WO2004050876A1 - Vaccin genique concernant un adenovirus recombinant convenant a la therapie et la prophylaxie de la maladie d'alzheimer, et utilisation correspondante - Google Patents
Vaccin genique concernant un adenovirus recombinant convenant a la therapie et la prophylaxie de la maladie d'alzheimer, et utilisation correspondante Download PDFInfo
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- WO2004050876A1 WO2004050876A1 PCT/CN2003/001019 CN0301019W WO2004050876A1 WO 2004050876 A1 WO2004050876 A1 WO 2004050876A1 CN 0301019 W CN0301019 W CN 0301019W WO 2004050876 A1 WO2004050876 A1 WO 2004050876A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- Recombinant adeno-associated virus gene vaccine for treating and preventing Alzheimer's disease with stem and use thereof
- the present invention relates to a recombinant adeno-associated virus gene vaccine for use in the treatment and prevention of Alzheimer's disease (AD) and its use. More specifically, the recombinant adeno-associated virus gene vaccine of the present invention comprises a recombinant DNA molecule containing a core sequence encoding a fusion protein of a cholera toxin B subunit and an A ⁇ peptide fragment. The invention also relates to a pharmaceutical composition comprising said recombinant DNA molecule or fusion protein. Background technique
- AD Alzheimer's disease
- presenile dementia is a common degenerative disease of the central nervous system. Its main clinical manifestations are progressive memory loss and cognitive impairment.
- the neuropathological features of AD are excessive extracellular ⁇ -amyloid precipitation, increased neurofibrillary tangles, decreased synaptic density, and decreased neuronal cells. Among them, the deposition of extracellular P amyloid, which forms scattered senile plaques and amyloid angiopathy, is considered to be a key link in the pathogenesis of the disease.
- Amyloid ⁇ protein is a ⁇ -amyloid precursor protein (APP) that is cleaved by ⁇ -secretase (BACE) and ⁇ -secretase and contains 39 to 43 amino acid residues. Enzymatic fragment. Under normal circumstances, most of APP is cleaved by ⁇ -secretase and ⁇ -secretase, does not form A ⁇ peptide, and cannot form extracellular ⁇ -amyloid precipitate. The occurrence of AD is related to several mutants of the AP protein.
- the APP V717I Goate et al, Nature 349; 704), APP V717G (Hai an et al, Nature 353; 844), APP V717F (MurreIl, Science 254; 97), APP lysine 595 - methionine Yue 596 changed into a double mutation of aspartic acid 595 -leucine 596 (Mullan et al, Nature Genet., 1; 345).
- the APP gene was sequenced and localized on chromosome 21 (Kang et al, Nature 325; 733). Expression of the APP gene produces several 695, 751, and 770 JJ3 ⁇ 4-containing isomers of A ⁇ . (Kang et al, Nature 325; 733, Kitaguchi et al, Nature 331; 530). Although there is evidence that APP has a role in mediating adhesion and growth of neurons (Schubert et al, Neuron 3; 689) and a role in G protein-linked signaling pathways (Nishimoto et al, Nature 362; 75), The exact function of APP in the nervous system is unclear.
- ⁇ -amyloid plays a key role in the occurrence of AD. All factors related to the disease either promote the deposition of ⁇ -amyloid or strengthen the pathological changes caused by ⁇ -amyloid. Therefore, the main target site currently developed by researchers for the treatment of AD drugs is to reduce the amount of beta amyloid deposits in the central nervous system (CNS). This type of research is mainly divided into two major areas, namely reducing the production of beta amyloid and increasing its clearance.
- patent application WO 98/44955 discloses a method for preventing or preventing Alzheimer's disease using a recombinant antibody specific to ⁇ -amyloid terminus, and this patent application protects a recombinant antibody specific to amyloid terminus and encoding Their DNA and how to use it.
- WO 99/27944 discloses the use of A ⁇ 42 peptide to produce anti-P amyloid antibody. The antibody can enter the brain tissue through the blood-brain barrier to bind to P amyloid protein, and clear P starch by phagocytosis of microglia. Like Protein deposition.
- the patent application protects the preparation of A & 42 peptide vaccine and its use method. Although the patent application mentions the potential of the AP 42 DNA fragment as a gene vaccine, it does not disclose how the gene vaccine is used in therapy.
- a ⁇ 42 peptide has a small molecule and low immunogenicity. In order to obtain an anti-Amyloid antibody that can clear senile plaques, repeated injections of A ⁇ 42 peptide are required. Adopting long-term subcutaneous immunization, the operation is tedious, and it also brings greater pain to AD patients. Secondly, the use of passive immunotherapy to directly inject anti-beta amyloid antibody into AD patients also has obvious shortcomings, that is, the half-life of the antibody in the body is very short. Not suitable for the treatment of chronic diseases like AD.
- a new genetic recombinant vaccine which contains a recombinant DNA sequence encoding a cholera toxin B subunit and an A ⁇ peptide fragment or a derivative thereof, and also contains a recombinant DNA molecule operatively linked to the recombinant DNA molecule Promoter capable of expressing fusion proteins (cholera toxin B subunit and A ⁇ peptide fragment) in eukaryotic cells.
- a leader sequence is also provided at the 5 end of the recombinant DNA molecule, which encodes the N-terminal signal peptide.
- the nucleotide sequence of the fusion protein encoding the cholera toxin beta subunit and the A ⁇ peptide fragment is shown in Figure 4 and the sequence listing (SEQ ID NO. 1).
- Another aspect of the present invention is to provide a recombinant DNA molecule containing a gene encoding a fusion protein of a cholera toxin B subunit and an A ⁇ peptide fragment.
- the fusion protein can induce the body to produce anti-beta amyloid antibodies.
- Yet another aspect of the present invention is to provide a recombinant viral vector comprising the recombinant DNA molecule of the present invention, the recombinant viral vector encoding A fusion protein is used to introduce the recombinant DNA molecule into a body cell.
- Suitable viral vectors include genovirus, adenovirus, herpes virus, retrovirus, vaccinia virus, fowlpox virus, Sindbis virus or fowlpox virus.
- the pharmaceutical composition contains a fusion protein called CB- ⁇ (sequence listing SEQ IDNO. 2).
- the protein CB- ⁇ is composed of a cholera toxin B subunit "head-A ⁇ peptide fragment.
- the protein CB- ⁇ may be an expression product of a recombinant DNA molecule in a prokaryotic cell, or a eukaryotic cell such as Yeast expression products.
- the fusion protein of the present invention is not limited to being formed from the cholera toxin B subunit and the A ⁇ peptide.
- the fusion protein can also be phycoglobin ⁇ first 3 peptides; in some pharmaceutical compositions, the fusion protein can also be a modified bacterial toxin-linker-A ⁇ peptide; in some pharmaceutical compositions, the fusion The protein may also be a 3 ⁇ 48 8 "head 0 peptide of HBV; in some pharmaceutical compositions, the fusion protein may also be a lipid or liposome- ⁇ peptide.
- the present invention also provides a pharmaceutical composition containing the recombinant DNA molecule of the present invention encoding a fusion protein (cholera toxin B subunit-linker-A ⁇ peptide) and a suitable gene transport vector.
- the composition is suitable for a variety of routes of administration for preventing or inhibiting the further development of Alzheimer's disease.
- the pharmaceutical composition can be administered orally, nasally, intradermally, subcutaneously, intramuscularly, topically or intravenously.
- Figure 1 shows the mechanism of beta amyloid production and the pathway of beta amyloid precursor protein (APP) hydrolysis.
- P-amyloid is a peptide fragment produced by ⁇ -amyloid precursor protein (APP) cleavage by ⁇ -secretase (BACE) and ⁇ -secretase.
- Figure 1 also shows the degradation process of APP after cleavage by ⁇ -secretase and ⁇ -secretase.
- Figure 2 shows a column of beta amyloid-producing regions in AP. Arrows indicate ⁇ -, Cleavage sites for ⁇ -, or ⁇ -secretase. Beta amyloid is a peptide fragment containing 39 to 43 amino acid residues.
- Figure 3 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Figure 3 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Embodiment 1 shows the technical route for the construction of genetically recombinant vaccines, including the cloning, ligation, sequencing, construction of plasmid plants, packaging and harvesting of CB and ⁇ 42 DNA.
- Figure 4 shows the DNA sequence encoding the fusion protein in the recombinant DNA molecule, which contains a total of 510 nucleotides.
- Figure 5 shows a 3 ⁇ 4 ⁇ column of the gene recombinant DNA molecule expression product-fusion protein, consisting of 170 amino acid residues.
- Figure 6 shows the titers of anti-beta amyloid antibodies in peripheral blood after immunizing PDAPP transgenic mice and non-transgenic mice with a genetically modified immune vaccine.
- A PDAPP transgenic mice in the treatment group
- B PDAPP transgenic mice in the prevention group
- C Non-transgenic mice in the treatment group
- D Non-transgenic mice in the prevention group.
- FIG. 7 shows the neutralizing effect of serum anti-A ⁇ IgG antibodies.
- rAAV / CB-A ⁇ 42 immune serum was diluted 1:10, the neurotoxic effect of A ⁇ 42 was partially neutralized.
