CN114107313B - Transgenic expression cassettes for the treatment of multiple sclerosis - Google Patents

Transgenic expression cassettes for the treatment of multiple sclerosis Download PDF

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CN114107313B
CN114107313B CN202111362629.7A CN202111362629A CN114107313B CN 114107313 B CN114107313 B CN 114107313B CN 202111362629 A CN202111362629 A CN 202111362629A CN 114107313 B CN114107313 B CN 114107313B
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吴侠
仲晨
肖啸
郑静
郑浩
陈慧
杜增民
蒋威
程诚
赵锴
王慧
李尚�
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Abstract

The present disclosure relates to nucleic acid molecules encoding cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), nucleic acid molecules encoding CD40 protein, transgenic expression cassettes comprising the same, medicaments comprising the same, and use of the transgenic expression cassettes in the preparation of medicaments for treating multiple sclerosis.

Description

Transgenic expression cassettes for the treatment of multiple sclerosis
Technical Field
The present disclosure is in the field of gene therapy. The present disclosure relates to nucleic acid molecules encoding cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), nucleic acid molecules encoding CD40 protein, transgenic expression cassettes comprising the same, medicaments comprising the same, and use of the transgenic expression cassettes in the preparation of medicaments for treating multiple sclerosis.
Background
Adeno-associated virus (AAV) technology is one of the important technologies currently widely used in the field of gene therapy. AAV has the advantages of low pathogenicity, high-efficiency long-term gene expression, easy gene operation and the like.
B7/CD28 and CD40/CD40L are two important co-stimulatory signal pathways, provide co-stimulatory signals during T cell and B cell activation, and play an important role in the occurrence and development of autoimmune diseases and transplant rejection. The main risk of autoimmune diseases is the hyperadaptive immune system. Cytotoxic T lymphocyte-associated antigen 4 (Cytotoxic T lymphocyte-associated antigen-4, CTLA-4) is a T cell transmembrane receptor that competitively binds with CD28 to B7 molecules, thereby blocking B7 binding to CD28 and exerting an immunonegative regulatory effect. CD40 is a transmembrane protein on the surface of antigen presenting cells and B cells. CD40 binds with its ligand CD40L, induces antigen presenting cells to express B7 molecules, promotes activation of the antigen presenting cells themselves, activates T cells, and plays an important role in B cell differentiation and maturation and antibody production. Thus, blocking the CD40/CD40L pathway inhibits T, B cell activation.
Multiple sclerosis (Multiple sclerosis, MS) is a chronic multifocal central nervous system inflammatory disease. The incidence and prevalence of multiple sclerosis is increasing in both developed and developing countries. Multiple sclerosis is a complex disease, the root cause of which is still uncertain, but is thought to be related to environmental factors such as vitamin D deficiency or exposure to ultraviolet B light (UVB), epstein-Barr virus (Epstein-Barr virus) infection, obesity and smoking. MS lesions are characterized by immune cell infiltration, demyelination, axonal degeneration, and astrocyte proliferation. Experimental Autoimmune Encephalomyelitis (EAE) is the most common and clinically relevant murine model of human Multiple Sclerosis (MS). At present, th1, th17 and B cells are known to be involved in the pathogenesis of multiple sclerosis. MS lesions are formed by CD4 + T cell activated butThe amplification and damage is caused by CD8 + T-cell mediated. Thus, blocking CD4 by blocking T cell costimulation of immune pathways + T、CD8 + Abnormal activation of T and B cells may contribute to the treatment of MS. The use of Abatacept for the treatment of Relapsing Remitting Multiple Sclerosis (RRMS) was reported in 2016, but the clinical effect was less than ideal (Khory S J, rochon J, ding L et al, ACCLAIM: A randomized trial of Abatacept (CTLA 4-Ig) for relapsing-remitting multiple scirosis, 2016,23 (5): 686).
Thus, it would be desirable to have CTLA-4 coding sequences and CD40 coding sequences with higher expression levels, and drugs with better therapeutic effects on multiple sclerosis.
Disclosure of Invention
In order to solve the above technical problems, first, the present inventors have optimally designed an original human CTLA4 sequence and an original human CD40 sequence, and obtained a codon-optimized CTLA4 sequence and CD40 sequence with higher expression level.
In a first aspect, the present disclosure provides a nucleic acid molecule encoding cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) having a nucleotide sequence identical to SEQ ID NO:1, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity.
In one embodiment, the nucleic acid molecule comprises SEQ ID NO:1, and a nucleotide sequence shown in the specification. In a preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is as set forth in SEQ ID NO: 1.
The nucleic acid molecules encoding CTLA-4 of the present disclosure comprise a codon-optimized human CTLA 4-encoding nucleic acid sequence having a higher CTLA4 expression level as compared to the original human CTLA 4-encoding nucleic acid sequence that is not codon-optimized.
In a second aspect, the present disclosure provides a nucleic acid molecule encoding a CD40 protein having a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO:2, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity.
In one embodiment, the nucleic acid molecule comprises SEQ ID NO:2, and a nucleotide sequence shown in the following formula. In a preferred embodiment, the nucleotide sequence of the above nucleic acid molecule is as set forth in SEQ ID NO: 2.
The nucleic acid molecules encoding CD40 proteins of the present disclosure comprise a codon-optimized human CD40 encoding nucleic acid sequence that has a higher level of CD40 protein expression than the original human CD40 encoding nucleic acid sequence that was 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, an Fc sequence, mini-polyA.
In one embodiment, the promoter is selected from: CB promoter, CAG promoter, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16, VP64 promoter, tuj1 promoter, GFAP promoter, vimentin promoter, RPE65 promoter, VMD2 promoter, synapsin promoter, VGAT promoter, DAT promoter, TH promoter, osteocalcin promoter, CMV promoter, PGK1 promoter, ubc promoter, TRE promoter, UAS promoter, ac5 promoter, caMKIIa promoter, GDS promoter, ADH1 promoter. In a preferred embodiment, the promoter is a CB promoter.
In one embodiment, the transgenic expression cassette further comprises: introns, such as Mini introns; signal peptides, such as albumin signal peptide, CTLA4 original signal peptide, FIB signal peptide and BM40 signal peptide, preferably CTLA4 original signal peptide, FIB signal peptide, BM40 signal peptide, yfkN signal peptide, bpr signal peptide, mpr signal peptide, phoB signal peptide, wapA signal peptide, abnA signal peptide, alpha factor signal peptide, acid phosphatase (PHO 5) signal peptide, sucrase (SUC 2 signal peptide), fibroin LC signal peptide, human insulin signal peptide, influenza hemagglutinin signal peptide, gaussia luc signal peptide, human IL-2 signal peptide, human trypsinogen-2 signal peptide, human IgG2H signal peptide, mouse Ig kappa signal peptide, VSV-G signal peptide and human OSM signal peptide; and/or two ITRs at both ends.
In one embodiment, the Fc sequence is as set forth in SEQ ID NO: 3. The Fc sequence disclosed by the disclosure is a sequence optimized by a codon, so that the half-life period of the transgene protein expressed in vivo can be prolonged more effectively, the protein stability is improved, and a better therapeutic effect is realized.
In one embodiment, the nucleotide sequence of the transgene expression cassette is set forth in SEQ ID NO: 4. 6, 8 or 10.
In a fourth aspect, the present disclosure provides a transgenic expression cassette comprising: a promoter, a nucleic acid molecule according to the second aspect, an Fc sequence, mini-polyA.
In one embodiment, the promoter is selected from: CB promoter, CAG promoter, EF1 promoter, ubiquitin promoter, T7 promoter, SV40 promoter, VP16, VP64 promoter, tuj1 promoter, GFAP promoter, vimentin promoter, RPE65 promoter, VMD2 promoter, synapsin promoter, VGAT promoter, DAT promoter, TH promoter, osteocalcin promoter, CMV promoter, PGK1 promoter, ubc promoter, TRE promoter, UAS promoter, ac5 promoter, caMKIIa promoter, GDS promoter, ADH1 promoter. In a preferred embodiment, the promoter is a CB promoter.
In one embodiment, the transgenic expression cassette further comprises: introns, such as mini-introns; signal peptides, such as albumin signal peptide, CD40 original signal peptide, FIB signal peptide, BM40 signal peptide, yfkN signal peptide, bpr signal peptide, mpr signal peptide, phoB signal peptide, wapA signal peptide, abnA signal peptide, alpha factor signal peptide, acid phosphatase (PHO 5) signal peptide, sucrase (SUC 2 signal peptide), fibroin LC signal peptide, human insulin signal peptide, influenza hemagglutinin signal peptide, gaussia luc signal peptide, human IL-2 signal peptide, human trypsinogen-2 signal peptide, human IgG2H signal peptide, mouse Ig kappa signal peptide, VSV-G signal peptide and human OSM signal peptide, preferably CD40 original signal peptide and FIB signal peptide; and/or two ITRs at both ends.
