ataggatgga gctcgagggc gcgagttacc aggtgccccc gcagccgaac 1560

ggcatgacca acaacctcca gggcagcaac acctatgccc tggagaacac tatgatcttc 1620

aacagccagc cggcgaaccc gggcaccacc gccacgtacc tcgagggcaa catgctcatc 1680

accagcgaga gcgagacgca gccggtgaac cgcgtggcgt acaacgtcgg cgggcagatg 1740

gccaccaaca accagagctc caccactgcc cccgcgaccg gcacgtacaa cctccaggaa 1800

atcgtgcccg gcagcgtgtg gatggagagg gacgtgtacc tccaaggacc catctgggcc 1860

aagatcccag agacgggggc gcactttcac ccctctccgg ccatgggcgg attcggactc 1920

aaacacccac cgcccatgat gctcatcaag aacacgcctg tgcccggaaa tatcaccagc 1980

ttctcggacg tgcccgtcag cagcttcatc acccagtaca gcaccgggca ggtcaccgtg 2040

gagatggagt gggagctcaa gaaggaaaac tccaagaggt ggaacccaga gatccagtac 2100

acaaacaact acaacgaccc ccagtttgtg gactttgccc cggacagcac cggggaatac 2160

agaaccacca gacctatcgg aacccgatac cttacccgac ccctt 2205

<210> 4

<211> 735

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAVT13 amino acid sequence

<400> 4

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ser Ala Gly Gly

195 200 205

Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val

225 230 235 240

Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro Ser Tyr Asn Asn His

245 250 255

Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp Arg Ser Asn Ala Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Ser His Trp Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val Lys Ile Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Asp Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys Leu Pro Ala Phe Pro

355 360 365

Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr Ala Thr Leu Asn Arg

370 375 380

Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser Phe Phe Cys Leu Glu

385 390 395 400

Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Thr

405 410 415

Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser Phe Ala Pro Ser Gln

420 425 430

Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp Gln Tyr Leu Tyr Arg

435 440 445

Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln Phe Asn Lys Asn Leu

450 455 460

Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp Phe Pro Gly Pro Thr

465 470 475 480

Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly Val Asn Arg Ala Ser

485 490 495

Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu Leu Glu Gly Ala Ser

500 505 510

Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr Asn Asn Leu Gln Gly

515 520 525

Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile Phe Asn Ser Gln Pro

530 535 540

Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu Gly Asn Met Leu Ile

545 550 555 560

Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg Val Ala Tyr Asn Val

565 570 575

Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser Thr Thr Ala Pro Ala

580 585 590

Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro Gly Ser Val Trp Met

595 600 605

Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro Glu

610 615 620

Thr Gly Ala His Phe His Pro Ser Pro Ala Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Met Met Leu Ile Lys Asn Thr Pro Val Pro Gly

645 650 655

Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu Trp Glu Leu Lys Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Asn Asn Tyr

690 695 700

Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp Ser Thr Gly Glu Tyr

705 710 715 720

Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 5

<211> 772

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CB promoter nucleotide sequence

<400> 5

acgcgtggta cctctggtcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 60

caacgacccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 120

actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 180

caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 240

tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctactcg 300

aggccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt 360

tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg gggggggcgc 420

gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg 480

gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg 540

cggccctata aaaagcgaag cgcgcggcgg gcgggagcgg gatcagccac cgcggtggcg 600

gccctagagt cgatcgagga actgaaaaac cagaaagtta actggtaagt ttagtctttt 660

tgtcttttat ttcaggtccc ggatccggtg gtggtgcaaa tcaaagaact gctcctcagt 720

ggatgttgcc tttacttcta ggcctgtacg gaagtgttac ttctgctcta aa 772

<210> 6

<211> 208

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> bGH POLYA nucleotide sequence