- FIG. 8 shows the mean latency (MeaniSEM) of PDAPP transgenic mice in the treatment group searching for hidden platforms in the water maze experiment for 1 to 6 days.
- A Non-treatment group and AAV / GFP group.
- B rAAV / A ⁇ 42 group.
- C rAAV / CB-A ⁇ 42 group.
- im indicates intramuscular injection, in indicates nasal feeding, and oral indicates intragastric administration (the following figures are the same).
- Figure 9 shows the average incubation period (Mean soil SEM) of PDAPP transgenic mice in the prevention group searching for hidden platforms in the water maze experiment for 1 to 6 days.
- Figure 10 shows the percentage of search time in the TQ quadrant of the PDAPP transgenic mice in each group in the treatment group (A) and the prevention group (B). * P ⁇ 0.05, ** P ⁇ 0.01, compared with non-treatment group or AAV-GFP group.
- FIG. 11 shows the number of times the mice of each group of the treatment group (A) and the prevention group (B) passed the platform position in 30 seconds in the exploration experiment.
- Figure 12 shows the mean latency (Mean SEM) of PD APP transgenic mice in the treatment group in a visible platform water maze test.
- Figure 13 shows the mean latency (MeaniSEM) of PDAPP transgenic mice in the prevention group in the visible platform water maze test.
- FIG. 14 Immunohistochemical detection of A ⁇ amyloid in the brain tissue of PDAPP transgenic mice in the treatment group (cerebral cortex).
- FIG. 15 Immunohistochemical detection of ⁇ amyloid in brain tissue of PDAPP transgenic mice in the treatment group (hippocampal region).
- FIG. 16 Immunohistochemical detection of A ⁇ amyloid in the brain tissue of PDAPP transgenic mice in the prevention group (cerebral cortex).
- FIG. 1 Immunohistochemical detection of ⁇ amyloid in brain tissue of PDAPP transgenic mice in the prevention group (Hippocampus region).
- FIG. 1 Congo red histochemical detection (brain cortex) of brain tissue from PDAPP transgenic mice in the treatment group.
- FIG. 19 Histochemical detection of Congo red brain tissue in hippocampal regions of PDAPP transgenic mice in the treatment group.
- FIG. 20 Congo red histochemical detection (brain cortex) of the brain tissue of PDAPP transgenic mice in the prevention group.
- FIG. 21 Congo red histochemical detection of brain tissue in PDAPP transgenic mice in the prevention group (Hippocampus region).
- Figure 22 GFAP (Glial fibrillary acidic) in brain tissue of PDAPP transgenic mice in the treatment group protein glial fibrillary acidic protein) immunohistochemical detection.
- FIG. 23 Immunohistochemical detection of GFAP in brain tissue of PDAPP transgenic mice in the prevention group.
- the genetically recombinant vaccine of the present invention can be used for the prevention and treatment of Alzheimer's disease (a disease characterized by ⁇ amyloid deposition).
- the recombinant DNA molecule of the present invention can express a polypeptide such as the cholera toxin B subunit ⁇ head-A ⁇ peptide, which refers to a nucleotide sequence that contains transcriptional and translational regulatory information, and this sequence is effectively related to encoding this polypeptide The nucleotide sequences are linked.
- Effective linking refers to the linking that regulates the DNA sequence and the way the DNA sequence is linked to cause gene expression.
- the regulatory regions required for gene expression include a promoter region and a DNA sequence that, when transcribed into RNA, will become the starting signal for protein synthesis. Such regions will typically include those 5, -noncoding sequences involved in transcription and translation.
- the promoter used is, for example, a CMV promoter, which is suitable for a variety of cells and can promote the expression of a gene of interest in a variety of cells.
- Vectors used for gene transfer include viral vectors (such as adeno-associated virus vectors), lipids / liposomes, and ligands for cell surface receptors.
- the recombinant DNA molecule needs to be combined with a gene transfer vector.
- the gene transfer vector in some viral vectors and the recombinant DNA molecule encoding a fusion protein are integrated into the DNA of the viral vector or packaged into virus particles; some lipids or lipids
- the plastid gene transfer vector can encapsulate the recombinant DNA molecule therein; some ligands of cell surface receptors are used as gene transfer vectors to combine with the recombinant DNA molecule in a coupling or other manner. Therefore, the term "combination" includes integration or packaging, compounding, coupling, and the like.
- Adeno-associated virus was originally contaminated in tissue culture It was later found that a non-pathogenic co-infection factor was isolated during the onset of adenovirus infection in children (Blacklow et al, 1986), which was called the eye related virus or adenovirus-associated virus.
- AAV is a single-stranded DNA virus of the genus Parvovirus, having a 4.7 kb genome.
- AAV is a replication-deficient virus that requires a co-infection of a helper virus (usually an adenovirus or herpes virus) to replicate efficiently and complete its life cycle.
- helper virus usually an adenovirus or herpes virus
- AAV becomes a latent form and undergoes stable integration at a high frequency, usually at a specific site on chromosome 19 (Kotin et al, 1992).
- the AAV genome has been sequenced, and the only sequence necessary for AAV integration was found to be an inverted terminal repeat (ITR) of 145 nucleotides.
- ITR inverted terminal repeat
- the clone capacity of AAV is about 4.7Kb (Muzyczka, 1992). Because the virus has its own superior characteristics (non-pathogenic, extensive host cells, efficient integration ability, heat resistance, acid resistance, and long-term efficient expression), it is particularly suitable for use as a gene transfer vector.
- AAV can infect cells and integrate onto the cell chromosome, express the fusion protein (cholera toxin B subunit head-A ⁇ peptide), and secrete it outside the cell for antigen presentation
- fusion protein cholera toxin B subunit head-A ⁇ peptide
- present antigens to T and B lymphocytes induce immune responses, and produce anti-beta amyloid antibodies.
- the antibody can enter the extracellular environment of neurons in brain tissue and combine with soluble or aggregated beta amyloid to form a beta amyloid-antibody complex.
- the antibodies regulate the beta amyloid-antibody complex through the opsonization of Fc receptors on the surface of small neuroglia shield cells to clear the deposition of amyloid beta, or the beta amyloid-antibody complex passes through The excretion of sagittal sinus arachnoid villi is cleared from the central nervous system, thereby avoiding the deposition of beta amyloid in brain tissue and the neurotoxic effects induced by it.
- the genetic recombination vaccine and the pharmaceutical composition of the present invention are not only suitable for patients who have apparently developed Alzheimer's disease pathological features, but also suitable for the general population to prevent the occurrence of Alzheimer's disease, so that the general population can obtain a response to this epidemic.
- Sexual and destructive disease immunity especially patients with Down's syndrome or people with familial Alzheimer's disease-related gene mutations who are prone to develop Alzheimer's disease.
- the best route of administration is subcutaneous, intradermal, intramuscular, oral, nasal or intravenous.
- a recombinant DNA molecule containing a fusion protein gene combined with a gene transfer vector can be used in the production or preparation of a pharmaceutical composition containing such a required effective amount of the recombinant DNA molecule.
- a suitable dose of virus particles in a pharmaceutical composition for treating or preventing Alzheimer's disease is about 5 ⁇ 10 4 to 1 ⁇ 10 12 virus particles.
- a ligand of a cell surface receptor is used as a gene transfer carrier, the amount of DNA molecules bound by the ligand used is about 0.5 to 100 ⁇ g.
- the amount of DNA molecules used is about 1 to 500 g.
- PDAPP transgenic mice refers to C57 mice transfected with APP V7171 gene mutants. The mice begin to have behavioral changes at 3 to 5 months and appear in brain tissues at 11 to 13 months. Similar to the pathological changes in patients with Alzheimer's disease, there is obvious deposition of amyloid and senile plaque.
- non-transgenic mice refers to C57 normal mice that are not transfected with the APP V71 "gene mutant.
- APP V717I , APP V717G, and APP V717F refer to mutations in the 717 site of the APP protein encoded by the human APP gene from the original valine to isoleucine, glycine, and phenylalanine, respectively.
- ⁇ peptide refers to AP peptide fragments containing acid residues 1-39, 1-40, 1-41, 1-42, and 1-43, mainly including AP 39 , ⁇ 40 , ⁇ 41 , 0 42 and 0 43 .
- "Alpha] [beta peptide” include ⁇ 39, ⁇ ⁇ 40, ⁇ 41, ⁇ derivatives ⁇ ⁇ 43 and ⁇ 42 a.
- a ⁇ 41 , A ⁇ 40 and A ⁇ 39 differ from A ⁇ 42 in that they lack the C-terminal Ala, Ala-Ile and Ala-Ile-Val, respectively.
- ⁇ 43 differs from ⁇ 42 in that it adds a Thr to the C-terminus.
- amyloid ⁇ protein is equivalent in meaning to the term “A ⁇ peptide” and includes A ⁇ 39 , A ⁇ 40 , A ⁇ 41 , A ⁇ 42 and A ⁇ 43 peptides.
- immune response refers to the generation of a response in a patient being treated against amyloid beta antibodies and / or ⁇ specific T cells or their secreted products.
- adjuvant refers to a substance that can enhance the body's immune response to an antigen when used in combination with an antigen, but does not produce an immune response to the antigen when used alone.
- adjuvants can enhance the immune response through several mechanisms, including aggregation of lymphocytes, activation of B cells and / or T cells, activation of macrophages Cell.