In one embodiment, the Fc sequence is as set forth in SEQ ID NO: 3.
In one embodiment, the nucleotide sequence of the transgene expression cassette is set forth in SEQ ID NO: 12. 14, 16 or 18.
In a fifth aspect, the present disclosure provides the use of a transgenic expression cassette according to the third or fourth aspect or a combination of both in the manufacture of a medicament for the treatment of multiple sclerosis.
In a sixth aspect, the present disclosure provides a medicament comprising: a transgenic expression cassette according to the third aspect, a transgenic expression cassette according to the fourth aspect, or a combination thereof; and AAV capsid proteins.
In one embodiment, the AAV capsid protein is a native AAV capsid protein or an 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-DJ, AAVXL32, AAVrh8, AAVrh39, AAVrh43, AAV32.33, AAVrh10, AAV3B, AAVv, aav.php.b. In a more preferred embodiment, the AAV is AAV8.
In one embodiment, the medicament further comprises an excipient. In one embodiment, the excipient comprises one or more of a polyol, a sugar, a buffer, a preservative, and an inorganic salt.
In one embodiment, the medicament is in the form of an injection such as an injection solution and a sterile powder for injection.
The medicaments of the present disclosure (AAV-mediated CTLA4-Ig, AAV-mediated CD40-Ig, and AAV-mediated CTLA4-Ig and CD40-Ig combination) have potent prophylactic and therapeutic effects on MS. Patients do not need to continuously take medicines to maintain the inhibition of inflammation and disease activities, and can generate lasting immunosuppression response.
In a seventh aspect, the present disclosure provides a method of treating multiple sclerosis comprising administering to a subject in need thereof a therapeutically effective amount of a medicament according to the sixth aspect.
In one embodiment, the drug is administered by systemic or local routes, such as intravenous, intramuscular, subcutaneous, oral, topical, intraperitoneal and intralesional.
Drawings
FIG. 1A shows schematic construction of AAV vector plasmids pAAV-CTLA4-Ig and pAAV-CD 40-Ig.
FIGS. 1B and 1C show the quantitative results of transgene protein expression. 45: CTLA 4-albumin signal peptide; 46: CTLA 4-original signal peptide; 47: CTLA4-FIB signal peptide; 48: CTLA4-BM40 signal peptide; 49: CD 40-original signal peptide; 50: CD 40-albumin signal peptide; 51: CD40-FIB signal peptide; 52: CD40-BM40 signal peptide.
FIG. 1D shows Western blot analysis of pAAV-CTLA4-Ig and pAAV-CD40-Ig expression in HUH-7 cells with optimized or original transgene sequences.
FIG. 1E is a gray scale statistical analysis of the Western blot analysis results of FIG. 1D, each value representing mean.+ -. SEM.
Fig. 2A shows the study design of in vivo prevention experiments.
FIG. 2B shows the levels of CTLA4-Ig and CD40-Ig in mouse serum as determined by ELISA. Each value represents the mean ± SEM.
Figure 2C shows the weight change and average clinical score of mice. Each value represents the mean ± SEM. P <0.05, P <0.01, P <0.001 compared to control.
Fig. 2D shows the results of the behavioural tension experiments of the mice. Each value represents the mean ± SEM. P <0.05, P <0.01, P <0.001 compared to control.
Figure 2E shows the survival rate of mice.
Fig. 3A shows representative images of astrocyte activation detected by immunofluorescence with the specific marker Glial Fibrillary Acidic Protein (GFAP). Scale bar: the upper row is 100 μm and the lower row is 50 μm. Each value represents the mean ± SEM. * P <0.05.
Fig. 3B shows a representative image of microglial activation detected by immunofluorescence with the specific marker IBA 1. Scale bar: the upper row is 100 μm and the lower row is 50 μm. Each value represents the mean ± SEM. * P <0.05.
Fig. 3C shows representative H & E staining for white matter inflammatory infiltrates (n=2). Scale bar: the upper row is 500 μm and the lower row is 100 μm.
Fig. 3D shows demyelinated Luxol Fast Blue (LFB) staining (n=2). Scale bar: the upper row is 500 μm and the lower row is 100 μm.
Fig. 3E shows CD4 immunohistochemistry (n=2). Scale bar: the upper row is 500 μm and the lower row is 100 μm.
Fig. 3F shows CD8 immunohistochemistry (n=2). Scale bar: the upper row is 500 μm and the lower row is 100 μm.
FIG. 4A shows MOG in ELISA-assayed mouse serum 35-55 Specific IgG levels. Each value represents the mean ± SEM. * P (P)<0.05，***P<0.001。
Fig. 4B shows the mRNA levels of pro-inflammatory and anti-inflammatory cytokines in spinal cord samples analyzed by real-time quantitative PCR. Each value represents the mean ± SEM.
Fig. 5A shows plasma ALT and AST levels at day 7 and day 35 after AAV injection.
Fig. 5B shows that the hematological toxicity quantified from peripheral blood at day 35 post EAE immunization included White Blood Cells (WBC), lymphocytes (Lymph), platelets (PLT), granulocytes (Granu), red Blood Cells (RBC), and Hemoglobin (HGB) (n=8 per group). * P <0.05, P <0.01, compared to healthy controls.
Fig. 6A shows the study design of in vivo experiments.
Figure 6B shows the change in body weight and average clinical score (MCS) of mice from day 7 post EAE induction. Each value represents the mean ± SEM. * P <0.05, < P <0.001, compared to control; # P <0.01, compared to betanaproxen (belatacept).
Figure 6C shows survival of mice on day 43.
Fig. 7A shows representative images of astrocyte activation detected by immunofluorescence with the specific marker Glial Fibrillary Acidic Protein (GFAP). Scale bar: the upper row is 100 μm and the lower row is 50 μm.
Fig. 7B shows representative images of microglial activation detected by immunofluorescence with the specific marker IBA 1. Scale bar: the upper row is 100 μm and the lower row is 50 μm.
Fig. 7C shows the result of quantification of fluorescence intensity (n=3-4). Each value represents the mean ± SEM. * P <0.05, < P <0.001, compared to control; #P <0.05, #P <0.01, compared to Betazepine.
Fig. 8A shows the study design of in vivo treatment experiments.
Fig. 8B shows the weight change and average clinical score (MCS) of mice from day 7 post EAE induction. Each value represents the mean ± SEM. * P <0.0001.
Figure 8C shows the survival rate of mice on day 59.
Fig. 8D shows demyelinated Luxol Fast Blue (LFB) staining. Scale bar: the upper row is 500 μm and the lower row is 100 μm.
FIG. 9A shows MOG35-55 specific IgG levels in mouse serum as determined by ELISA. Each value represents the mean ± SEM.
Fig. 9B shows the mRNA levels of pro-inflammatory cytokines in spinal cord samples analyzed by real-time quantitative PCR. Each value represents the mean ± SEM. * P <0.05, < P <0.01, < P <0.001.
Fig. 9C shows the mRNA levels of pro-inflammatory cytokines in spleen samples analyzed by real-time quantitative PCR. Each value represents the mean ± SEM. * P <0.05, < P <0.01, < P <0.001.
FIG. 10 shows the codon-optimized human CTLA4 base sequence (SEQ ID NO: 1).
FIG. 11 shows the codon-optimized human CD40 base sequence (SEQ ID NO: 2).
FIG. 12 shows the codon-optimized human FC base sequence (SEQ ID NO: 3).
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 sequences listed herein are in single stranded form, oriented from 5 'to 3', left to right. The nucleotides and amino acids provided herein are in the format suggested by the IUPACIUB biochemical nomenclature committee, and for amino acids are in a single letter code.
Unless otherwise indicated, "nucleic acid" refers to polymeric forms of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. Nucleic acids may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.
In this document, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to").
The terms "patient" and "subject" are used interchangeably herein and in their conventional sense to refer to an organism suffering from or susceptible to a disorder that can be prevented or treated by administration of the medicaments of the present disclosure, and include humans and non-human animals (e.g., rodents or other mammals).
Herein, the term "treatment" includes: (1) Inhibiting a condition, disease, or disorder, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence or the progression of at least one clinical or sub-clinical symptom thereof; or (2) alleviating the disease, i.e., causing regression of at least one of the condition, disease, or disorder, or a clinical or subclinical symptom thereof.
Herein, the term "prevention" 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 have or be susceptible to the condition, disease or disorder but who has not yet experienced or exhibited the clinical or subclinical symptom of the condition, disease or disorder.