<400> 6

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctgggga 208

<210> 7

<211> 2142

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of B36 expression cassette

<400> 7

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggaatt cacgcgtggt 120

acgatctgaa ttcggtacaa ttcacgcgtg gtacctctgg tcgttacata acttacggta 180

aatggcccgc ctggctgacc gcccaacgac cccgcccatt gacgtcaata atgacgtatg 240

ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 300

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 360

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 420

ctacttggca gtacatctac tcgaggccac gttctgcttc actctcccca tctccccccc 480

ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 540

gggggggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 600

gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 660

ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 720

cgggatcagc caccgcggtg gcggccctag agtcgatcga ggaactgaaa aaccagaaag 780

ttaactggta agtttagtct ttttgtcttt tatttcaggt cccggatccg gtggtggtgc 840

aaatcaaaga actgctcctc agtggatgtt gcctttactt ctaggcctgt acggaagtgt 900

tacttctgct ctaaaagctg cggaattgta cccgcggccg atccaccggt gccaccatga 960

aatgggtcac ctttatcagc ctgctgttcc tgttcagcag cgcctacagc cacagccaca 1020

gagacttcca gcctgtgctg catctggtgg ccctgaactc tccactgagt ggtggcatga 1080

gaggcatcag aggggctgac ttccagtgct tccagcaggt gagtatctca gggatccaga 1140

catggggata tgggaggtgc ctctgatccc agggctcact gtgggtctct ctgttcacag 1200

gccagagctg ttggactggc tggaaccttc agagccttcc tgagcagcag actgcaggac 1260

ctgtacagca ttgtcagaag ggcagacaga gctgctgtgc ccattgtgaa cctgaaggat 1320

gaactgctgt tccctagctg ggaagccctg ttctctggct ctgagggacc tctgaaacct 1380

ggggccagaa tcttcagctt tgatggcaag gatgtgctga gacaccccac ctggcctcag 1440

aaatctgtgt ggcatggctc tgaccccaat ggcagaaggc tgacagagtc ctactgtgaa 1500

acttggagaa cagaggcccc atctgccaca ggccaggcca gttcacttct tggaggtaga 1560

ctgctgggcc agtctgcagc ctcttgtcac catgcctaca ttgtgctgtg cattgagaac 1620

agcttcatga cagccagcaa gtgaaagctt atcaggtgag tggcgggccc tgagctgggg 1680

ggcgggggtg ttggctctgg aggctgggtc tgagcgtaat tttgcacccc cgcgtccctg 1740

caggataccg tcgactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 1800

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 1860

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 1920

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 1980

tggggagaga tcgatctagg aacccctagt gatggagttg gccactccct ctctgcgcgc 2040

tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 2100

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aa 2142

<210> 8

<211> 201

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B36 protein product amino acid sequence

<400> 8

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala

20 25 30

Leu Asn Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp

35 40 45

Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe

50 55 60

Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg

65 70 75 80

Arg Ala Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu

85 90 95

Leu Phe Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu

100 105 110

Lys Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg

115 120 125

His Pro Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn

130 135 140

Gly Arg Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala

145 150 155 160

Pro Ser Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu

165 170 175

Gly Gln Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile

180 185 190

Glu Asn Ser Phe Met Thr Ala Ser Lys

195 200

<210> 9

<211> 3450

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of B110 expression cassette

<400> 9

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacctctggt cgttacataa cttacggtaa 240