- the term "patient” includes humans and other mammalian subjects who receive prophylactic or therapeutic treatments.
- the term "fusion protein” is referred to herein as containing a new fusion protein shield called CB-Ap.
- the new protein (CB- ⁇ ) consists of the cholera toxin B subunit head- ⁇ peptide fragment.
- the linker amino acid sequence is glycine-proline-glycine-proline.
- recombinant DNA molecule refers to a nucleotide sequence containing derivatives encoding the cholera toxin beta subunit and A ⁇ peptide fragments, and derivatives thereof.
- derivative refers to a sequence containing a nucleotide that encodes a polypeptide that substantially retains the biological function or activity of human amyloid. Or refers to a polypeptide that substantially retains the biological function or activity of human beta amyloid.
- promote sequence refers to the part of a DNA molecule that can specifically bind to RNA polymerase, that is, the part where transcription starts.
- gene transfer vector refers to any technology and / or substance suitable for introducing DNA molecules into the body.
- the AAV gene recombinant vaccine of the present invention After the AAV gene recombinant vaccine of the present invention is administered to a patient, it ectopically expresses a fusion protein molecule in the patient's body, and induces the body to produce an antibody against beta amyloid.
- the antibody is capable of neuron extracellular environment in brain tissue, and Soluble or aggregated beta amyloid binds to form a beta amyloid-antibody complex.
- the antibodies promote the phagocytosis of ⁇ -amyloid-antibody complex by the opsonization of Fc receptors on the surface of microglia to clear the deposition of P amyloid, or the ⁇ -amyloid-antibody complex passes through The excretion of sagittal sinus arachnoid villi is cleared from the central nervous system, thereby avoiding the deposition of beta amyloid in brain tissue and the neurotoxic effects induced by it.
- the amyloid proteins involved in the pathological process of Alzheimer's disease namely A ⁇ 39 , A ⁇ 4 ⁇ A ⁇ 41 , AP 42 and A ⁇ 43 are the main clearance targets. Therefore, the genetic recombinant vaccine of the present invention can be used to prevent or treat Alzheimer's disease, or to inhibit the further development of Alzheimer's disease.
- beta amyloid eggs can be prevented or eliminated
- the invention also avoids the problems caused by long-term repeated use of pharmaceutical preparations that need to cross the blood-brain barrier.
- experiments show that the structural design of the fusion protein of the present invention and the selection of the linker allow the two polypeptides linked by the linker to maintain their respective activities in the body, thereby pre-paying or treating the Alzheimer's gene recombinant vaccine and the fusion protein of the present invention. Symptoms provide a guarantee.
- the content of the present invention can be more easily understood by referring to the following examples, which are only for further explanation and are not meant to limit the scope of the present invention.
- Upstream primer 5, -GGTCCTGGTCCTGATGCAGAATTCCGACATG AC-3, (SEQIDNO. 3),
- Downstream primer 5, -GGAAGATCTTTACTACGCTATGACAACACCGCCC-3, (SEQIDNO. 4).
- CB cholera toxin B subunit
- the universal AAV shield particle vector pSNAV (Chinese patent Application number: 99119038.6, Title of invention: Construction and application of a series of general-purpose adenovirus-associated virus vectors, Publication No .: CN 1252450A), after double digestion with Kpn I and Bgl ll, large fragments were recovered to obtain pSNAV vectors containing sticky ends Fragment.
- the digested pSNAV vector fragment and the CB-A P 42 DNA fragment were ligated into the pSNAV-CB-A 0 42 plasmid. Then, the ligation product was transformed, and a small amount of purified plasmid DNA was extracted.
- pSNAV-A ⁇ 42 plasmid was constructed, and pSNAV-A ⁇ 42 plasmid and pSNAV-GFP plasmid were used as controls for pSNAV-CB-A ⁇ 42 plasmid.
- the pSNAV-GFP plasmid was previously constructed by Wu Xiaobing et al. (Chinese patent application number: 99119038.6, invention name: Construction and use of a series of general-purpose adenovirus-associated virus vectors).
- the pSNAV-A ⁇ 42 plasmid contains only A ⁇ 42 DNA fragments and is preceded by an AAP leader sequence, but does not contain the CB gene DNA fragment.
- the green fluorescent protein (GFP) gene was inserted into the pSNAV-GFP plasmid.
- the pSNAV-CB-A P 42 plasmid obtained in the above Example 1 was packaged into a recombinant AAV virus (rAAV / CB-A ⁇ 42 virus) containing a CB-A ⁇ 42 expression cassette, that is, a genetic recombinant vaccine of the present invention.
- rAAV / CB-A ⁇ 42 virus a recombinant AAV virus
- CB-A ⁇ 42 expression cassette that is, a genetic recombinant vaccine of the present invention.
- It is the method of the patent applied by Wu Xiaobing et al. Choinese patent application number: 98120033.8, invention name: Production and use of fully functional helper virus for recombinant adeno-associated virus production; Chinese patent application number: 99119039.4, invention name: available Production method and use of recombinant adenovirus-associated virus produced in Da Mo; Chinese Patent Application No. 99123723.4, Title of Invention: A method and
- Step 1 Establishment of rAAV / CB-A ⁇ 42 virus vector cell line BHK cells were transfected with the pSNAV-CB-A P 42 plasmid, and the rAAV / CB-A ⁇ 42 virus vector cell line BHK / pSNAV-CB-A ⁇ 42 was obtained by selective culture.
- BHK cells were cultured in RPMI1640 medium 37 containing 10% fetal bovine serum.
- the pSNAV-CB-A ⁇ 42 plasmid was transfected into BHK cells with lipofectamine (GIBCO BRL), digested 24 hours later, and passaged at 1: 2 to 5.
- Apparently resistant cell clones formed after 10 days. Cell clones were singled out for further culture and cryopreserved.
- the cloned and cultured virus vector cells obtained are infected with a full-function helper virus HSVl-rc (Chinese patent application number: 98120033.8, invention name: Production and use of full-function helper virus for recombinant adeno-associated virus production)
- HSVl-rc Full-function helper virus
- the cells and their culture fluids were harvested after the cells became virulent.
- the results of SDS-PAGE electrophoresis showed three characteristic electrophoretic bands of AAV virus.
- Southern hybridization results showed that the rAAV virus contained the CB-A ⁇ 42 nucleic acid sequence.
- the titer of rAAV / CB-A ⁇ 42 virus was determined.
- a cell line with high rAAV virus titer was selected as the AAV-GFP virus plant cell line.
- the cloned cultured virus vector cells obtained above were further cultured to a large number of roller bottle culture scales with RPMI1640 medium containing 10% fetal bovine serum. After the cells reached a certain number, the full-function helper virus HSVl-rc was used to expand the culture Cell.
- the cells and their culture fluids are harvested, and the recombinant rAAV / CB-A ⁇ 42 virus is isolated and purified using the following steps: 1) chloroform disrupts the cells, inactivates HSV helper viruses, and denatures a large number of cellular proteins; 2) Treat cell lysate with DNasel and RNase to degrade nucleic acid; 3) Add NaCI to separate rAAV from cell debris and centrifuge to remove cell debris; 4) Precipitate rAAV with PEG / NaCl; 5) Extract with chloroform to remove impurities and residues 6) dialysis and desalting; 7) further purification of rAAV by density gradient centrifugation or affinity chromatography.
- the titer of rAAV / CB-A ⁇ 42 virus was determined by the method of Roche's digoxin DNA labeling and detection kit (Cat. No. 1093657), and serially dried plasmid pSNAV-CB-A P 42 plasmid As a standard control, southern hybridization was performed, and the hybridization signal of the sample was compared and quantified with the standard control. The results showed that the rAAV virus contained CB-A ⁇ 42 Nucleic acid sequence. rAAV virus titer of 2 X 10 12 - 13 viral particles / ml.
- the electrophoretic purity of rAAV / CB-A p 42 virus was determined by SDS-PAGE method, with a sample volume of 5 g, and the gel was stained with Coomassie blue after electrophoresis. See the three main bands of AAV-2 coat protein VP1, VP2, and VP3 After scanning with a scanner, calculate the percentage of total VP1, VP2, and VP3 proteins to the total protein. The results showed three characteristic electrophoretic bands of AAV virus.
- the packaged virus was orally, nasally, and intramuscularly injected to immunize 1- and 11-month-old PDAPP transgenic mice (PDAPP V7171 transgenic mice, see literature: Qin Chuan, Chang Yang, etc.).
- peripheral blood was collected from mice, and serum was isolated.
- ELISA was used to detect the titer of anti- ⁇ antibody in serum.
- rAAV / CB-A P 42 immunized mice were able to produce high titers of anti-AP l g G antibodies in either the treatment group or the prevention group, whether in transgenic mice or non-transgenic mice.
- the titer of anti-AP lgG antibody was the highest in the serum (the antibody titer was between 5,000-15,000, which is equivalent to 10 times the antibody titer produced by the corresponding mice immunized with rAAV / AP 42).
- Antibody (antibody titer below 1: 64). It was also found that the titers of antibody production were different between the three pathways of rAAV / CB-A P 42 immunized mice. At two months, the antibody titer was significantly higher than that of the gavage group and the nasal feeding group, and there was no significant difference between the gavage group and the nasal feeding group. The rAAV / CB-A P 42 immunized mice had a lower anti-A ⁇ I g G antibody titer in the peripheral blood of the treatment group than the prevention group (P ⁇ 0.05).