In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cattle, 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, wild birds and game birds such as chickens, turkeys and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.
As used herein, the term "therapeutically effective amount" refers to the dosage administered that produces a prophylactic or therapeutic effect. For example, a therapeutically effective amount of a drug suitable for treating multiple sclerosis is an amount that is capable of preventing or ameliorating one or more symptoms associated with multiple sclerosis.
In this context, the term "improvement" 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.
In some embodiments, the transgene expression cassette is packaged into an AAV particle to deliver the transgene product into the target cell. In addition to the sequences encoding the protein products, the transgenic expression cassettes may also include other regulatory elements, such as Inverted Terminal Repeats (ITRs), specific promoters/enhancers, polyas, regulatory introns, etc., to enhance or attenuate expression of the transgenic products.
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. ITRs herein include, but are not limited to, terminal repeats from type 1-11 AAV (avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). Furthermore, AAV terminal repeats 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 with therapeutic effects.
In this context, the term "codon optimization" refers to a nucleic acid sequence modified from its native form. Such modifications result in differences of one or more base pairs, with or without changes in the corresponding amino acid sequence.
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 type 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV) and other engineered AAV capsid proteins (e.g., capsid proteins of engineered AAV type 1-11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV). Genomic sequences, ITR sequences, rep and Cap proteins of different AAV serotypes are known in the art. These sequences may be found in the literature or in public databases, such as the GenBank database. For example, specific sequences of AAV-DJ have been described in WO 2021050970 A1; specific sequences of AAVXL32 have been described in US 2019/036676 A1; AAVrh8 and AAVrh10 can be seen in Choudhury SR et al, in Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene therapy. Mol Ther.,2016, 24 (7): 1247-57; AAVrh39, AAVrh43, AAV32.33, AAV3B, AAVv and AAV.PHP.B can be found in Hsu HL. et al, structural characterization of a novel human adeno-associated virus capsid with neurotropic properties. Nat Commun 11,3279 (2020).
It is well known in the art that B7/CD28 and CD40/CD40L co-stimulatory signaling pathways are critical for T cell activation, and blocking these signaling pathways is effective in treating diseases associated with abnormal T cell activation (Kimberly C et al, abul K. Abbas, the Journal of Immunology,2000,164 (9) 4465-4470). CTLA-4 can competitively bind to the B7 molecule with CD28, thereby blocking the B7/CD28 signaling pathway. CD40-Ig fusion proteins act as specific blockers of the CD40/CD40L interaction and selectively inhibit T-cell and/or B-cell activation. Thus, the medicaments of the present disclosure may also be used for the treatment of diseases associated with abnormal activation of T cells, such as cancer, autoimmune diseases, transplant rejection, etc.
The present disclosure is described in further detail below with reference to the 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 procedure, in which specific conditions are not specified in the examples, is performed according to conventional conditions known in the art or according to conditions recommended by the manufacturer.
Examples
Example 1: plasmid construction (codon optimization and Signal peptide screening)
First, AAV expression humanized transgenic vectors pAAV-CTLA4-Ig and pAAV-CD40-Ig were constructed (FIG. 1A). Next, to investigate the effect of different signal peptides on protein secretion, pAAV-CTLA4-Ig and pAAV-CD40-Ig with different signal peptides were transiently transfected into HEK293 and HUH-7 cells with PEI. Transgenic protein expression was quantified and GAPDH was used as a reference protein, with the results shown in fig. 1B. CTLA4-Ig and CD40-Ig levels in cell supernatants were determined by ELISA (FIG. 1C).
The results show that for humanized CTLA4-Ig, protein secretion is significantly increased when the self-contained signal peptide (original signal peptide), FIB or BM40 signal peptide is used as compared to the albumin signal peptide. For humanized CD40-Ig, the protein secretion amount is large when the self-contained signal peptide (original signal peptide) and FIB signal peptide are used.
It was also observed that there was no statistical difference in secretion amounts of the original signal peptide, FIB signal peptide and BM40 signal peptide (p > 0.05) for humanized CTLA 4-Ig. For humanized CD40-Ig, there was no statistical difference in secretion amounts of the original signal peptide and the FIB signal peptide (p > 0.05). Thus, since replacement of the signal peptide carried by itself may lead to a potential immune response, it is more advantageous to retain its original signal peptide without significant statistical differences in the secretion of the protein of interest.
In addition, the expression of pAAV-CTLA4-Ig and pAAV-CD40-Ig with optimized or original transgene sequences in HUH-7 cells was analyzed by Western blotting, and the results are shown in FIG. 1D and FIG. 1E.
The results show that the codon optimized CTLA4-Ig and CD40-Ig have increased protein expression in both cell supernatants and cells compared to the original sequence.
EXAMPLE 2 prevention of the effects of AAV-expressed CTLA4-Ig and CD40-Ig on MS
In studying the prophylactic effect of AAV-mediated CTLA4-Ig and CD40-Ig expression on MS mice, the mice were set to AAV8-CTLA4-Ig (abbreviated herein as AAV-CTLA 4), AAV8-CD40-Ig (abbreviated herein as AAV-CD 40), and in combination AAV8-CTLA4-ig+aav8-CD40-Ig (abbreviated herein as AAV-CTLA4/CD 40) (n=8), model group (control, n=4).
C57BL/6 female mice (9-12 weeks old, n=8-10) were given 2×10 intravenous injections 12 vg/kg AAV-CTLA4, AAV-CD40 or AAV-CTLA4/CD40 (i.e., 2X 10) 12 vg/kg AAV8-CTLA4+2×10 12 vg/kg AAV8-CD40 Viral vector). After two weeks, an MS model (EAE) was established as follows: MOG for C57BL/6 mice 35-55 The preparation of emulsion with Complete Freund's Adjuvant (CFA) induced followed by pertussis toxin administration, one day after immunization. Control mice induced EAE without AAV injection (n=4). Healthy controls were untreated mice (n=4).
On days 21 and 42 from the virus injection, mice were bled to obtain serum to examine the expression of the target proteins CTLA4-Ig and CD40-Ig in vivo, and smooth expression of the target proteins was observed (FIG. 2B). Body weight and average clinical score (MCS) were recorded daily from day 7 post EAE induction and the results are shown in figure 2C. On day 21 after EAE induction, the mice were subjected to an behavioural tensile test, the results of which are shown in fig. 2D. Mice survival was determined on day 35 and the results are shown in figure 2E.
The results show that mice from both the single administration group (AAV-CTLA 4 and AAV-CD 40) and the combination administration group (AAV-CTLA 4/CD 40) have increased weight relative to the mice from the model group (control group). Behavioural scores also indicate that both dosing groups have good prophylactic effects on MS, with AAV-CTLA4 and AAV-CTLA4/CD40 being better effective than AAV-CD40 groups. The results of the behavioural tensile experiments showed that the motor function of mice in the administration group was superior to that of mice in the model group, wherein the motor functions of mice in the AAV-CTLA4 and AAV-CTLA4/CD40 groups were significantly superior to that of mice in the model group (FIG. 2D). The statistics of the survival rate data show that one of the mice in the model group died at the 15 day peak, while none of the animals in the treatment group died (FIG. 2E), indicating that AAV-mediated CTLA4-Ig and CD40-Ig expression had a prophylactic effect on MS mice and increased survival rate.
The behavioural scoring criteria are briefly as follows: EAE clinical symptoms were examined daily with clinical scores of 0-5 points. 0: no clinical signs; 0.5: tail part lameness; 1.0: tail paralysis; 2.0: movement loss coordination, hind limb paralysis; 2.5: paralysis of one hind limb; 3.0: paralysis of the two hind limbs; 3.5: paralysis of the hind limbs and weakness of the front limbs; 4.0: paralysis of the forelimbs (quadriplegia); and 5.0: the dying struggles.
EXAMPLE 3 pathological evaluation of the effects of AAV-expressed CTLA4-Ig and CD40-Ig on MS prevention
On day 35, all groups of mice died. Histological analysis of the lumbar spinal cord was performed. To observe activation of spinal cord astrocytes and microglia, astrocytes (astroglias) were labeled with a specific marker GFAP (glial acidic protein) (fig. 3A), microglia (microglia) were labeled with Iba1 (surface glycoprotein) (fig. 3B), and fluorescence intensity was observed with confocal microscopy. The integrated fluorescence intensities were quantified (n=2), and statistics showed that the astrocytes and microglia activation numbers were smaller for AAV-CTLA4, AAV-CD40 and AAV-CTLA4/CD40 groups than for the model group (control group).