atggcccgcc tggctgaccg cccaacgacc ccgcccattg acgtcaataa tgacgtatgt 300

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 420

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 480

tacttggcag tacatctact cgaggccacg ttctgcttca ctctccccat ctcccccccc 540

tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 600

gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 660

aggcggaaag gtgcggcggc agccaatcaa agcggcgcgc tccgaaagtt tccttttatg 720

gcaaggcggc ggcggcggcg gccctataaa aagcaaaccg cgcggcgggc gggagcggga 780

tcagccaccg cggtggcggc ctaaagtcga cgaggaactg aaaaaccaga aagttaactg 840

gtaagttaag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 900

agaactgctc ctcagtggat gttgccttta cttctaggcc tgtacggaag tgttacttct 960

gctctaaaag ctgcggaatt gtacccgcgg ccggccgcca ccatgtggtg gcgtctgtgg 1020

tggctgctgt tgctgctgct tctgctgtgg cctatggtct gggctgacta caaagacgat 1080

gacgacaagc acagccacag agacttccag cctgtgctgc atctggtggc cctgaactct 1140

ccactgagtg gtggcatgag aggcatcaga ggggctgact tccagtgctt ccagcaggcc 1200

agagctgttg gactggctgg aaccttcaga gccttcctga gcagcagact gcaggacctg 1260

tacagcattg tcagaagggc agacagagct gctgtgccca ttgtgaacct gaaggatgaa 1320

ctgctgttcc ctagctggga agccctgttc tctggctctg agggacctct gaaacctggg 1380

gccagaatct tcagctttga tggcaaggat gtgctgagac accccacctg gcctcagaaa 1440

tctgtgtggc atggctctga ccccaatggc agaaggctga cagagtccta ctgtgaaact 1500

tggagaacag aggccccatc tgccacaggc caggccagtt cacttcttgg aggtagactg 1560

ctgggccagt ctgcagcctc ttgtcaccat gcctacattg tgctgtgcat tgagaacagc 1620

ttcatgacag ccagcaagag aaagcggaga agcggatctg gcgcccctgt gaaacagacc 1680

ctgaacttcg acctgctgaa gctggctggc gacgtggaaa gcaaccctgg acctatgtgg 1740

tggcggctct ggtggttgtt gctccttctc ctgctgctct ggcccatggt ttgggccaac 1800

cggaagtcca gcatcatcat ccgcatgcgc gacgtggtgc tgttcgagaa gaaggtgtac 1860

ctgagcgagt gcaagaccgg caacggcaag aactacagag gcaccatgag caagaccaag 1920

aacggcatca cctgtcagaa gtggtccagc acaagccctc acagacccag attcagcccc 1980

gccacacatc caagcgaagg cctggaagag aactactgca gaaaccccga caacgaccct 2040

caaggccctt ggtgctacac caccgatcct gagaagagat acgactactg cgacatcctg 2100

gaatgcgaag aggaatgcat gcactgcagc ggcgagaact acgacggcaa gatctccaag 2160

accatgagcg gactggaatg ccaggcttgg gacagccagt ctcctcacgc tcacggctac 2220

atccccagca agttccccaa caagaacctg aagaagaatt actgtcggaa ccccgaccgc 2280

gagctgaggc cttggtgttt taccacagat ccaaacaagc gctgggagct gtgcgacatc 2340

cccagatgca caacccctcc acctagcagc ggccctacct accaatgtct gaaaggcacc 2400

ggcgagaatt accggggcaa tgtggctgtg accgtgtccg gccatacctg ccaacattgg 2460

agcgcccaga cacctcacac acacaacaga acccctgaga acttcccctg caagaatctg 2520

gacgaaaact actgtaggaa tcccgatggc aagagggccc catggtgtca caccaccaac 2580

agccaagtcc gctgggagta ctgcaagatc cccagctgtg atagcagccc cgtgtctaca 2640

gaacagctgg cccctacagc tcctcctgag ctgacacctg tggtgcagga ttgctatcac 2700

ggcgacggcc agtcctatag aggcacaagc agcaccacca caaccggcaa gaagtgccag 2760

agctggtcct ccatgacacc ccaccggcac cagaaaaccc cagaaaacta ccccaatgcc 2820

ggcctgacca tgaactattg ccggaatcct gacgccgaca aaggcccctg gtgtttcaca 2880

actgacccca gcgtcagatg ggaatactgt aatctgaaga agtgcagcgg caccgaggcc 2940

tctgttgttg ctcctcctcc ttacccatac gatgttcctg actatgcggg ctatccctat 3000

gacgtcccgg actatgcata aatgcacttc gaattaaacc gctgatcagc ctcgactgtg 3060

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 3120

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 3180

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 3240

gacaatagca ggcatgctgg ggactcgagt agataagtag catggcgggt taatcattaa 3300

ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 3360

tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 3420

cgagcgagcg cgcagcctta attaacctaa 3450

<210> 10

<211> 672

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B110 protein product amino acid sequence