- Example 4 Example 4
- the SH-S ⁇ 5 ⁇ human neuroblastoma cells purchased from Chinese Peking Union Medical University cells) per well in 100 ⁇ containing 10 4 cells were seeded in 96-well plates, culture medium serum-free medium DMEM / F12 (available (Gibicol, USA).
- a ⁇ 42 peptide (Sigma Company, USA) was diluted to 0.12 ⁇ M, and incubated at 37 ° C for 1 week to produce fibril filaments. Then, mix 10: 1 and 50: 1 with the immune serum, and incubate for 24h at 7. Meanwhile, non-immune serum was used as a control. Each sample was duplicated. This reaction mixture was added to culture wells containing SH-SY5Y human neuroblastoma cells, 37. C.
- rAAV-GFP intramuscular group and non-treated groups were statistically analyzed with rAAV / A ⁇ 42 immunized intramuscularly in groups rAAV / CB-A ⁇ 42 intramuscularly Immunohistochemistry showed rAAV / CB-A ⁇ 42
- the average latency of the intramuscularly immunized PDAPP transgenic mice group was significantly shorter than that of the rAAV / GFP group (p ⁇ 0.01) and the untreated group at 5-6 days.
- the rAAV / CB-A ⁇ 42 gene recombinant vaccine was used in one-month-old mice. Behavioral indicators of mice were observed after 12 months. Since the ⁇ ⁇ / 42 gene recombinant vaccine was found in pre-experiments to produce anti-A ⁇ antibody titers in PDAPP transgenic mice was very low, and the vaccine did not significantly improve the behavior of transgenic mice in the treatment group, so we The rAAV / A ⁇ 42 group was not added to the prevention group.
- the percentage of search time in the TQ quadrant of rAAV / CB-A ⁇ 42 intramuscularly immunized mice was 37.5%, compared with 27.2% in the rAAV / GFP group and 25.9% in the untreated group. There were significant differences between the AAV-GFP group and the untreated group and the rAAV / CB-A ⁇ 42 intramuscular injection group (p ⁇ 0.05). rAAV-A P 42 42 intramuscularly immunized mice had a search time percentage in the TQ quadrant and the average number of times they passed through the platform were 28.9%, respectively.
- the r AAV / A ⁇ 42 intramuscular injection immunization group averaged 0.66 ⁇ 0.25 in the 30-second search time, and the rAAV / GFP group There was no significant difference between the treatment group and the non-treatment group (both P> 0.05), and it was also significantly less than the rAAV / CB-A ⁇ 42 intramuscular injection group (PO.01). There was no significant difference in the percentage of search time in the TQ quadrant and the number of times to pass through the platform position among non-transgenic mice in each group.
- the average latency of the rAAV / CB-A ⁇ 42 intramuscular injection group on the fourth day was 15.1 ⁇ l.ls, while the rAAV / GFP group, the untreated group, and the rAAV / A ⁇ 42 intramuscular injection group were respectively It was 19.4 ⁇ 3.1s, 21 ⁇ 3.6s, and 19.9 ⁇ 3.3s.
- rAAV / CB-A ⁇ 42 was injected into the immunized group and AAV-GFP group, untreated group and rAAV / A ⁇ 42 muscle. There were significant differences between the injection groups (all P values were less than 0.05).
- a ⁇ 42 levels were significantly lower than those in the treatment group.
- rAAV / CB-Ap 42 showed no significant differences among the three immune pathways, the contents of A ⁇ 40 and A ⁇ 42 in the brain tissue of PDAPP transgenic mice immunized with rAAV / CB-Ap 42 were significantly lower than those in the other groups.
- rAAV / CB-Ap 42 was 90.1 ⁇ 13.2 ng / g in the brain tissue of PDAPP transgenic mice in the immunized PDAPP transgenic mice, and the content of A ⁇ 42 was 40.2 ⁇ 10.3 ng / g, which were significantly lower than those in non- Treatment group (A ⁇ 40 was 130.6 ⁇ 17.9 ng / g, A 42 was 51.9 ⁇ 10.2 ng / g) and rAAV / GFP group (127.8 ⁇ 18.6 ng /, A ⁇ 42 was 55.3 ⁇ 13.5 ng / g) (all p values were Less than 0.05).
- Example 7 Immunohistochemical staining of amyloid P in brain tissue of PDAPP mice