HE staining was performed to observe inflammatory infiltration of spinal white matter. The results showed that no significant inflammatory infiltrate was observed in the model group, whereas no significant inflammatory infiltrate was observed in the dosing group (fig. 3C). LFB staining was performed and spinal cord demyelination was observed. The results showed that the spinal cord demyelination was significant in the model group mice, whereas the spinal cord demyelination was significantly less in the dosing group mice (fig. 3D). The results of CD4 and CD8 immunohistochemistry showed that the spinal cord CD4 of the mice in the model group + T cells and CD8 + T cells were significantly increased, whereas no apparent T, B cell activation was observed in the AAV-dosed group (fig. 3E and 3F).
The results indicated that AAV-mediated CTLA4-Ig and CD40-Ig expression have a prophylactic effect on mouse MS disease and can effectively prevent pathological changes of spinal cord tissues.
EXAMPLE 4 exploration of the immune mechanism of AAV-expressed CTLA4-Ig and CD40-Ig against MS prophylaxis
Mice were sacrificed on day 35 post EAE immunization and spinal cord and spleen were isolated. To further explore the immune-related effects of AAV-mediated expression of CTLA4-Ig and CD40-Ig on MS prophylaxis, mouse sera were taken on day 35 for MOG 35-55 And (5) detecting specific antibodies. Results show (FIG. 4A), MOG of the dosing group relative to the model group (control group) 35-55 The specific antibody level is significantly reduced, and the AAV-CTLA4 and AAV-CTLA4/CD40 effects are superior to those of AAV-CD40 group.
mRNA was extracted from the spinal cord of mice and QPCR was performed to detect the expression of the pro-inflammatory factors TNF- α, IFN- γ, IL 1. Beta., IL6, IL17A, IL, F4/80, TGF- β. The results showed (fig. 4B) that the mice in the dosing group had reduced polio factor relative to the model group. The above results demonstrate that AAV-mediated CTLA4-Ig and CD40-Ig expression in mice with MS is associated with their reduced inflammatory response.
Example 5 evaluation of safety
To assess the safety of long-term expression of CTLA4-Ig and CD40-Ig, chronic hepatotoxicity, including plasma ALT and AST levels, was determined on days 7 and 35 after AAV injection.
The results showed that at day 7 post-injection, ALT/AST was significantly elevated in AAV-CTLA4 group compared to mice not injected with AAV (healthy control), and AST levels were significantly elevated in AAV-CD40 group compared to healthy control. At day 35 post-injection, only ALT was significantly elevated in AAV-CD40 groups relative to healthy controls, which may be associated with sustained high expression of CD40-Ig protein. ALT/AST values were below 1.0pg/ul for each group except AAV-CD 40.
On day 35 post injection, mouse whole blood was taken for hematological evaluation. The results show that there was no significant difference between the various indicators of the mice in each group. From the results of the toxicology evaluation in mice, AAV-mediated CTLA4-Ig and CD40-Ig expression was safe in MS mice.
Example 6 AAV-mediated CTLA4-Ig and CD40-Ig effects on light MS treatment
Mice were injected with AAV on day 4 post-model: c57BL/6 female mice (9-12 weeks old, n=8-10) were injected 5×10 intravenously 12 vg/kg AAV-CTLA4, AAV-CD40 or AAV-CTLA4/CD40 (FIG. 6A). Meanwhile, on day 4 after modeling, mice were treated with betanaproxen, administered 100 μg every other day. Weighing and behavioural scoring were performed daily and the results are shown in figure 6B.
The results showed that the weight of mice in AAV-CTLA4 group and AAV-CTLA4/CD40 group was always increasing; AAV-CD40 mice and model group (control group) mice began to lose weight one week after modeling, AAV-CD40 group mice began to quickly return to weight around day 17 of modeling, and model group mice began to return to weight around day 25 of modeling; the commercial drug betanap group mice began to dip around 17 days of modeling and began to rise around 22 days.
The mice were scored behaviorally, and the results showed that the AAV-CTLA4, AAV-CD40 and AAV-CTLA4/CD40 groups were significantly lower than the model groups, wherein the AAV-CTLA4 and AAV-CTLA4/CD40 effects were superior to the AAV-CD40 groups, and the behaviorally scores of the commercial protein drug, betaxostat, were not significantly different from that of the model groups.
The survival rate of the mice was determined on day 43. The mice survival data statistics showed good survival of the mice in the AAV-CTLA4 and AAV-CTLA4/CD40 groups (FIG. 6C).
The results show that AAV mediated CTLA4-Ig and CD40-Ig expression has good therapeutic effect on mild MS mice, can improve the survival rate of MS mice, and has better therapeutic effect than commercial CTLA4 recombinant protein-beta-sephadex.
EXAMPLE 7 pathological investigation of the effects of AAV-expressed CTLA4-Ig and CD40-Ig on the treatment of mild MS
Histological analysis of the lumbar spinal cord was performed. The integrated fluorescence intensity was quantified, and the result is shown in fig. 7C.
From immunofluorescence statistics, the star gum activation of AAV-CTLA4 and AAV-CTLA4/CD40 groups was significantly less than that of the model group (control group) and the Betazep treatment group, and there was no significant difference between the microglial cell numbers of each group. The above results indicate that AAV-mediated CTLA4-Ig and CD40-Ig expression can significantly reduce aberrant activation of mild MS mouse astrocytes.
Example 8 AAV-mediated CTLA4-Ig and CD40-Ig effects on severe MS treatment
MOG35-55 induced EAE model in mice. AAV injections were then performed at a mouse behavioural score of 1.5-2.5, with the injection lasting 11-17 days due to non-uniform time of onset of the mice. Tail vein injection of C57BL/6 female mice (9-12 weeks old, n=8-10) was 5×10 12 vg/kg AAV-CTLA4, AAV-CD40 or AAV-CTLA4/CD40 (FIG. 8A). Control mice were immunized with EAE and were not injected with AAV (n=7). Healthy control mice were untreated (n=8).
The mice body weight and behavioural data indicated that after AAV injection treatment, the mice in AAV-CTLA4, AAV-CD40 and AAV-CTLA4/CD40 groups began to gradually return to their body weight, the behavioural score gradually decreased, while the mice in the model group (control group) had a small magnitude of return, but the behavioural score was significantly higher than that of the treatment group (fig. 8B). Furthermore, AAV mediated CD40-Ig therapeutic efficacy is lower than AAV-CTLA4 and AAV-CTLA4/CD40 groups.
The survival rate of the mice was determined on day 59. The mice survival data statistics showed that AAV-CTLA4/CD40 mice had the best survival (FIG. 8C).
LFB staining observed spinal cord demyelination. The results showed that demyelination of the spinal cord white matter areas was evident in the mice of the model group (fig. 8D). Only slight white matter demyelination was observed in AAV-CD40 groups, no demyelination was observed in AAV-CTLA4 and AAV-CTLA4/CD40 groups.
The results show that AAV expresses CTLA4-Ig and CD40-Ig and has better therapeutic effect on severe MS.
Example 9 study of the immune mechanism of AAV-mediated CTLA4-Ig and CD40-Ig on the treatment of severe MS
Mice were sacrificed on day 35 post EAE immunization and spinal cord and spleen were isolated. To explore the relevant immune mechanisms of AAV-mediated expression of CTLA4-Ig and CD40-Ig for the therapeutic effect on heavy MS, mouse serum was taken at day 60 for MOG 35-55 And (5) detecting specific antibodies. The results showed that the MOG of AAV-CTLA4/CD40 group relative to the model group (control group) 35-55 The specific antibody levels were significantly reduced (fig. 9A).
mRNA was extracted from spinal cord and spleen of mice, and QPCR was performed to detect expression of pro-inflammatory factors TNF-. Alpha., IFN-. Gamma., IL 1. Beta., IL4, IL6, IL17, IL23, and TGF-. Beta.in mice. The results showed that the levels of spinal cord and spleen inflammatory factors were lower in the AAV-CTLA4 and AAV-CTLA4/CD40 mice than in the model group (FIG. 9B).
The above results indicate that AAV expression of CTLA4-Ig and CD40-Ig can reduce the inflammatory levels in severe MS mice.
While the present disclosure has been shown and described with respect to certain preferred embodiments thereof, it will be understood by those of ordinary skill in the art that the foregoing is a further detailed description of the present disclosure with reference to specific embodiments and is not intended to limit the practice of the present disclosure to such descriptions. Various changes in form and detail may be made therein by those skilled in the art, including a few simple inferences or alternatives, without departing from the spirit and scope of the present disclosure.