<400> 10

Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp

1 5 10 15

Pro Met Val Trp Ala Asp Tyr Lys Asp Asp Asp Asp Lys His Ser His

20 25 30

Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn Ser Pro Leu

35 40 45

Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln

50 55 60

Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser

65 70 75 80

Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala

85 90 95

Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp

100 105 110

Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg

115 120 125

Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro

130 135 140

Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr

145 150 155 160

Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly

165 170 175

Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala

180 185 190

Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met

195 200 205

Thr Ala Ser Lys Arg Lys Arg Arg Ser Gly Ser Gly Ala Pro Val Lys

210 215 220

Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

225 230 235 240

Asn Pro Gly Pro Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu

245 250 255

Leu Leu Leu Trp Pro Met Val Trp Ala Asn Arg Lys Ser Ser Ile Ile

260 265 270

Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr Leu Ser

275 280 285

Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys

290 295 300

Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His

305 310 315 320

Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu

325 330 335

Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr

340 345 350

Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys

355 360 365

Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile

370 375 380

Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser

385 390 395 400

Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu

405 410 415

Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys

420 425 430

Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg

435 440 445

Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys

450 455 460

Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly

465 470 475 480

His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg

485 490 495

Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg

500 505 510

Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln

515 520 525

Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val

530 535 540

Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val

545 550 555 560

Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser

565 570 575

Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr

580 585 590

Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu

595 600 605

Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys

610 615 620

Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys

625 630 635 640

Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Tyr Pro Tyr

645 650 655

Asp Val Pro Asp Tyr Ala Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

660 665 670

<210> 11

<211> 3255

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of B111 expression cassette

<400> 11

ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct 180

aggaagatcg gaattctcta gaacgcgtgg tacctctggt cgttacataa cttacggtaa 240

atggcccgcc tggctgaccg cccaacgacc ccgcccattg acgtcaataa tgacgtatgt 300

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 420

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 480

tacttggcag tacatctact cgaggccacg ttctgcttca ctctccccat ctcccccccc 540

tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 600

gggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg gggcggggcg 660

aggcggaaag gtgcggcggc agccaatcaa agcggcgcgc tccgaaagtt tccttttatg 720

gcaaggcggc ggcggcggcg gccctataaa aagcaaaccg cgcggcgggc gggagcggga 780

tcagccaccg cggtggcggc ctaaagtcga cgaggaactg aaaaaccaga aagttaactg 840

gtaagttaag tctttttgtc ttttatttca ggtcccggat ccggtggtgg tgcaaatcaa 900

agaactgctc ctcagtggat gttgccttta cttctaggcc tgtacggaag tgttacttct 960

gctctaaaag ctgcggaatt gtacccgcgg ccggccgcca ccatgtctgc acttctgatc 1020

ctagctcttg ttggagctgc agttgctgac tacaaagacg atgacgacaa gcacacccac 1080

caggatttcc agcctgtgct gcatctggtg gccctgaaca cacctctgtc tggcggcatg 1140

agaggcatca gaggcgccga cttccagtgt ttccagcagg ctagagctgt gggcctgagc 1200

ggaaccttca gagccttcct gtctagcaga ctgcaggacc tgtacagcat cgtgcggaga 1260

gccgatagag gcagcgtgcc aatcgtgaac ctgaaggacg aggtgctgag ccctagctgg 1320

gactctctgt ttagcggctc tcagggacag ctgcagcctg gcgctagaat cttcagcttc 1380

gacggcaggg acgtgctgag acatcctgcc tggcctcaga aatctgtgtg gcacggctct 1440

gatcctagcg gcagacggct gatggaaagc tactgcgaga catggcggac cgagacaaca 1500

ggcgctacag gacaggcaag ctctctgctg agtggcagac tgctggaaca gaaggccgcc 1560

agctgtcaca acagctacat cgtgctgtgc atcgagaaca gcttcatgac cagcttcagc 1620

aagagaaagc ggagaagcgg atctggcgcc acgaacttct ctctgttaaa gcaagcagga 1680

gacgtggaag aaaaccccgg tccgatgtct gcacttctga tcctagctct tgttggagct 1740

gcagttgctg tgtacctgag cgagtgcaag acaggcatcg gcaacggcta cagaggcacc 1800

atgagcagga caaagtctgg cgtggcctgt cagaagtggg gcgctacatt tcctcacgtg 1860

cccaactaca gccccagcac acaccctaac gaaggcctgg aagagaacta ctgcagaaac 1920

cccgacaacg acgagcaagg cccttggtgc tacaccaccg atcctgacaa gagatacgac 1980

tactgcaaca tccccgagtg cgaagaggaa tgcatgtact gcagcggcga gaagtacgag 2040

ggcaagatca gcaagaccat gagcggcctg gattgtcagg cctgggactc tcagtctcct 2100

cacgctcacg gctacatccc cgctaagttc cccagcaaga acctgaagat gaattactgt 2160

aggaaccctg acggcgagcc cagaccatgg tgcttcacaa cagaccccac caagagatgg 2220

gagtactgtg acatccccag atgcaccaca cctccaccac ctccatctcc aacctaccag 2280

tgcctgaaag gcagaggcga gaactaccgg ggcacagtgt ctgtgaccgt gtctggcaag 2340

acatgccagc gttggagcga gcagacaccc cacagacaca atagaacccc tgagaacttc 2400

ccctgcaaaa acctcgagga aaactactgc cgcaatccag acggcgagac agccccatgg 2460

tgttatacca cagacagcca gctgcgctgg gagtattgcg agatccctag ctgcgagagc 2520

agcgcctctc ctgaccagag cgattcttct gtgcctcctg aggaacagac acccgtggtg 2580

caagagtgct accagtctga cggccagagc tacaggggca caagcagcac aaccatcacc 2640

ggcaagaagt gccagagctg ggccgctatg ttccctcacc ggcactctaa gacacccgag 2700

aactttccag acgccggcct cgagatgaac tattgccgga atcctgatgg cgacaaaggc 2760

ccctggtacc catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat 2820

gcataaatgc acttcgaatt aaaccgctga tcagcctcga ctgtgccttc tagttgccag 2880

ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2940

gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 3000

ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 3060

gctggggact cgagtagata agtagcatgg cgggttaatc attaactaca aggaacccct 3120

agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 3180

aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 3240

ccttaattaa cctaa 3255

<210> 12

<211> 607

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> B111 protein product amino acid sequence