- the CB-A P 42 and A ⁇ 42 DNA fragments were amplified by a polymerization reaction (PCR) (the specific steps are the same as those in Example 1), and they were respectively loaded into pET30a (Novagen) and pET42a (Novagen) prokaryotic cells. Expression vector. Then, it was transformed into an E. coli BL21 (DE3) -expressing strain and induced by IPTG to obtain a fusion protein. The fusion protein was purified through a His Trap purification column, and then the CB-A ⁇ 42 protein and the A ⁇ 42 peptide fragment were respectively cut with thrombin Xa and purified, lyophilized, and stored at -20.
- PCR polymerization reaction
- the lyophilized CB-A ⁇ 42 protein and AP 42 peptide obtained in Example 10 were separately dissolved in phosphate buffered saline (PBS), and Balb / c mice were immunized subcutaneously without adjuvant, and crusted Shows that after one month of immunization, the CB-A ⁇ 42 protein immunization group can produce high titers of anti-P amyloid
- the antibody (antibody titer is 1: 10,000 ⁇ 20,000), while the A ⁇ 42 peptide immunization group produced only very low anti-beta amyloid antibodies (antibody titer: 1: 200 ⁇ 500). This shows that CB-AP 42 protein can also be used as a drug for the prevention and treatment of Alzheimer's disease.
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Description
用干治疗和预防阿尔茨海默病的重组腺 相关病毒基因疫苗及其用途
发明领域
本发明涉及一种用于阿尔茨海默病 （Alzheimer's disease, AD ) 的治疗 和预防的重组腺相关病毒基因疫苗及其用途。 更具体地， 本发明的重组腺 相关病毒基因疫苗包含一种重組 DNA分子， 该重组 DNA分子含有编码一 种霍乱毒素 B亚单位和 Αβ肽片段的融合蛋白的核^^列。本发明还涉及包 含所述重组 DNA分子或融合蛋白的药物组合物。 背景技术
阿尔茨海默病（ Alzheimer's disease, AD )又称早老性痴呆（ Presenile dementia ) ， 是一种常见的中枢神经系统进行性变性疾病， 其主要临床表 现为进行性记忆减退和认知障碍。 AD的神经病理学特征为过度的细胞外 β 淀粉样蛋白沉淀、 神经纤维缠结增多、 突触密度减少、 神经元细胞减少等。 其中细胞外 Ρ淀粉样蛋白的沉积， 形成分散的老年斑和淀粉样血管病， 被 认为是该病发病机制中的关键环节。 β淀粉样蛋白 （amyloid β protein ) 是由 β淀粉样前体蛋白 （ β amyloid precursor protein, APP )经过 β-分泌酶 ( BACE )和 γ-分泌酶裂解后产生的含 39 ~ 43个氨基酸残基的酶解片段。 正常情况下， ΑΡΡ大部分经过 α-分泌酶和 γ-分泌酶裂解， 不形成 Αβ肽， 不 能形成细胞外 β淀粉样蛋白沉淀。 AD 的发生与 ΑΡΡ蛋白的几种突变体有 关。如 APPV717I( Goate et al, Nature 349;704 )、 APPV717G(Hai an et al, Nature 353;844)、 APPV717F(MurreIl, Science 254;97)、 APP的赖氨酸 595-曱硫氨酸 596变为天冬氨酸 595-亮氨酸 596的双突变 ( Mullan et al, Nature Genet., 1 ;345 ) 。 这些突变改变了 APP的酶切位点， 使大部分 APP经过 β-分泌酶 和 Υ -分泌酶裂解， 产生 39 ~ 43个氨基酸残基的 Αβ肽， 形成细胞外 β淀粉 样蛋白聚集沉淀。特别是这些突变增加了 Αβ42肽和 Αβ43肽的产生量， 由于
Αβ42肽和 Αβ43肽羧基末端延伸部位的构象更难溶于水， 易于聚集形成反向 平行的 β折叠片层优先沉淀 (Joachim et al, Nature 325;733、 Halverson et al, Biochemistry 29;2639、 Barrow et al， J. Mol. Bio. 225; 1075)。 然而， Αβ42肽 的聚集可以促进 Αβ4。肽聚集， 从而形成老年斑。
ΑΡΡ基因巳被测序， 并定位于第 21号染色体上 (Kang et al, Nature 325;733)。 APP基因的表达产生几种 695， 751和 770个 JJ¾的含有 Αβ 的异构体。（Kang et al, Nature 325;733 , Kitaguchi et al， Nature 331 ;530)。 虽然有证据表明 APP具有介导神经元的粘连与生长的作用 (Schubert et al, Neuron 3;689)以及在 G蛋白质连接的信号传导途径中起作用（Nishimoto et al, Nature 362;75), 但是 APP在神经系统中的确切功能还尚不清楚。
目前普遍认为， β淀粉样蛋白的沉积在 AD的发生 ^过程中起关键性 作用， 所有与该疾病相关的因素要么促进 β淀粉祥蛋白的沉积， 要么强化由 β淀粉样蛋白引发的病理变化。 因此， 目前研究人员开发用于治疗 AD药物 的主要靶位点是降低中枢神经系统 (CNS)中 β淀粉祥蛋白沉积的数量。 此类 研究主要分为两大领域， 即降低 β淀粉祥蛋白的产生和增加它的清除。
对于第一个领域， 从药物学的角度来说， 降低 β淀粉祥蛋白的产生的最 直接的方式是通过直接抑制 β或 Υ分泌酶。但是， 目前对 β-分泌酶（ BACE ) 和 Υ -分泌酶在人体内的其它功能尚未研究清楚， 而且它们存在于细胞的内 质网或内体系统的内膜上， 现在还难以找到既能通过血脑屏障， 又能通过 细胞膜和内质网膜的药物来抑制它们的酶活性的特异性抑制物。
对于第二个领域， 尤其对于已发展成为 AD的病人来说， 清除脑组织内 Ρ淀粉样蛋白的沉积更为重要。 目前这一领域的研究比较热。 其中， 专利 申请 WO 98 / 44955公开了使用特异于 β -淀粉样蛋白末端的重组抗体防止 或预防阿尔茨海默病的方法， 该专利申请保护的是特异于 淀粉样蛋白末 端的重组抗体和编码它们的 DNA及其使用方法。 WO 99 / 27944公开了使 用 Α β 42肽产生抗 Ρ淀粉样蛋白的抗体，该抗体能够通过血脑屏障进入脑组 织与 Ρ淀粉样蛋白结合， 并通过小神经胶质细胞的吞噬作用清除 Ρ淀粉样
蛋白的沉积。该专利申请保护的是 A & 42肽疫苗的制备及其使用方法。 虽然 该专利申请中提及到有关 A P 42 DNA片段作为基因疫苗的潜在的可能性， 但没有公开基因疫苗是如何在治疗中使用的具体内容。
虽然上述肽疫苗或抗体被动免疫较以往 AD 的治疗方法有了较大的突 破， 但是仍不够完善。 首先， Αβ42肽分子较小， 免疫原性低， 为得到能清 除老年斑的抗 Ρ淀粉样蛋白抗体， 需要反复注射 Αβ42肽。 采用长期的皮下 免疫， 操作繁瑣， 同时也给 AD病人带来较大的痛苦。 其次， 采用被动免 疫治疗方法给 AD病人直接注射抗 β淀粉样蛋白抗体， 也存在着明显的缺 点， 即抗体在体内的半衰期非常短。 不适用于象 AD这类慢性疾病的治疗。 再次， 从目前所取得的实验结果来看， 虽然疗效显著， 但仍未达到最理想 的程度， 如特异性抗体的滴度仍不是 4艮高， 仍有部分老年斑形成等。 因此， AD的免疫治疗需要进一步改进。 目前国际上 AD免疫治疗的发展趋势是： ( 1 )增强 Αβ42肽疫苗的免疫原性， 进一步提高特异性抗体的滴度； （2 ) 改进免疫途径和方法； （3 )减少操作的复杂性和成本， 便于大量制备， 广 泛使用。 发明内容
根据本发明的一个方面， 提供了一种新的基因重组疫苗， 该疫苗含有 编码霍乱毒素 Β亚单位和 Αβ肽片段的重组 DNA序列或其衍生物， 也含有 与该重组 DNA分子有效连接的并能够在真核细胞中表达融合蛋白（霍乱毒 素 Β亚单位和 Αβ肽片段）的启动子。 同时， 为了能够使融合蛋白顺利地从 表达细胞中分泌出来，在重组 DNA分子的 5，端也提供了一个前导序列，编 码 Ν-末端信号肽。 编码霍乱毒素 Β亚单位和 Αβ肽片段的融合蛋白核苷酸 序列如图 4和序列表 ( SEQIDNO. 1 )所示。
本发明的另一个方面是提供一种重组 DNA分子，它含有编码一种霍乱 毒素 Β亚单位和 Αβ肽片段的融合蛋白的基因，该融合蛋白能够诱发机体产 生抗 β淀粉样蛋白抗体。
本发明的又一个方面是提供一种包含本发明的重组 DNA 分子的重组 病毒载体，该重组病毒栽体编码能够有效诱导对 β淀粉样蛋白产生免疫应答 的霍乱毒素 Β亚单位和 Αβ肽片段的融合蛋白， 用于将所述重组 DNA分子 导入机体细胞。 适合的病毒载体包括 目关病毒、 腺病毒、 疱疹病毒、 逆 转录病毒、 痘苗病毒、 禽痘病毒、 新培斯病毒或鸡痘病毒。