Sequence listing
<110> Shanghai Ding also Biotech Co., ltd
<120> transgenic expression cassettes for the treatment of multiple sclerosis
<130> PCNCNN219329G
<160> 23
<170> PatentIn version 3.5
<210> 1
<211> 378
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> codon optimized human CTLA4 base sequence
<400> 1
aaagcaatgc atgtggccca gcctgctgtg gtgctggcct cttctagagg cattgccagc 60
tttgtgtgtg aatatgcctc tcctggcaag gccactgaag tcagagtgac agtgctgaga 120
caggctgaca gccaagtgac tgaagtctgt gctgccacct acatgatggg caatgagctg 180
accttcctgg atgacagcat ctgcacaggc accagctctg gcaatcaagt gaacctgacc 240
atccagggcc tgagagccat ggacactggc ctgtacatct gcaaggtgga actgatgtac 300
cctcctcctt actaccttgg cattggcaat ggcacccaga tctatgtgat tgaccctgag 360
ccttgtcctg actctgat 378
<210> 2
<211> 519
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> codon optimized human CD40 base sequence
<400> 2
gaacctccta cagcctgcag agagaagcag tacctgatca acagccagtg ctgcagcctg 60
tgccagcctg gccagaaact ggtgtctgac tgcacagagt tcacagagac agagtgcctg 120
ccttgtgggg agtctgagtt cctggacacc tggaacagag agacacactg ccaccagcac 180
aagtactgtg accccaacct gggcctgaga gtgcagcaga agggcacctc tgagacagac 240
accatctgca cctgtgaaga aggatggcac tgtacctctg aggcctgtga aagctgtgtg 300
ctgcacagaa gctgcagccc aggctttgga gtgaagcaga ttgccactgg tgtttctgat 360
accatctgtg aaccttgtcc tgtgggcttc ttcagcaatg tgtcctctgc ctttgagaag 420
tgtcacccct ggaccagctg tgaaacaaag gacctggtgg tgcagcaggc tgggaccaac 480
aagacagatg ttgtgtgtgg accccaggac agactgaga 519
<210> 3
<211> 685
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> codon optimized human FC base sequence
<400> 3
gagcccaaga gctgtgacaa gacccacacc tgtcctccat gtcctgctcc agagctgctt 60
ggaggcccct ctgtgttcct gtttcctcca aagcctaagg acaccctgat gatcagcaga 120
acccctgaag tgacctgtgt ggtggtggat gtgtcccatg aggacccaga agtgaagttc 180
aattggtatg tggatggggt tgaagtgcac aatgctaaga ccaagcctag agaggaacag 240
tacaacagca cctacagagt ggtgtcagtg ctgactgtgc tgcaccagga ctggctgaat 300
ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc ctgctcctat tgagaaaacc 360
atcagcaagg ccaagggcca gcctagggaa ccccaggttt acacactgcc accaagcaga 420
gaagagatga ccaagaacca ggtgtccctg acctgcctgg tcaagggctt ctacccctct 480
gacattgctg tggaatggga gagcaatggc cagcctgaga acaactacaa gacaacccct 540
cctgtgctgg attctgatgg ctcattcttc ctgtacagca agctgacagt ggacaagagc 600
agatggcagc agggcaatgt gttcagctgc tctgtgatgc atgaggccct gcacaaccac 660
tacacccaga aatccctgtc tctga 685
<210> 4
<211> 1955
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 45 pAAV-alb-CTLA4-Ig base sequence (CTLA 4-albumin signal peptide)
<400> 4
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgaagtgggt aacctttatt tcccttcttt 780
ttctctttag ctcggcttat tccaaagcaa tgcatgtggc ccagcctgct gtggtgctgg 840
cctcttctag aggcattgcc agctttgtgt gtgaatatgc ctctcctggc aaggccactg 900
aagtcagagt gacagtgctg agacaggctg acagccaagt gactgaagtc tgtgctgcca 960
cctacatgat gggcaatgag ctgaccttcc tggatgacag catctgcaca ggcaccagct 1020
ctggcaatca agtgaacctg accatccagg gcctgagagc catggacact ggcctgtaca 1080
tctgcaaggt ggaactgatg taccctcctc cttactacct tggcattggc aatggcaccc 1140
agatctatgt gattgaccct gagccttgtc ctgactctga tgagcccaag agctgtgaca 1200
agacccacac ctgtcctcca tgtcctgctc cagagctgct tggaggcccc tctgtgttcc 1260
tgtttcctcc aaagcctaag gacaccctga tgatcagcag aacccctgaa gtgacctgtg 1320
tggtggtgga tgtgtcccat gaggacccag aagtgaagtt caattggtat gtggatgggg 1380
ttgaagtgca caatgctaag accaagccta gagaggaaca gtacaacagc acctacagag 1440
tggtgtcagt gctgactgtg ctgcaccagg actggctgaa tggcaaagag tacaagtgca 1500
aggtgtccaa caaggccctg cctgctccta ttgagaaaac catcagcaag gccaagggcc 1560
agcctaggga accccaggtt tacacactgc caccaagcag agaagagatg accaagaacc 1620
aggtgtccct gacctgcctg gtcaagggct tctacccctc tgacattgct gtggaatggg 1680
agagcaatgg ccagcctgag aacaactaca agacaacccc tcctgtgctg gattctgatg 1740
gctcattctt cctgtacagc aagctgacag tggacaagag cagatggcag cagggcaatg 1800
tgttcagctg ctctgtgatg catgaggccc tgcacaacca ctacacccag aaatccctgt 1860
ctctgagccc tggcaagtga aagcttatcg ataccgtcga caggcctaat aaagagctca 1920
gatgcatcga tcagagtgtg ttggtttttt gtgtg 1955
<210> 5
<211> 376
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 45 pAAV-alb-CTLA4-Ig amino acid sequence (CTLA 4-albumin signal peptide)
<400> 5
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser
20 25 30
Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys
35 40 45
Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val
50 55 60
Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe
65 70 75 80
Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn
85 90 95
Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys
100 105 110
Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn
115 120 125
Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
130 135 140
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
145 150 155 160
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
165 170 175
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
180 185 190
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
195 200 205
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
225 230 235 240
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
245 250 255
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
260 265 270
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
275 280 285
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
290 295 300
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
305 310 315 320
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
340 345 350
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
355 360 365
Ser Leu Ser Leu Ser Pro Gly Lys
370 375
<210> 6
<211> 2006
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 46 pAAV-org-CTLA4-Ig base sequence (CTLA 4-original Signal peptide)
<400> 6
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tggcttgcct tggatttcag cggcacaagg 780
ctcagctgaa cctggctacc aggacctggc cctgcactct cctgtttttt cttctcttca 840
tccctgtctt ctgcaaagca atgcatgtgg cccagcctgc tgtggtgctg gcctcttcta 900
gaggcattgc cagctttgtg tgtgaatatg cctctcctgg caaggccact gaagtcagag 960
tgacagtgct gagacaggct gacagccaag tgactgaagt ctgtgctgcc acctacatga 1020
tgggcaatga gctgaccttc ctggatgaca gcatctgcac aggcaccagc tctggcaatc 1080
aagtgaacct gaccatccag ggcctgagag ccatggacac tggcctgtac atctgcaagg 1140
tggaactgat gtaccctcct ccttactacc ttggcattgg caatggcacc cagatctatg 1200
tgattgaccc tgagccttgt cctgactctg atgagcccaa gagctgtgac aagacccaca 1260
cctgtcctcc atgtcctgct ccagagctgc ttggaggccc ctctgtgttc ctgtttcctc 1320
caaagcctaa ggacaccctg atgatcagca gaacccctga agtgacctgt gtggtggtgg 1380
atgtgtccca tgaggaccca gaagtgaagt tcaattggta tgtggatggg gttgaagtgc 1440
acaatgctaa gaccaagcct agagaggaac agtacaacag cacctacaga gtggtgtcag 1500
tgctgactgt gctgcaccag gactggctga atggcaaaga gtacaagtgc aaggtgtcca 1560
acaaggccct gcctgctcct attgagaaaa ccatcagcaa ggccaagggc cagcctaggg 1620
aaccccaggt ttacacactg ccaccaagca gagaagagat gaccaagaac caggtgtccc 1680
tgacctgcct ggtcaagggc ttctacccct ctgacattgc tgtggaatgg gagagcaatg 1740
gccagcctga gaacaactac aagacaaccc ctcctgtgct ggattctgat ggctcattct 1800
tcctgtacag caagctgaca gtggacaaga gcagatggca gcagggcaat gtgttcagct 1860
gctctgtgat gcatgaggcc ctgcacaacc actacaccca gaaatccctg tctctgagcc 1920
ctggcaagtg aaagcttatc gataccgtcg acaggcctaa taaagagctc agatgcatcg 1980
atcagagtgt gttggttttt tgtgtg 2006
<210> 7
<211> 393
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 46 pAAV-org-CTLA4-Ig amino acid sequence (CTLA 4-original Signal peptide)
<400> 7
Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala
1 5 10 15
Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro
20 25 30
Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala
35 40 45
Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly
50 55 60
Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln
65 70 75 80
Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr
85 90 95
Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
100 105 110
Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile
115 120 125
Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly
130 135 140
Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser
145 150 155 160
Asp Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
165 170 175
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
180 185 190
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
195 200 205
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
210 215 220
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
225 230 235 240
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
245 250 255
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
260 265 270
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
275 280 285
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
290 295 300
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
305 310 315 320
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
325 330 335
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
340 345 350
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
355 360 365
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
370 375 380
Lys Ser Leu Ser Leu Ser Pro Gly Lys