<400> 12

Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val Ala Asp

1 5 10 15

Tyr Lys Asp Asp Asp Asp Lys His Thr His Gln Asp Phe Gln Pro Val

20 25 30

Leu His Leu Val Ala Leu Asn Thr Pro Leu Ser Gly Gly Met Arg Gly

35 40 45

Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala Val Gly

50 55 60

Leu Ser Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln Asp Leu

65 70 75 80

Tyr Ser Ile Val Arg Arg Ala Asp Arg Gly Ser Val Pro Ile Val Asn

85 90 95

Leu Lys Asp Glu Val Leu Ser Pro Ser Trp Asp Ser Leu Phe Ser Gly

100 105 110

Ser Gln Gly Gln Leu Gln Pro Gly Ala Arg Ile Phe Ser Phe Asp Gly

115 120 125

Arg Asp Val Leu Arg His Pro Ala Trp Pro Gln Lys Ser Val Trp His

130 135 140

Gly Ser Asp Pro Ser Gly Arg Arg Leu Met Glu Ser Tyr Cys Glu Thr

145 150 155 160

Trp Arg Thr Glu Thr Thr Gly Ala Thr Gly Gln Ala Ser Ser Leu Leu

165 170 175

Ser Gly Arg Leu Leu Glu Gln Lys Ala Ala Ser Cys His Asn Ser Tyr

180 185 190

Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ser Phe Ser Lys Arg

195 200 205

Lys Arg Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln

210 215 220

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Ala Leu Leu Ile

225 230 235 240

Leu Ala Leu Val Gly Ala Ala Val Ala Val Tyr Leu Ser Glu Cys Lys

245 250 255

Thr Gly Ile Gly Asn Gly Tyr Arg Gly Thr Met Ser Arg Thr Lys Ser

260 265 270

Gly Val Ala Cys Gln Lys Trp Gly Ala Thr Phe Pro His Val Pro Asn

275 280 285

Tyr Ser Pro Ser Thr His Pro Asn Glu Gly Leu Glu Glu Asn Tyr Cys

290 295 300

Arg Asn Pro Asp Asn Asp Glu Gln Gly Pro Trp Cys Tyr Thr Thr Asp

305 310 315 320

Pro Asp Lys Arg Tyr Asp Tyr Cys Asn Ile Pro Glu Cys Glu Glu Glu

325 330 335

Cys Met Tyr Cys Ser Gly Glu Lys Tyr Glu Gly Lys Ile Ser Lys Thr

340 345 350

Met Ser Gly Leu Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala

355 360 365

His Gly Tyr Ile Pro Ala Lys Phe Pro Ser Lys Asn Leu Lys Met Asn

370 375 380

Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr

385 390 395 400

Asp Pro Thr Lys Arg Trp Glu Tyr Cys Asp Ile Pro Arg Cys Thr Thr

405 410 415

Pro Pro Pro Pro Pro Ser Pro Thr Tyr Gln Cys Leu Lys Gly Arg Gly

420 425 430

Glu Asn Tyr Arg Gly Thr Val Ser Val Thr Val Ser Gly Lys Thr Cys

435 440 445

Gln Arg Trp Ser Glu Gln Thr Pro His Arg His Asn Arg Thr Pro Glu

450 455 460

Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp

465 470 475 480

Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr Asp Ser Gln Leu Arg Trp

485 490 495

Glu Tyr Cys Glu Ile Pro Ser Cys Glu Ser Ser Ala Ser Pro Asp Gln

500 505 510

Ser Asp Ser Ser Val Pro Pro Glu Glu Gln Thr Pro Val Val Gln Glu

515 520 525

Cys Tyr Gln Ser Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr

530 535 540

Ile Thr Gly Lys Lys Cys Gln Ser Trp Ala Ala Met Phe Pro His Arg

545 550 555 560

His Ser Lys Thr Pro Glu Asn Phe Pro Asp Ala Gly Leu Glu Met Asn

565 570 575

Tyr Cys Arg Asn Pro Asp Gly Asp Lys Gly Pro Trp Tyr Pro Tyr Asp

580 585 590

Val Pro Asp Tyr Ala Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

595 600 605

<210> 13

<211> 2214

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAVXL32 coding sequence

<400> 13

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc 480

ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540

gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga 600

cctaatacaa tggcttcagg cggtggcgca ccaatggcag acaataacga aggcgccgac 660

ggagtgggta atgcctcagg aaattggcat tgcgattcca catggctggg cgacagagtc 720

atcaccacca gcacccgaac atgggccttg cccacctata acaaccacct ctacaagcaa 780

atctccagtg cttcaacggg ggccagcaac gacaaccact acttcggcta cagcaccccc 840