本发明的另一方面是提供一种用于防止或抑制阿尔茨海默病的进一步 发展的新的药物组合物。 该药物组合物包含一种融合蛋白质， 称为 CB-Αβ (序列表 SEQ IDNO. 2 )。 该蛋白质 CB-Αβ由霍乱毒素 Β亚单位 "^头 -Αβ 肽片段组成。 在一些药物组合物中该蛋白质 CB-Αβ可以是重组 DNA分子 在原核细胞中表达产物， 也可以是真核细胞如酵母的表达产物。
应理解的是， 本发明的融合蛋白不仅限于由霍乱毒素 Β亚单位与 Αβ肽 形成。 在一些药物组合物中， 融合蛋白也可以^ 疫球蛋白 ~ 头 3肽； 在一些药物组合物中， 融合蛋白也可以是改造的细菌毒素 -接头 -Αβ肽； 在 一些药物组合物中， 融合蛋白也可以是 HBV的 1¾8 8" 头 0肽； 在一 些药物组合物中， 融合蛋白也可以是脂类或脂质体 -Αβ肽。
本发明还提供了含编码融合蛋白（霍乱毒素 Β亚单位 -接头 -Αβ肽）的本 发明的重组 DNA分子及合适的基因转运载体的药物组合物。所述组合物适 用于多种给药途径的， 用于防止或抑制阿尔茨海默病的进一步发展。 该药 物组合物的给药途径包括口服、 鼻饲、 皮内、 皮下、 肌内、 局部或静脉内 给药。 附图简要说明
图 1 表示 β淀粉样蛋白产生的机制和 β淀粉样前体蛋白 （ΑΡΡ )酶解的 途径。 p淀粉样蛋白是由 β淀粉样前体蛋白 （ ΑΡΡ )经过 β-分泌酶（ BACE ) 和 Υ -分泌酶裂解后产生的肽片段。 同时， 图 1也显示了 ΑΡΡ经 α-分泌酶和 Υ -分泌酶裂解后降解的过程。
图 2表示在 ΑΡΡ产生 β淀粉样蛋白区域的 列。 箭头表示 α-、
β -、 或 Υ -分泌酶的切割位点。 β淀粉样蛋白是含 39 ~ 43个氣基酸残基的肽 片段。
图 3表示基因重组疫苗的构建技术路线， 主要包括 CB和 Αβ42 DNA 的克隆、 连接、 测序、 质粒栽体的构建、 病毒的包装与收获等过程。 具体 内容参照实施例 1。
图 4表示基因重组 DNA分子中编码融合蛋白的 DNA序列，共含有 510 个核苷酸组成。
图 5表示基因重组 DNA分子表达产物 -融合蛋白的 ¾ ^列， 由 170个氨基酸残基组成。
图 6表示基因重组免疫疫苗免疫 PDAPP转基因小鼠和非转基因小鼠后 外周血抗 β淀粉样蛋白抗体的效价。 （Α) 治疗组 PDAPP转基因小鼠； （Β ) 预防组 PDAPP转基因小鼠； （C )治疗组非转基因小鼠； （D )预防组非 转基因小鼠。
图 7表示血清抗 Α β IgG抗体的中和作用， 在 rAAV/CB-A β 42免疫血 清 1: 10稀幹度时， 能够部分中和 A β 42的神经毒性作用。
图 8表示治疗组 PDAPP转基因小鼠在水迷宫实验中 1 ~ 6天搜索隐蔽 平台的平均潜伏期（ MeaniSEM )。 A. 非处理组和 AAV/GFP组。 B. rAAV/A β 42组。 C. rAAV/CB-A β 42组。 其中 im表示肌肉注射， in表示鼻饲， oral 表示灌胃 （以下各图相同） 。
图 9表示预防组 PDAPP转基因小鼠在水迷宫实验中 1 ~ 6天搜索隐蔽 平台的平均潜伏期 （Mean土 SEM ) 。 A.非处理组和 AAV-GFP 组。 B. AAV-CB-A β 42三种免疫途径。
图 10表示治疗组 (Α)和预防组 (Β)中各组 PDAPP转基因小鼠在探索实 验中在 TQ象限中搜索时间的所占百分比。 * P<0.05，** P<0.01， 与非治疗组 或 AAV-GFP组相比较。
图 11表示治疗组 (A)和预防组 (B)各组小鼠在探索实验中在 30秒内经过 平台位置的次数。
图 12表示治疗组 PD APP转基因小鼠在可见平台水迷宫试验中的平均 潜伏期 （Mean士 SEM ) 。 A. 非处理组和 AAV/GFP组。 B. rAAV/CB-A β 42组。 C. rAAV/A P 42组。
图 13表示预防组 PDAPP转基因小鼠在可见平台水迷宫试验中的平均 潜伏期 （MeaniSEM ) 。 Α· 非处理组和 AAV/GFP组。 B. rAAV/CB-A β 42组。
图 14.治疗组 PDAPP转基因小鼠脑组织内 Αβ淀粉样蛋白免疫组织化学 检测（大脑皮层）。 A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ CB-AP42 肌注组。
图 15. 治疗组 PDAPP转基因小鼠脑组织内 Αβ淀粉样蛋白免疫组织化 学检测（海马区域）。 A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ ΟΒ-Αβ42 肌注组。
图 16.预防组 PDAPP转基因小鼠脑组织内 Αβ淀粉样蛋白免疫组织化 学检测 (大脑皮层）。 A. Control. B. rAAV/ GFP肌注组 C. rAAV/CB-A 42 肌注组。
图 17.预防组 PDAPP转基因小鼠脑组织内 Αβ淀粉样蛋白免疫组织化 学检测（海马区域）。 A. Control. B. rAAV/ GFP肌注组 C. rAAV/CB-Ap42 肌注组。
图 18. 治疗组 PDAPP转基因小鼠脑组织刚果红组织化学检测（大脑皮 层） 。 A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ Β-Αβ42肌注组。
图 19. 治疗组 PDAPP转基因小鼠脑组织刚果红组织化学检测（海马区 域） 。 A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ CB-A 42 肌注组。
图 20.预防组 PDAPP转基因小鼠脑组织刚果红组织化学检测（大脑皮 层） 。 A. Control. B. rAAV/ Αβ42肌注组 . C. rAAV/ CB-AP42肌注组。
图 21.预防组 PDAPP转基因小鼠脑组织刚果红组织化学检测（海马区 域） 。 A. Control. B， rAAV/ Αβ42肌注组 . C. rAAV/ CB-AP42肌注组。
图 22. 治疗组 PDAPP转基因小鼠脑组织 GFAP( Glial fibrillary acidic
protein 神经胶质元纤维酸性蛋白）免疫组织化学检测。 A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ CB-Ap42 肌注组。
图 23.预防组 PDAPP转基因小鼠脑组织 GFAP免疫组织化学检测。
A. Control. B. rAAV/ Αβ42肌注组 C. rAAV/ CB-Ap42 肌注组。 具体实施方案 本发明的基因重组疫苗可以用于预防和治疗阿尔茨海默病 （有 Αβ淀粉 样蛋白沉积特征疾病）。本发明的重组 DNA分子可以表达一种多肽如霍乱 毒素 Β亚单位 ^头 -Αβ肽，是指它含有转录和翻译调节信息的核苷酸序列， 并且这种序列是有效地与编码这种多肽的核苷酸序列连接的。 有效连接是 指这样一种连接， 即调节 DNA序列和表达基因 DNA序列的连接方式可以 引起基因的表达。 总之， 基因表达所需要的调节区域包括一种启动子区域 和一种 DNA序列， 该序列当被转录成 RNA时, 将成为蛋白质合成的起始 信号。 这种区域将通常包括那些参与转录和翻译的 5，-非编码序列。 在本发 明的一个实施方案中， 所用启动子例如是 CMV启动子， 该启动子适用于 多种细胞， 可以在多种细胞内促使目的基因的表达。
为了能够将带有编码霍乱毒素 Β 亚单位和 Αβ肽片段的本发明的重组 DNA分子导入患者体内，需要不同的用于基因转运的载体，与该重组 DNA 分子组合使用。 用于基因转运的载体包括病毒栽体（如腺相关病毒载体）， 脂类 /脂质体， 细胞表面受体的配体等。该重组 DNA分子需要与基因转运 载体组合在一起， 例如一些病毒载体中的基因转运载体与编码融合蛋白的 重組 DNA分子被整合到病毒载体的 DNA中或包装到病毒颗粒中； 一些脂 类或脂质体基因转运载体可以将重组 DNA分子包裹在其中；一些细胞表面 受体的配体作为基因转运栽体与重组 DNA分子以偶联或其它方式结合。因 此， 术语 "组合" 包括整合或包装、 复合、 偶联等方式。
目关病毒 ( adeno-associated virus, AAV )最初是在组织培养的污染
物中分离出来的， 后来发现在儿童感染腺病毒发作期分离到一种非致病性 共感染因子（ Blacklow et al, 1986 )，称其为 目关病毒或腺病毒伴随病毒。
AAV是一种细小病毒属的单链 DNA病毒，具有 4.7kb基因组。 AAV是一 种复制缺陷型病毒， 需要辅助病毒（通常为腺病毒或疱疹病毒）共感染才 能进行有效的复制，完成其生命周期。在缺乏辅助病毒感染的情况下， AAV 变成潜伏的形式， 并以高频率进行稳定的整合， 通常是在第 19号染色体的 特定位点 （ Kotin et al， 1992 ) 。 AAV基因组已被测序， 发现对于 AAV整 合所必须的、 唯一的序列是 145个核苷酸的反向末端重复序列 （ITR ) 。
AAV 的克隆容量大约为 4.7Kb ( Muzyczka,1992 ) 。 由于该病毒具有自身 的优越特点（非致病性、 广泛的宿主细胞、 高效的整合能力、 耐热、 耐酸、 可以长期高效表达）使其特别适合用作基因转运载体。