385 390
<210> 8
<211> 1973
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 47 pAAV-FIB-CTLA4-Ig base sequence (CTLA 4-FIB Signal peptide)
<400> 8
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgctcagggg tccgggaccc gggcggctgc 780
tgctgctagc agtcctgtgc ctggggacat cggtgcgctg caaagcaatg catgtggccc 840
agcctgctgt ggtgctggcc tcttctagag gcattgccag ctttgtgtgt gaatatgcct 900
ctcctggcaa ggccactgaa gtcagagtga cagtgctgag acaggctgac agccaagtga 960
ctgaagtctg tgctgccacc tacatgatgg gcaatgagct gaccttcctg gatgacagca 1020
tctgcacagg caccagctct ggcaatcaag tgaacctgac catccagggc ctgagagcca 1080
tggacactgg cctgtacatc tgcaaggtgg aactgatgta ccctcctcct tactaccttg 1140
gcattggcaa tggcacccag atctatgtga ttgaccctga gccttgtcct gactctgatg 1200
agcccaagag ctgtgacaag acccacacct gtcctccatg tcctgctcca gagctgcttg 1260
gaggcccctc tgtgttcctg tttcctccaa agcctaagga caccctgatg atcagcagaa 1320
cccctgaagt gacctgtgtg gtggtggatg tgtcccatga ggacccagaa gtgaagttca 1380
attggtatgt ggatggggtt gaagtgcaca atgctaagac caagcctaga gaggaacagt 1440
acaacagcac ctacagagtg gtgtcagtgc tgactgtgct gcaccaggac tggctgaatg 1500
gcaaagagta caagtgcaag gtgtccaaca aggccctgcc tgctcctatt gagaaaacca 1560
tcagcaaggc caagggccag cctagggaac cccaggttta cacactgcca ccaagcagag 1620
aagagatgac caagaaccag gtgtccctga cctgcctggt caagggcttc tacccctctg 1680
acattgctgt ggaatgggag agcaatggcc agcctgagaa caactacaag acaacccctc 1740
ctgtgctgga ttctgatggc tcattcttcc tgtacagcaa gctgacagtg gacaagagca 1800
gatggcagca gggcaatgtg ttcagctgct ctgtgatgca tgaggccctg cacaaccact 1860
acacccagaa atccctgtct ctgagccctg gcaagtgaaa gcttatcgat accgtcgaca 1920
ggcctaataa agagctcaga tgcatcgatc agagtgtgtt ggttttttgt gtg 1973
<210> 9
<211> 382
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 47 pAAV-FIB-CTLA4-Ig amino acid sequence (CTLA 4-FIB Signal peptide)
<400> 9
Met Leu Arg Gly Pro Gly Pro Gly Arg Leu Leu Leu Leu Ala Val Leu
1 5 10 15
Cys Leu Gly Thr Ser Val Arg Cys Lys Ala Met His Val Ala Gln Pro
20 25 30
Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu
35 40 45
Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg
50 55 60
Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met
65 70 75 80
Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser
85 90 95
Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp
100 105 110
Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr
115 120 125
Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu
130 135 140
Pro Cys Pro Asp Ser Asp Glu Pro Lys Ser Cys Asp Lys Thr His Thr
145 150 155 160
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
165 170 175
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
180 185 190
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
195 200 205
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
210 215 220
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
225 230 235 240
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
245 250 255
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
260 265 270
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
275 280 285
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
290 295 300
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
305 310 315 320
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
325 330 335
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
340 345 350
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
355 360 365
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375 380
<210> 10
<211> 1952
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 48 pAAV-BM40-CTLA4-Ig base sequence (CTLA 4-BM40 Signal peptide)
<400> 10
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgagggcttg gatctttttc ctgctgtgcc 780
tggctggacg ggctctggct aaagcaatgc atgtggccca gcctgctgtg gtgctggcct 840
cttctagagg cattgccagc tttgtgtgtg aatatgcctc tcctggcaag gccactgaag 900
tcagagtgac agtgctgaga caggctgaca gccaagtgac tgaagtctgt gctgccacct 960
acatgatggg caatgagctg accttcctgg atgacagcat ctgcacaggc accagctctg 1020
gcaatcaagt gaacctgacc atccagggcc tgagagccat ggacactggc ctgtacatct 1080
gcaaggtgga actgatgtac cctcctcctt actaccttgg cattggcaat ggcacccaga 1140
tctatgtgat tgaccctgag ccttgtcctg actctgatga gcccaagagc tgtgacaaga 1200
cccacacctg tcctccatgt cctgctccag agctgcttgg aggcccctct gtgttcctgt 1260
ttcctccaaa gcctaaggac accctgatga tcagcagaac ccctgaagtg acctgtgtgg 1320
tggtggatgt gtcccatgag gacccagaag tgaagttcaa ttggtatgtg gatggggttg 1380
aagtgcacaa tgctaagacc aagcctagag aggaacagta caacagcacc tacagagtgg 1440
tgtcagtgct gactgtgctg caccaggact ggctgaatgg caaagagtac aagtgcaagg 1500
tgtccaacaa ggccctgcct gctcctattg agaaaaccat cagcaaggcc aagggccagc 1560
ctagggaacc ccaggtttac acactgccac caagcagaga agagatgacc aagaaccagg 1620
tgtccctgac ctgcctggtc aagggcttct acccctctga cattgctgtg gaatgggaga 1680
gcaatggcca gcctgagaac aactacaaga caacccctcc tgtgctggat tctgatggct 1740
cattcttcct gtacagcaag ctgacagtgg acaagagcag atggcagcag ggcaatgtgt 1800
tcagctgctc tgtgatgcat gaggccctgc acaaccacta cacccagaaa tccctgtctc 1860
tgagccctgg caagtgaaag cttatcgata ccgtcgacag gcctaataaa gagctcagat 1920
gcatcgatca gagtgtgttg gttttttgtg tg 1952
<210> 11
<211> 375
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 48 pAAV-BM40-CTLA4-Ig amino acid sequence (CTLA 4-BM40 Signal peptide)
<400> 11
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser
20 25 30
Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala
35 40 45
Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr
50 55 60
Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu
65 70 75 80
Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu
85 90 95
Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys
100 105 110
Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly
115 120 125
Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Glu
130 135 140
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
145 150 155 160
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
165 170 175
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
180 185 190
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
195 200 205
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
210 215 220
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
225 230 235 240
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
245 250 255
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
260 265 270
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
275 280 285
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
290 295 300
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
305 310 315 320
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
325 330 335
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
340 345 350
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
355 360 365
Leu Ser Leu Ser Pro Gly Lys
370 375
<210> 12
<211> 2147
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 49 pAAV-org-CD40-Ig base sequence (CD 40-original Signal peptide)
<400> 12
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tggcttgcct tggatttcag cggcacaagg 780
ctcagctgaa cctggctacc aggacctggc cctgcactct cctgtttttt cttctcttca 840
tccctgtctt ctgcgaacct cctacagcct gcagagagaa gcagtacctg atcaacagcc 900
agtgctgcag cctgtgccag cctggccaga aactggtgtc tgactgcaca gagttcacag 960
agacagagtg cctgccttgt ggggagtctg agttcctgga cacctggaac agagagacac 1020
actgccacca gcacaagtac tgtgacccca acctgggcct gagagtgcag cagaagggca 1080
cctctgagac agacaccatc tgcacctgtg aagaaggatg gcactgtacc tctgaggcct 1140
gtgaaagctg tgtgctgcac agaagctgca gcccaggctt tggagtgaag cagattgcca 1200
ctggtgtttc tgataccatc tgtgaacctt gtcctgtggg cttcttcagc aatgtgtcct 1260
ctgcctttga gaagtgtcac ccctggacca gctgtgaaac aaaggacctg gtggtgcagc 1320
aggctgggac caacaagaca gatgttgtgt gtggacccca ggacagactg agagagccca 1380
agagctgtga caagacccac acctgtcctc catgtcctgc tccagagctg cttggaggcc 1440
cctctgtgtt cctgtttcct ccaaagccta aggacaccct gatgatcagc agaacccctg 1500
aagtgacctg tgtggtggtg gatgtgtccc atgaggaccc agaagtgaag ttcaattggt 1560
atgtggatgg ggttgaagtg cacaatgcta agaccaagcc tagagaggaa cagtacaaca 1620
gcacctacag agtggtgtca gtgctgactg tgctgcacca ggactggctg aatggcaaag 1680
agtacaagtg caaggtgtcc aacaaggccc tgcctgctcc tattgagaaa accatcagca 1740
aggccaaggg ccagcctagg gaaccccagg tttacacact gccaccaagc agagaagaga 1800
tgaccaagaa ccaggtgtcc ctgacctgcc tggtcaaggg cttctacccc tctgacattg 1860
ctgtggaatg ggagagcaat ggccagcctg agaacaacta caagacaacc cctcctgtgc 1920
tggattctga tggctcattc ttcctgtaca gcaagctgac agtggacaag agcagatggc 1980
agcagggcaa tgtgttcagc tgctctgtga tgcatgaggc cctgcacaac cactacaccc 2040
agaaatccct gtctctgagc cctggcaagt gaaagcttat cgataccgtc gacaggccta 2100
ataaagagct cagatgcatc gatcagagtg tgttggtttt ttgtgtg 2147
<210> 13
<211> 425
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 49 pAAV-org-CD40-Ig amino acid sequence (CD 40-original Signal peptide)
<400> 13
Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr
1 5 10 15
Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu
20 25 30
Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val
35 40 45
Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
50 55 60
Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His