tgggggtatt ttgatttcaa cagattccac tgccatttct caccacgtga ctggcagcga 900

ctcatcaaca acaattgggg attccggccc aagagactca acttcaagct cttcaacatc 960

caagtcaagg aggtcacgac gaatgatggc gtcacgacca tcgctaataa ccttaccagc 1020

acggttcaag tcttctcgga ctcggagtac cagttgccgt acgtcctcgg ctctgcgcac 1080

cagggctgcc tccctccgtt cccggcggac gtgttcatga ttccgcaata cggctacctg 1140

acgctcaaca atggcagcca agccgtggga cgttcatcct tttactgcct ggaatatttc 1200

ccttctcaga tgctgagaac gggcaacaac tttaccttca gctacacctt tgaggaagtg 1260

cctttccaca gcagctacgc gcacagccag agcctggacc ggctgatgaa tcctctcatc 1320

gaccagtacc tgtattacct gaacagaact cagaatcagt ccggaagtgc ccaaaacaag 1380

gacttgctgt ttagccgtgg gtctccagct ggcatgtctg ttcagcccaa aaactggcta 1440

cctggaccct gttaccggca gcagcgcgtt tctaaaacaa aaacagacaa caacaacagc 1500

aactttacct ggactggtgc ttcaaaatat aacctcaatg ggcgtgaatc catcatcaac 1560

cctggcactg ctatggcctc acacaaagac gacaaagaca agttctttcc catgagcggt 1620

gtcatgattt ttggaaagga gagcgccgga gcttcaaaca ctgcattgga caatgtcatg 1680

atcacagacg aagaggaaat caaagccact aaccccgtgg ccaccgaaag atttgggact 1740

gtggcagtca atctccagag cagcagcaca gaccctgcga ccggagatgt gcatgttatg 1800

ggagccttac ctggaatggt gtggcaagac agagacgtat acctgcaggg tcctatttgg 1860

gccaaaattc ctcacacgga tggacacttt cacccgtctc ctctcatggg cggctttgga 1920

cttaagcacc cgcctcctca gatcctcatc aaaaacacgc ctgttcctgc gaatcctccg 1980

gcagagtttt cggctacaaa gtttgcttca ttcatcaccc agtattccac aggacaagtg 2040

agcgtggaga ttgaatggga gctgcagaaa gaaaacagca aacgctggaa tcccgaagtg 2100

cagtatacat ctaactatgc aaaatctgcc aacgttgatt ttactgtgga caacaatgga 2160

ctttatactg agcctcgccc cattggcacc cgttacctca cccgtcccct gtaa 2214

<210> 14

<211> 737

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> AAVXL32 amino acid sequence

<400> 14

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ser Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn

435 440 445

Arg Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe

450 455 460

Ser Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp

485 490 495

Asn Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu

500 505 510

Asn Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His

515 520 525

Lys Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe

530 535 540

Gly Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met

545 550 555 560

Ile Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu

565 570 575

Arg Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro

580 585 590

Ala Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly

705 710 715 720

Leu Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro

725 730 735

Leu

<210> 15

<211> 549

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> codon optimized human endostatin coding nucleic acid sequence

<400> 15

cacagccaca gagacttcca gcctgtgctg catctggtgg ccctgaactc tccactgagt 60

ggtggcatga gaggcatcag aggggctgac ttccagtgct tccagcaggc cagagctgtt 120

ggactggctg gaaccttcag agccttcctg agcagcagac tgcaggacct gtacagcatt 180

gtcagaaggg cagacagagc tgctgtgccc attgtgaacc tgaaggatga actgctgttc 240

cctagctggg aagccctgtt ctctggctct gagggacctc tgaaacctgg ggccagaatc 300

ttcagctttg atggcaagga tgtgctgaga caccccacct ggcctcagaa atctgtgtgg 360

catggctctg accccaatgg cagaaggctg acagagtcct actgtgaaac ttggagaaca 420

gaggccccat ctgccacagg ccaggccagt tcacttcttg gaggtagact gctgggccag 480

tctgcagcct cttgtcacca tgcctacatt gtgctgtgca ttgagaacag cttcatgaca 540

gccagcaag 549

<210> 16

<211> 1164

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> codon optimized human angiostatin coding nucleic acid sequence