利用 AAV包装的重组 DNA分子， 一旦转运到患者体内， AAV即可感 染细胞并整合到细胞染色体上，表达融合蛋白（霍乱毒素 B亚单位 头 -Αβ 肽） ， 并分泌到细胞外， 被抗原呈递细胞所识别、 吞噬、 处理和抗原呈递 作用， 将抗原呈递给 Τ和 Β淋巴细胞， 诱发机体免疫应答， 产生抗 β淀粉 样蛋白抗体。 该抗体能够进入脑组织内的神经元细胞外环境， 与可溶性或 聚集的 β淀粉样蛋白结合， 形成 β淀粉样蛋白-抗体复合物。 在脑组织内抗体 通过小神经胶盾细胞表面 Fc受体的调理作用促进其吞噬 β淀粉样蛋白 -抗体 复合物， 以清除 Ρ淀粉样蛋白的沉积， 或者 β淀粉样蛋白-抗体复合物通过 上矢状窦蛛网膜绒毛的排泄作用被清除出中枢神经系统，从而避免了 β淀粉 样蛋白在脑组织内的沉积和由它诱发的神经毒性作用。
本发明的基因重组疫苗和药物组合物不仅适用于已经明显发展成具有 阿尔茨海默病病理特征的患者， 而且适用于普通人群预防阿尔茨海默病的 发生， 使普通人群获得对这种流行性而具有破坏力的疾病免疫力， 特别是 患有唐氏综合征 ( Down's syndrome )的患者或具有家族性阿尔茨海默病相 关基因突变易于发展成阿尔茨海默病的人群。 最佳施药途径是皮下、 皮内、 肌内、 口服、 鼻饲或静脉。
与基因转运载体组合在一起的含有融合蛋白基因的重组 DNA分子可被 用于生产或制备药物组合物， 这种药物组合物包含所需要的有效量的重组 DNA分子。 例如， 基因转运栽体是 AAV载体时，用于治疗或预防阿尔茨海 默病的药物组合物中病毒颗粒的适当剂量大约为 5X104至 l x lO12个病毒 颗粒。 当以一种细胞表面受体的配体作为基因转运载体时， 所使用的配体 结合的 DNA分子的量大约为 0.5 ~ 100 μ g。 如果以脂类或脂质体作为基因 转运栽体时， 所使用的 DNA分子的量大约为 l ~500 g。
在本发明中， 所使用的术语定义如下：
术语 "PDAPP转基因小鼠" 是指转染了 APPV7171基因突变体的 C57小 鼠， 该小鼠在 3 ~5个月时就开始有行为学改变， 在 11 ~ 13个月时脑组织 内出现类似阿尔茨海默病病人的病理改变， 有明显的淀粉样蛋白的沉积， 老年斑的沉积。
术语"非转基因小鼠，，是指未转染 APPV71"基因突变体的 C57正常小鼠。 术语 "APPV717I、 APPV717G和 APPV717F，， 分别指由人 APP基因编码的 APP蛋白的 717位点由原来的缬氨酸突变为异亮氨酸、甘氨酸和苯丙氨酸。
术语 "Αβ肽" 是指含 酸残基 1-39、 1-40、 1-41、 1-42和 1-43的 A P肽片段， 主要包括 A P 39、 Αβ40、 Αβ41、 042和 043。 "Αβ肽" 也包 括 Αβ39、 Α β40、 Αβ41、 Α β 42和 Α β 43的衍生物。 Αβ41、 Α β 40和 Α β 39 与 Α β 42 的不同之处在于， 它们分别缺失 C-末端的 Ala、 Ala-Ile 和 Ala-Ile-Val。 Αβ43与 Αβ42的不同之处是它在 C-末端加了一个 Thr。
术语 " β淀粉样蛋白（amyloid β protein ) "在意思上等同于术语 "A β肽" ， 包括 Αβ39、 Αβ40、 Αβ41、 Αβ42和 Αβ43肽。
术语 "免疫应答"是指在接受治疗的患者体内产生对抗 Ρ淀粉样蛋白抗 体和 /或 Α β特异性 Τ细胞或其分泌产物的应答。
术语 "佐剂" 指当与抗原结合使用时能够增强机体对抗原的免疫应答， 但是当单独使用时不产生对抗原的免疫应答的物质。 佐剂可以通过几种机 制增强免疫应答， 包括聚集淋巴细胞、 激活 Β细胞和 /或 Τ细胞、 激活巨
细胞。
术语"患者"包括接受预防性或治疗性处理的人和其他哺乳动物受试者。 术语 "融合蛋白" 在本文中是指含一种新的融合蛋白盾， 称为 CB-Ap。 该新的蛋白质 （CB-Αβ ) 由霍乱毒素 B亚单位 ^头 -Αβ肽片段组成。 在一 个优选的实施方案中， 所述接头氨基酸序列为甘氨酸-脯氨酸-甘氨酸 -脯氨 酸。
术语 "重组 DNA分子" 是指含有编码霍乱毒素 Β亚单位和 Αβ肽片段 的核苷酸序列及其衍生物。
术语 "衍生物"是指含有编码基本上保持人 Ρ淀粉样蛋白生物学功能或 活性多肽的核苷酸序列。 或者是指基本上保持人 β淀粉样蛋白生物学功能 或活性的多肽。 术语 "启动子序列" 是指 DNA分子可以与 RNA聚合酶特异结合的部 位， 也就是使转录开始的部位。
术语 "基因转运载体" 是指任何一种适用于将 DNA分子的导入体内的 技术和 /或物质。
本发明的 AAV基因重組疫苗在给予患者后，其在患者体内异位表达一 种融合蛋白分子，诱发机体产生抗 β淀粉样蛋白的抗体， 该抗体能够 脑 组织内的神经元细胞外环境， 与可溶性或聚集的 β淀粉样蛋白结合， 形成 β 淀粉样蛋白-抗体复合物。 在脑组织内抗体通过小神经胶质细胞表面 Fc受 体的调理作用促进其吞噬 β淀粉样蛋白-抗体复合物， 以清除 Ρ淀粉样蛋白 的沉积， 或者 β淀粉样蛋白 -抗体复合物通过上矢状窦蛛网膜绒毛的排泄作 用被清除出中枢神经系统，从而避免了 β淀粉样蛋白在脑组织内的沉积和由 它诱发的神经毒性作用。与阿尔茨海默病的病理过程中有关的淀粉样蛋白， 即 Α β 39、 Α β 4ο Α β 41、 A Ρ 42和 Α β 43是主要清除目标。 由此， 本发明的 基因重组疫苗可用于预防或治疗阿尔茨海默病， 或者用于抑制阿尔茨海默 病进一步发展。
利用本发明的基因重组疫苗或药物组合物， 可以防止或清除 β淀粉样蛋
白在神经元细胞外环境中的沉积， 从而防止神经元的损害与丟失， 并以此 作为预防或治疗阿尔茨海默病的关键点。 本发明也避免了长期重复使用需 要穿越血脑屏障的药物制剂所带来的问题。 另外， 实验表明， 本发明的融 合蛋白的结构的设计 , 和接头的选择使得由接头连接的两个多肽在体内保 持各自的活性， 从而为本发明基因重组疫苗及融合蛋白预付或治疗阿尔茨 海默病提供了保证。 通过参阅下述实施例可以更容易地了解本发明的内容，这些实施例只 是为进一步说明， 并不意味着限定本发明的范围。 实施例 1
基因重组疫苗的构建
基因重组疫苗的构建技术路线如图 3 所示。 首先， 通过聚合酶链反应 ( PCR )将 A P 42 DNA片段 脑组织 cDNA文库， 中克隆出来，扩增引 物为：
上游引物： 5，-GGTCCTGGTCCTGATGCAGAATTCCGACATG AC-3， ( SEQIDNO. 3 )，
下游引物： 5，-GGAAGATCTTTACTACGCTATGACAACACCGCCC-3， ( SEQIDNO. 4 )。 通过 RT-PCR技术将霍乱毒素 B亚单位（ CB ) 的基因 克隆出来， 扩增引物为 ：
上游： 5，-CCGGGGTACCCCACCATGATTAAATTAAAATTTGGTG -3' ( SEQIDNO. 5 )，
下游： 5，-CTGCATCAGGACCAGGACCATTTGCCATACTAATTGCG-3， ( SEQIDNO. 6 )。
然后利用重叠 PCR技术将 CB与 A β 42 DNA连接起来，中间加一段接 头 DNA (序列为 GGTCCTGGTCCT, SEQ ID NO.7 ). 该接头 DNA编码 一段柔性的氨基酸短链， 使表达出来的霍乱毒素 B亚单位（ CB )和 A P 42
肽片段可以维持各自的生物学活性。 将 CB-A P 42基因片段装入 pGEM-T Easy质粒（Promega公司生产）后测序。 测序结果如图 4所示。 将带有 CB-A β 42 DNA片段的 pGEM-T Easy质粒以 Kpn I和 Bgl II双酶切后，回 收含 CB-A β 42 DNA片段； 同时， 将通用型 AAV盾粒载体 pSNAV (中国 专利申请号： 99119038.6， 发明名称： 系列通用型腺病毒伴随病毒载体的构 建及用途， 公开号： CN 1252450A ), 以 Kpn I 和 Bgl ll双酶切后， 回收大 片段， 得到含有粘性末端的 pSNAV载体片段。 将酶切后的 pSNAV载体片 段与含 CB-A P 42 DNA片段连接成 pSNAV-CB-A 0 42质粒。 然后， 进行连 接产物的转化、 小量提取纯化质粒 DNA。
同时构建了 pSNAV-A β 42质粒，并将 pSNAV-A β 42质粒和 pSNAV-GFP 质粒作为 pSNAV-CB-A β 42质粒的对照。 pSNAV-GFP质粒为吴小兵等先前 构建（中国专利申请号： 99119038.6， 发明名称： 系列通用型腺病毒伴随病 毒载体的构建及用途）。 pSNAV-A β 42质粒中仅含有 Α β 42 DNA片段， 并在 其前面加了一个 AAP的前导序列，但不含 CB基因 DNA片段。 pSNAV-GFP 质粒中插入了绿色荧光蛋白 （GFP )基因。 实施例 2
rAAV/CB-A β 42病毒的包装及其滴度的测定
将上述实施例 1 获得的 pSNAV-CB-A P 42质粒包装成含有 CB-A β 42 表达盒的重组 AAV病毒（ rAAV/ CB-A β 42病毒）， 即本发明的基因重组 疫苗， 采用的是吴小兵等所申请的专利的方法 （中国专利申请号： 98120033.8,发明名称： 用于重组腺伴随病毒生产的全功能辅助病毒的产生 及其用途； 中国专利申请号： 99119039.4， 发明名称： 可用于大 莫生产的 重组腺病毒伴随病毒生产方法及用途； 中国专利申请号： 99123723.4， 发明 名称： 一种快速高效分离和纯化重组腺病毒相关病毒的方法和用途） 。 具 体步骤如下：
步骤 1. rAAV/CB-A β 42病毒载体细胞株的建立
用 pSNAV-CB-A P 42质粒转染 BHK细胞经选择培养得到 rAAV/CB-A β 42病毒载体细胞林 BHK/pSNAV-CB-A β 42。