65 70 75 80
Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95
Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr
100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly
115 120 125
Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu
130 135 140
Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys
145 150 155 160
Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln
165 170 175
Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu
180 185 190
Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
195 200 205
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
210 215 220
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
225 230 235 240
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
245 250 255
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
260 265 270
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
275 280 285
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
290 295 300
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
305 310 315 320
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
325 330 335
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
340 345 350
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
355 360 365
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
370 375 380
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
385 390 395 400
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
405 410 415
Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 14
<211> 2096
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 50 pAAV-alb-CD40-Ig base sequence (CD 40-albumin signal peptide)
<400> 14
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgaagtgggt aacctttatt tcccttcttt 780
ttctctttag ctcggcttat tccgaacctc ctacagcctg cagagagaag cagtacctga 840
tcaacagcca gtgctgcagc ctgtgccagc ctggccagaa actggtgtct gactgcacag 900
agttcacaga gacagagtgc ctgccttgtg gggagtctga gttcctggac acctggaaca 960
gagagacaca ctgccaccag cacaagtact gtgaccccaa cctgggcctg agagtgcagc 1020
agaagggcac ctctgagaca gacaccatct gcacctgtga agaaggatgg cactgtacct 1080
ctgaggcctg tgaaagctgt gtgctgcaca gaagctgcag cccaggcttt ggagtgaagc 1140
agattgccac tggtgtttct gataccatct gtgaaccttg tcctgtgggc ttcttcagca 1200
atgtgtcctc tgcctttgag aagtgtcacc cctggaccag ctgtgaaaca aaggacctgg 1260
tggtgcagca ggctgggacc aacaagacag atgttgtgtg tggaccccag gacagactga 1320
gagagcccaa gagctgtgac aagacccaca cctgtcctcc atgtcctgct ccagagctgc 1380
ttggaggccc ctctgtgttc ctgtttcctc caaagcctaa ggacaccctg atgatcagca 1440
gaacccctga agtgacctgt gtggtggtgg atgtgtccca tgaggaccca gaagtgaagt 1500
tcaattggta tgtggatggg gttgaagtgc acaatgctaa gaccaagcct agagaggaac 1560
agtacaacag cacctacaga gtggtgtcag tgctgactgt gctgcaccag gactggctga 1620
atggcaaaga gtacaagtgc aaggtgtcca acaaggccct gcctgctcct attgagaaaa 1680
ccatcagcaa ggccaagggc cagcctaggg aaccccaggt ttacacactg ccaccaagca 1740
gagaagagat gaccaagaac caggtgtccc tgacctgcct ggtcaagggc ttctacccct 1800
ctgacattgc tgtggaatgg gagagcaatg gccagcctga gaacaactac aagacaaccc 1860
ctcctgtgct ggattctgat ggctcattct tcctgtacag caagctgaca gtggacaaga 1920
gcagatggca gcagggcaat gtgttcagct gctctgtgat gcatgaggcc ctgcacaacc 1980
actacaccca gaaatccctg tctctgagcc ctggcaagtg aaagcttatc gataccgtcg 2040
acaggcctaa taaagagctc agatgcatcg atcagagtgt gttggttttt tgtgtg 2096
<210> 15
<211> 423
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 50 pAAV-alb-CD40-Ig amino acid sequence (CD 40-albumin signal peptide)
<400> 15
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn
20 25 30
Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp
35 40 45
Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu
50 55 60
Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr
65 70 75 80
Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu
85 90 95
Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu
100 105 110
Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly
115 120 125
Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys
130 135 140
Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His
145 150 155 160
Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly
165 170 175
Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Glu
180 185 190
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
195 200 205
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
210 215 220
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
225 230 235 240
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
245 250 255
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
260 265 270
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
275 280 285
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
290 295 300
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
305 310 315 320
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
325 330 335
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
340 345 350
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
355 360 365
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
370 375 380
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
385 390 395 400
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
405 410 415
Leu Ser Leu Ser Pro Gly Lys
420
<210> 16
<211> 2114
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 51 pAAV-FIB-CD40-Ig base sequence (CD 40-FIB Signal peptide)
<400> 16
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgctcagggg tccgggaccc gggcggctgc 780
tgctgctagc agtcctgtgc ctggggacat cggtgcgctg cgaacctcct acagcctgca 840
gagagaagca gtacctgatc aacagccagt gctgcagcct gtgccagcct ggccagaaac 900
tggtgtctga ctgcacagag ttcacagaga cagagtgcct gccttgtggg gagtctgagt 960
tcctggacac ctggaacaga gagacacact gccaccagca caagtactgt gaccccaacc 1020
tgggcctgag agtgcagcag aagggcacct ctgagacaga caccatctgc acctgtgaag 1080
aaggatggca ctgtacctct gaggcctgtg aaagctgtgt gctgcacaga agctgcagcc 1140
caggctttgg agtgaagcag attgccactg gtgtttctga taccatctgt gaaccttgtc 1200
ctgtgggctt cttcagcaat gtgtcctctg cctttgagaa gtgtcacccc tggaccagct 1260
gtgaaacaaa ggacctggtg gtgcagcagg ctgggaccaa caagacagat gttgtgtgtg 1320
gaccccagga cagactgaga gagcccaaga gctgtgacaa gacccacacc tgtcctccat 1380
gtcctgctcc agagctgctt ggaggcccct ctgtgttcct gtttcctcca aagcctaagg 1440
acaccctgat gatcagcaga acccctgaag tgacctgtgt ggtggtggat gtgtcccatg 1500
aggacccaga agtgaagttc aattggtatg tggatggggt tgaagtgcac aatgctaaga 1560
ccaagcctag agaggaacag tacaacagca cctacagagt ggtgtcagtg ctgactgtgc 1620
tgcaccagga ctggctgaat ggcaaagagt acaagtgcaa ggtgtccaac aaggccctgc 1680
ctgctcctat tgagaaaacc atcagcaagg ccaagggcca gcctagggaa ccccaggttt 1740
acacactgcc accaagcaga gaagagatga ccaagaacca ggtgtccctg acctgcctgg 1800
tcaagggctt ctacccctct gacattgctg tggaatggga gagcaatggc cagcctgaga 1860
acaactacaa gacaacccct cctgtgctgg attctgatgg ctcattcttc ctgtacagca 1920
agctgacagt ggacaagagc agatggcagc agggcaatgt gttcagctgc tctgtgatgc 1980
atgaggccct gcacaaccac tacacccaga aatccctgtc tctgagccct ggcaagtgaa 2040
agcttatcga taccgtcgac aggcctaata aagagctcag atgcatcgat cagagtgtgt 2100
tggttttttg tgtg 2114
<210> 17
<211> 429
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 51 pAAV-FIB-CD40-Ig amino acid sequence (CD 40-FIB Signal peptide)
<400> 17
Met Leu Arg Gly Pro Gly Pro Gly Arg Leu Leu Leu Leu Ala Val Leu
1 5 10 15
Cys Leu Gly Thr Ser Val Arg Cys Glu Pro Pro Thr Ala Cys Arg Glu
20 25 30
Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly
35 40 45
Gln Lys Leu Val Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu
50 55 60
Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His
65 70 75 80
Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln
85 90 95
Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly
100 105 110
Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser
115 120 125
Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp
130 135 140
Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser
145 150 155 160
Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu
165 170 175
Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro
180 185 190
Gln Asp Arg Leu Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
195 200 205
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
210 215 220
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
225 230 235 240
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
245 250 255
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
260 265 270
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
275 280 285
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
290 295 300
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
305 310 315 320
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
325 330 335
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
340 345 350
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
355 360 365
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
370 375 380
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
385 390 395 400
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
405 410 415
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
420 425
<210> 18
<211> 2093
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 52 pAAV-BM40-CD40-Ig base sequence (CD 40-BM40 Signal peptide)