<400> 16

aaccggaagt ccagcatcat catccgcatg cgcgacgtgg tgctgttcga gaagaaggtg 60

tacctgagcg agtgcaagac cggcaacggc aagaactaca gaggcaccat gagcaagacc 120

aagaacggca tcacctgtca gaagtggtcc agcacaagcc ctcacagacc cagattcagc 180

cccgccacac atccaagcga aggcctggaa gagaactact gcagaaaccc cgacaacgac 240

cctcaaggcc cttggtgcta caccaccgat cctgagaaga gatacgacta ctgcgacatc 300

ctggaatgcg aagaggaatg catgcactgc agcggcgaga actacgacgg caagatctcc 360

aagaccatga gcggactgga atgccaggct tgggacagcc agtctcctca cgctcacggc 420

tacatcccca gcaagttccc caacaagaac ctgaagaaga attactgtcg gaaccccgac 480

cgcgagctga ggccttggtg ttttaccaca gatccaaaca agcgctggga gctgtgcgac 540

atccccagat gcacaacccc tccacctagc agcggcccta cctaccaatg tctgaaaggc 600

accggcgaga attaccgggg caatgtggct gtgaccgtgt ccggccatac ctgccaacat 660

tggagcgccc agacacctca cacacacaac agaacccctg agaacttccc ctgcaagaat 720

ctggacgaaa actactgtag gaatcccgat ggcaagaggg ccccatggtg tcacaccacc 780

aacagccaag tccgctggga gtactgcaag atccccagct gtgatagcag ccccgtgtct 840

acagaacagc tggcccctac agctcctcct gagctgacac ctgtggtgca ggattgctat 900

cacggcgacg gccagtccta tagaggcaca agcagcacca ccacaaccgg caagaagtgc 960

cagagctggt cctccatgac accccaccgg caccagaaaa ccccagaaaa ctaccccaat 1020

gccggcctga ccatgaacta ttgccggaat cctgacgccg acaaaggccc ctggtgtttc 1080

acaactgacc ccagcgtcag atgggaatac tgtaatctga agaagtgcag cggcaccgag 1140

gcctctgttg ttgctcctcc tcct 1164

<210> 17

<211> 552

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> codon-optimized nucleic acid sequence encoding murine endostatin

<400> 17

cacacccacc aggatttcca gcctgtgctg catctggtgg ccctgaacac acctctgtct 60

ggcggcatga gaggcatcag aggcgccgac ttccagtgtt tccagcaggc tagagctgtg 120

ggcctgagcg gaaccttcag agccttcctg tctagcagac tgcaggacct gtacagcatc 180

gtgcggagag ccgatagagg cagcgtgcca atcgtgaacc tgaaggacga ggtgctgagc 240

cctagctggg actctctgtt tagcggctct cagggacagc tgcagcctgg cgctagaatc 300

ttcagcttcg acggcaggga cgtgctgaga catcctgcct ggcctcagaa atctgtgtgg 360

cacggctctg atcctagcgg cagacggctg atggaaagct actgcgagac atggcggacc 420

gagacaacag gcgctacagg acaggcaagc tctctgctga gtggcagact gctggaacag 480

aaggccgcca gctgtcacaa cagctacatc gtgctgtgca tcgagaacag cttcatgacc 540

agcttcagca ag 552

<210> 18

<211> 1017

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> codon optimized mouse source angiostatin coding nucleic acid sequence

<400> 18

gtgtacctga gcgagtgcaa gacaggcatc ggcaacggct acagaggcac catgagcagg 60

acaaagtctg gcgtggcctg tcagaagtgg ggcgctacat ttcctcacgt gcccaactac 120

agccccagca cacaccctaa cgaaggcctg gaagagaact actgcagaaa ccccgacaac 180

gacgagcaag gcccttggtg ctacaccacc gatcctgaca agagatacga ctactgcaac 240

atccccgagt gcgaagagga atgcatgtac tgcagcggcg agaagtacga gggcaagatc 300

agcaagacca tgagcggcct ggattgtcag gcctgggact ctcagtctcc tcacgctcac 360

ggctacatcc ccgctaagtt ccccagcaag aacctgaaga tgaattactg taggaaccct 420

gacggcgagc ccagaccatg gtgcttcaca acagacccca ccaagagatg ggagtactgt 480

gacatcccca gatgcaccac acctccacca cctccatctc caacctacca gtgcctgaaa 540

ggcagaggcg agaactaccg gggcacagtg tctgtgaccg tgtctggcaa gacatgccag 600

cgttggagcg agcagacacc ccacagacac aatagaaccc ctgagaactt cccctgcaaa 660

aacctcgagg aaaactactg ccgcaatcca gacggcgaga cagccccatg gtgttatacc 720

acagacagcc agctgcgctg ggagtattgc gagatcccta gctgcgagag cagcgcctct 780

cctgaccaga gcgattcttc tgtgcctcct gaggaacaga cacccgtggt gcaagagtgc 840

taccagtctg acggccagag ctacaggggc acaagcagca caaccatcac cggcaagaag 900

tgccagagct gggccgctat gttccctcac cggcactcta agacacccga gaactttcca 960

gacgccggcc tcgagatgaa ctattgccgg aatcctgatg gcgacaaagg cccctgg 1017

<210> 19

<211> 549

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> original (non-codon optimized) human endostatin-encoding nucleic acid sequence

<400> 19

cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60

ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120

gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180

gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240

cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300

ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360

catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420

gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480

agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540

gcctccaag 549

<210> 20

<211> 1164

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> original (non-codon optimized) human angiostatin-encoding nucleic acid sequence

<400> 20

aacaggaagt cctccataat cattaggatg agagatgtag ttttatttga aaagaaagtg 60

tatctctcag agtgcaagac tgggaatgga aagaactaca gagggacgat gtccaaaaca 120

aaaaatggca tcacctgtca aaaatggagt tccacttctc cccacagacc tagattctca 180

cctgctacac acccctcaga gggactggag gagaactact gcaggaatcc agacaacgat 240

ccgcaggggc cctggtgcta tactactgat ccagaaaaga gatatgacta ctgcgacatt 300

cttgagtgtg aagaggaatg tatgcattgc agtggagaaa actatgacgg caaaatttcc 360

aagaccatgt ctggactgga atgccaggcc tgggactctc agagcccaca cgctcatgga 420

tacattcctt ccaaatttcc aaacaagaac ctgaagaaga attactgtcg taaccccgat 480

agggagctgc ggccttggtg tttcaccacc gaccccaaca agcgctggga actttgtgac 540

atcccccgct gcacaacacc tccaccatct tctggtccca cctaccagtg tctgaaggga 600

acaggtgaaa actatcgcgg gaatgtggct gttaccgtgt ccgggcacac ctgtcagcac 660

tggagtgcac agacccctca cacacataac aggacaccag aaaacttccc ctgcaaaaat 720

ttggatgaaa actactgccg caatcctgac ggaaaaaggg ccccatggtg ccatacaacc 780

aacagccaag tgcggtggga gtactgtaag ataccgtcct gtgactcctc cccagtatcc 840

acggaacaat tggctcccac agcaccacct gagctaaccc ctgtggtcca ggactgctac 900

catggtgatg gacagagcta ccgaggcaca tcctccacca ccaccacagg aaagaagtgt 960

cagtcttggt catctatgac accacaccgg caccagaaga ccccagaaaa ctacccaaat 1020

gctggcctga caatgaacta ctgcaggaat ccagatgccg ataaaggccc ctggtgtttt 1080

accacagacc ccagcgtcag gtgggagtac tgcaacctga aaaaatgctc aggaacagaa 1140

gcgagtgttg tagcacctcc gcct 1164

<210> 21

<211> 552

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> original (non-codon optimized) nucleic acid sequence encoding murine endostatin

<400> 21

catactcatc aggactttca gccagtgctc cacctggtgg cactgaacac ccccctgtct 60

ggaggcatgc gtggtatccg tggagcagat ttccagtgct tccagcaagc ccgagccgtg 120

gggctgtcgg gcaccttccg ggctttcctg tcctctaggc tgcaggatct ctatagcatc 180

gtgcgccgtg ctgaccgggg gtctgtgccc atcgtcaacc tgaaggacga ggtgctatct 240

cccagctggg actccctgtt ttctggctcc cagggtcaac tgcaacccgg ggcccgcatc 300

ttttcttttg acggcagaga tgtcctgaga cacccagcct ggccgcagaa gagcgtatgg 360

cacggctcgg accccagtgg gcggaggctg atggagagtt actgtgagac atggcgaact 420

gaaactactg gggctacagg tcaggcctcc tccctgctgt caggcaggct cctggaacag 480

aaagctgcga gctgccacaa cagctacatc gtcctgtgca ttgagaatag cttcatgacc 540

tctttctcca aa 552

<210> 22

<211> 1017

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> original (non-codon optimized) murine angiostatin-encoding nucleic acid sequences

<400> 22

gtgtatctgt cagaatgtaa gaccggcatc ggcaacggct acagaggaac catgtccagg 60

acaaagagtg gtgttgcctg tcaaaagtgg ggtgccacgt tcccccacgt acccaactac 120

tctcccagta cacatcccaa tgagggacta gaagagaact actgtaggaa cccagacaat 180

gatgaacaag ggccttggtg ctacactaca gatccggaca agagatatga ctactgcaac 240

attcctgaat gtgaagagga atgcatgtac tgcagtggag aaaagtatga gggcaaaatc 300

tccaagacca tgtctggact tgactgccag gcctgggatt ctcagagccc acatgctcat 360

ggatacatcc ctgccaaatt tccaagcaag aacctgaaga tgaattattg ccacaaccct 420

gacggggagc caaggccctg gtgcttcaca acagacccca ccaaacgctg ggaatactgt 480

gacatccccc gctgcacaac acccccgccc ccacccagcc caacctacca atgtctgaaa 540

ggaagaggtg aaaattaccg agggaccgtg tctgtcaccg tgtctgggaa aacctgtcag 600

cgctggagtg agcaaacccc tcataggcac aacaggacac cagaaaattt cccctgcaaa 660

aatctggaag agaactactg ccggaaccca gatggagaaa ctgctccctg gtgctatacc 720

actgacagcc agctgaggtg ggagtactgt gagattccat cctgcgagtc ctcagcatca 780

ccagaccagt cagattcctc agttccacca gaggagcaaa cacctgtggt ccaggaatgc 840

taccagagcg atgggcagag ctatcggggt acatcgtcca ctaccatcac agggaagaag 900

tgccagtcct gggcagctat gtttccacac aggcattcga agaccccaga gaacttccca 960

gatgctggct tggagatgaa ctactgcagg aacccggatg gtgacaaggg cccttgg 1017