<400> 18
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 cgcgcggtgg gcggggttga agctaacaaa gaccacgacg 600
atatcacggt cgtggtctca aagaacaaca aacaacaaag tccgactgag aaggtgagta 660
tctcagggat ccagacatgg ggatatggga ggtgcctctg atcccagggc tcactgtggg 720
tctctctgtt cacaggaacc ggtgccacca tgagggcttg gatctttttc ctgctgtgcc 780
tggctggacg ggctctggct gaacctccta cagcctgcag agagaagcag tacctgatca 840
acagccagtg ctgcagcctg tgccagcctg gccagaaact ggtgtctgac tgcacagagt 900
tcacagagac agagtgcctg ccttgtgggg agtctgagtt cctggacacc tggaacagag 960
agacacactg ccaccagcac aagtactgtg accccaacct gggcctgaga gtgcagcaga 1020
agggcacctc tgagacagac accatctgca cctgtgaaga aggatggcac tgtacctctg 1080
aggcctgtga aagctgtgtg ctgcacagaa gctgcagccc aggctttgga gtgaagcaga 1140
ttgccactgg tgtttctgat accatctgtg aaccttgtcc tgtgggcttc ttcagcaatg 1200
tgtcctctgc ctttgagaag tgtcacccct ggaccagctg tgaaacaaag gacctggtgg 1260
tgcagcaggc tgggaccaac aagacagatg ttgtgtgtgg accccaggac agactgagag 1320
agcccaagag ctgtgacaag acccacacct gtcctccatg tcctgctcca gagctgcttg 1380
gaggcccctc tgtgttcctg tttcctccaa agcctaagga caccctgatg atcagcagaa 1440
cccctgaagt gacctgtgtg gtggtggatg tgtcccatga ggacccagaa gtgaagttca 1500
attggtatgt ggatggggtt gaagtgcaca atgctaagac caagcctaga gaggaacagt 1560
acaacagcac ctacagagtg gtgtcagtgc tgactgtgct gcaccaggac tggctgaatg 1620
gcaaagagta caagtgcaag gtgtccaaca aggccctgcc tgctcctatt gagaaaacca 1680
tcagcaaggc caagggccag cctagggaac cccaggttta cacactgcca ccaagcagag 1740
aagagatgac caagaaccag gtgtccctga cctgcctggt caagggcttc tacccctctg 1800
acattgctgt ggaatgggag agcaatggcc agcctgagaa caactacaag acaacccctc 1860
ctgtgctgga ttctgatggc tcattcttcc tgtacagcaa gctgacagtg gacaagagca 1920
gatggcagca gggcaatgtg ttcagctgct ctgtgatgca tgaggccctg cacaaccact 1980
acacccagaa atccctgtct ctgagccctg gcaagtgaaa gcttatcgat accgtcgaca 2040
ggcctaataa agagctcaga tgcatcgatc agagtgtgtt ggttttttgt gtg 2093
<210> 19
<211> 422
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> 52 pAAV-BM40-CD40-Ig amino acid sequence (CD 40-BM40 Signal peptide)
<400> 19
Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu
1 5 10 15
Ala Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser
20 25 30
Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys
35 40 45
Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe
50 55 60
Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys
65 70 75 80
Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr
85 90 95
Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala
100 105 110
Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val
115 120 125
Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro
130 135 140
Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro
145 150 155 160
Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr
165 170 175
Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Glu Pro
180 185 190
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
195 200 205
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
210 215 220
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
225 230 235 240
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
245 250 255
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
260 265 270
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
275 280 285
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
290 295 300
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
305 310 315 320
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
325 330 335
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
340 345 350
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
355 360 365
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
370 375 380
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
385 390 395 400
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
405 410 415
Ser Leu Ser Pro Gly Lys
420
<210> 20
<211> 483
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> original humanized CTLA4 base sequence
<400> 20
atggcttgcc ttggatttca gcggcacaag gctcagctga acctggctac caggacctgg 60
ccctgcactc tcctgttttt tcttctcttc atccctgtct tctgcaaagc aatgcacgtg 120
gcccagcctg ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat 180
gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc tgacagccag 240
gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat 300
tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 360
gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac 420
ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 480
gac 483
<210> 21
<211> 161
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> original humanized CTLA4 amino acid sequence
<400> 21
Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala
1 5 10 15
Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro
20 25 30
Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala
35 40 45
Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly
50 55 60
Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln
65 70 75 80
Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr
85 90 95
Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val
100 105 110
Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile
115 120 125
Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly
130 135 140
Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser
145 150 155 160
Asp
<210> 22
<211> 579
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> original humanized CD40 base sequence
<400> 22
atggttcgtc tgcctctgca gtgcgtcctc tggggctgct tgctgaccgc tgtccatcca 60
gaaccaccca ctgcatgcag agaaaaacag tacctaataa acagtcagtg ctgttctttg 120
tgccagccag gacagaaact ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt 180
ccttgcggtg aaagcgaatt cctagacacc tggaacagag agacacactg ccaccagcac 240
aaatactgcg accccaacct agggcttcgg gtccagcaga agggcacctc agaaacagac 300
accatctgca cctgtgaaga aggctggcac tgtacgagtg aggcctgtga gagctgtgtc 360
ctgcaccgct catgctcgcc cggctttggg gtcaagcaga ttgctacagg ggtttctgat 420
accatctgcg agccctgccc agtcggcttc ttctccaatg tgtcatctgc tttcgaaaaa 480
tgtcaccctt ggacaagctg tgagaccaaa gacctggttg tgcaacaggc aggcacaaac 540
aagactgatg ttgtctgtgg tccccaggat cggctgaga 579
<210> 23
<211> 193
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<220>
<223> original humanized CD40 amino acid sequence
<400> 23
Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr
1 5 10 15
Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu
20 25 30
Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val
35 40 45
Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu
50 55 60
Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His
65 70 75 80
Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr
85 90 95
Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr
100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly
115 120 125
Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu
130 135 140
Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys
145 150 155 160
Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln
165 170 175
Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu
180 185 190
Arg

Claims (8)

1. A transgenic expression cassette having a nucleotide sequence selected from the group consisting of:
(I) SEQ ID NO: 6. 8 or 10;
(II) SEQ ID NO:12 or 16.
2. Use of a transgene expression cassette in the manufacture of a medicament for the treatment of multiple sclerosis, wherein the transgene expression cassette is a transgene expression cassette selected from group (I) of claim 1, a transgene expression cassette selected from group (II) of claim 1, or a combination thereof.
3.A medicament comprising:
a transgenic expression cassette selected from group (I) of claim 1, a transgenic expression cassette selected from group (II) of claim 1, or a combination thereof; and
AAV capsid proteins.
4.A medicament according to claim 3, wherein the AAV capsid protein is a native AAV capsid protein or an engineered AAV capsid protein.
5. A medicament according to claim 3, wherein the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAVXL32, AAVrh8, AAVrh39, AAVrh43, AAV32.33, AAVrh10, AAV3B, AAVv, and AAV.
6.A medicament according to claim 3, wherein the AAV is AAV8.
7. The medicament of any one of claims 3 to 6, wherein the medicament is administered by systemic or local route.
8. The medicament of any one of claims 3 to 6, wherein the medicament is administered intravenously, intramuscularly, subcutaneously, orally, topically, intraperitoneally, or intralesionally.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564187A (en) * 2021-07-30 2021-10-29 上海信致医药科技有限公司 AAV-based anti-angiogenic gene delivery system and uses thereof
CN113584043A (en) * 2021-07-30 2021-11-02 上海信致医药科技有限公司 Transgene expression cassette for treating retinal diseases and cancers

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564187A (en) * 2021-07-30 2021-10-29 上海信致医药科技有限公司 AAV-based anti-angiogenic gene delivery system and uses thereof
CN113584043A (en) * 2021-07-30 2021-11-02 上海信致医药科技有限公司 Transgene expression cassette for treating retinal diseases and cancers

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