BHK 细胞用含 10 %胎牛血清的 RPMI1640 培养液 37 培养。 将 pSNAV-CB-A β 42质粒用脂质体 lipofectamine ( GIBCO BRL )转染 BHK 细胞， 24hr后消化， 1:2~5传代。 加 G418 800 μ g/ml选择培养。 10天后可 形成明显抗性细胞克隆。 将细胞克隆挑出继续培养并进行冻存保种。 用全 功能辅助病毒 HSVl-rc (中国专利申请号： 98120033.8， 发明名称： 用于重 组腺伴随病毒生产的全功能辅助病毒的产生及其用途）感染所获得的克隆 化培养的病毒载体细胞， 待细胞出毒后收获细胞及其培养液， SDS-PAGE 电泳检测结果显示 AAV病毒的三条特征性电泳带， Southern杂交结果显 示该 rAAV病毒包含有 CB-A β 42核酸序列，该 rAAV病毒是 rAAV/CB-A β 42病毒。测定 rAAV/CB-A β 42病毒的滴度。挑选出 rAAV病毒滴度高的 细胞株作为 AAV-GFP病毒栽体细胞株。
步骤 2. 制备重组 rAAV/ CB-A β 42病毒
将上述获得的克隆化培养的病毒载体细胞用含 10 %胎牛血清的 RPMI1640培养液继续于 培养至大量滚瓶培养规模， 在细胞达到一定 数量后， 用全功能辅助病毒 HSVl-rc感染扩大培养的细胞。 待细胞出毒后 收获细胞及其培养液， 采用下述步骤进行重组 rAAV/ CB-A β 42病毒的分 离和纯化： 1 )氯仿破碎细胞、 灭活 HSV辅助病毒及使大量细胞蛋白变性 沉淀； 2 )用 DNasel和 RNase处理细胞裂解液以降解核酸； 3 )加 NaCI 促使 rAAV与细胞碎片分离， 离心去除细胞碎片； 4 )用 PEG/NaCl 沉淀 rAAV; 5 )用氯仿抽提去除杂蛋白和残余的 PEG; 6 )透析除盐； 7 )用密 度梯度离心法或亲和层析法进一步纯化 rAAV。
rAAV/ CB-A β 42病毒的滴度测定： 以 Roche公司地高辛 DNA标记和 检测试剂盒 （ Cat.No.1093657 ) 的方法进行， 以系列稀幹的质粒 pSNAV-CB-A P 42质粒为标准对照， 进行 southern杂交， 将样品的杂交信 号与标准对照进行比较、 定量。 结果显示该 rAAV 病毒包含有 CB-A β 42
核酸序列。 rAAV病毒的滴度为 2 X 1012-13病毒颗粒 /ml。
rAAV/CB-A p 42病毒的电泳纯度测定： 采用 SDS-PAGE法， 加样量为 5 g, 电泳后凝胶进行考马斯亮蓝染色， 见 AAV-2外壳蛋白 VP1、 VP2、 VP3三条主带， 用扫描仪扫描后计算 VP1、 VP2、 VP3蛋白总量占总蛋白 量的百分比。 结果显示 AAV病毒的三条特征性电泳带。
rAAV/A β 42病毒的包装及其滴度的测定方法与 rAAV/CB-A β 42病毒相 同。 实施例 3
外周血 A Ρ特异性 IgG抗体滴度检测
将包装好的病毒分别通过口服、 鼻饲和肌内注射， 免疫 1月龄和 11月 龄 PDAPP转基因小鼠（PDAPPV7171转基因小鼠， 参见文献: 秦川， 常洋 等。 阿尔茨海默病转基因动物模型的建立。 解剖学报； 2000； 31 ( 2 )： 144 - 147 )和 C57非转基因小鼠。 在免疫后 1、 2、 5、 12个月， 釆集小鼠外周 血， 分离血清。 采用 ELISA方法检测血清中抗 Α β抗体的滴度。 如图 6所 示， rAAV/CB-A Ρ 42免疫小鼠无论在治疗组还是预防组， 无论是在转基因 小鼠还是在非转基因小鼠，均能够产生高滴度的抗 A P lgG抗体，在免疫 2 个月时， 血清中抗 A P lgG抗体的滴度最高 （抗体的滴度在 5,000-15,000 之间，相当于 10倍 rAAV/A P 42免疫相应小鼠产生的抗体滴度） 。 在治疗 組免疫 5个月和预防组免疫 12个月后，仍然能够检测到高滴度的抗 A β IgG 抗体； rAAV-A β 42免疫转基因小鼠后仅仅产生非常低的抗 Α β IgG抗体 (抗 体的滴度在 200-500之间）； rAAV/CB-A β 42在免疫转基因小鼠与非转基 因小鼠之间没有明显的差异， 而 rAAV-A β 42免疫非转基因小鼠后产生抗 Α β IgG抗体的滴度明显高于转基因小鼠组（Ρ<0.05 )； rAAV-GFP免疫 组和非处理组外周血清中无论在转基因小鼠还是在非转基因小鼠均未检测 到抗 IgG抗体（抗体滴度低于 1: 64 )。 另外还发现， rAAV/CB-A P 42免疫小鼠的三种途径之间抗体产生的滴度有所不同，肌肉注射组在第一、
二个月时， 抗体的滴度明显高于灌胃组和鼻饲组， 而灌胃組与鼻飼组之间 无明显差异。 rAAV/CB-A P 42免疫小鼠， 治疗组外周血中抗 A β IgG抗体 的滴度低于预防组（P<0.05 ) 。 实施例 4
血清抗 A β IgG抗体对 Α β介导的神经细胞毒性作用的中和作用
将 SH-S Υ5Υ人神经母细胞瘤细胞 (购买于中国协和医科大学细胞中心) 按每孔 100 μΐ含 104 个细胞接种于 96孔板中， 培养基为无血清培养基 DMEM/F12 (购于美国 Gibicol公司）。将 Α β 42肽 (美国 Sigma公司）稀释成 0.12 μΜ， 37 Ό孵育 1周， 使其产生纤维丝。 然后， 按 10: 1和 50: 1与 免疫血清混匀， 7 孵育 24h。 同时， 以非免疫血清做对照。 每一样本做 双复孔。将这种反应混合物加到含 SH-SY5Y人神经母细胞瘤细胞的培养孔 中， 37 。C , 孵育 2天。 细胞的存活率采用 MTT实验检测。 每孔加入 15μ1 的 5mg/ml的 MTT, 37 孵育 4小时。小心吸出培养上清，每孔加入 DMSO 200ul, 用移液枪充分吹打。 在全自动酶标仪 (美国宝生物公司生产)上， 使 用检测波长 570nm测定光密度（ OD. )值。
检测结果显示， rAAV/CB-A β 42免疫小鼠的血清能够部分中和 Α β 42介 导的对 SH-SY5Y人神经母细胞瘤细胞的神经毒性作用。 如图 7所示， 在 rAAV/CB-A β 42免疫血清 1: 10稀幹度时， 能够部分中和 A Ρ 42的神经毒 性作用 （70 %的细胞存活率） ， 在 1: 50稀释度时， 则无明显中和 Α β 42 神经毒性的作用。 而非免疫血清则无明显作用。 实施例 5
免疫小鼠的行为学检测 （水迷宫实验）
( 1 ) 隐蔽平台实验（ Hidden water maze )
为了研究 rAAV/CB-A P 42基因重组疫苗对 PDAPP 转基因小鼠认知 和记忆力的改善作用，首先采用 Morris水迷宫隐蔽平台实! ^价各组小鼠
的学习和记忆能力 （ Moiris RGM. 1981 ) 。 在实验过程中， 记录了小鼠搜 索隐蔽平台的潜伏期、 搜索距离和速度。 经分析各组小鼠之间的搜索速度 无显著性差异， 潜伏期与搜索距离之间具有非常明显的相关性， 所以， 以 小鼠搜索平台的潜伏期作为评价小鼠空间学习和记忆能力的指标。 如图 8 所示， 治疗组在 6天的训练过程中， 尽管各组小鼠搜索平台的潜伏期之间 的存在差异， 但是潜伏期均有明显的缩短。 采用 post hoc分析各组的平均 潜伏期发现 rAAV/CB-A β 42免疫 PDAPP转基因小鼠的三种免疫途径之间 没有明显的差异（ρ>0.05 )， 同样 rAAV/A P 42在三种免疫途径之间也没有 明显的差异（ρ〉0.05 ) 。 因此， 我们以 rAAV/CB-A Ρ 42肌肉注射免疫组与 rAAV/A β 42肌肉注射免疫组、 rAAV-GFP肌肉注射组和非处理组进行统计 学分析， 结果显示 rAAV/CB-A β 42肌肉注射免疫 PDAPP转基因小鼠组的 平均潜伏期在第 5 ~ 6天时明显短于 rAAV/GFP 组（p<0.01 )和非处理组
( p<0.01 ) 。 而 rAAV/A β 42 免疫转基因小鼠组的潜伏期在 6 天内与 AAV-GFP组（p>0.05 )和非处理组（p>0.05 )均无明显的差异。 同样在第 5 ~ 6 天时， rAAV/CB-A P 42 免疫 PDAPP 转基因小鼠组的平均潜伏期
( 24.2s )与 rAAV/A P 42组（ 30.5s )也有明显的差异（ρ<0·05 )。 各组非转 基因小鼠之间在实验 1 ~ 6天均无明显的差异。
为了进一步研究 rAAV/CB-A β 42基因重组疫苗是否能够对 PDAPP转基 因小鼠的认知和记忆损伤有预防作用， 将 rAAV/CB-A β 42基因重组疫苗用 于 1月龄小鼠， 在第 12个月后观察小鼠行为学指标。 由于 ^ ¥/ 42基 因重组疫苗在预实验中发现在 PDAPP转基因小鼠体内产生抗 Α β抗体的 滴度非常低， 而且该疫苗在治疗组对转基因小鼠的行为学无明显的改善， 所以我们在预防组没有加入 rAAV/A β 42组。
在预防组， 如图 9所示， 我们发现在第 6 天时 rAAV/CB-A β 42免疫 PDAPP转基因小鼠的平均潜伏期明显比 rAAV/GFP组（ p<0.05 )和非处理 组（p<0.05 )缩短。 各组非转基因小鼠之间在实验 1 ~ 6天均无明显的差异 (p>0.05)。
( 2 )探索实验（Probe test ) 由于小鼠在搜索隐蔽平台过程中， 可能有小鼠偶尔、 无意识或随机的碰 到平台后上台的， 为了消除这种随机性， 我们将平台从水池中拿走， 让小 鼠在水池内搜索平台 60 秒， 观察小鼠在原来含有平台的象限（Target quadrant, TQ ) 中的搜索时间和经过平台位置次数， 通过比较在 TQ象限 中搜索时间的百分比和经过平台的次数， 评价小鼠空间学习和记忆能力。 同时， 考虑到小鼠可能由于在平台位置搜索不到平台而影响小鼠后面的行 为， 所以， 我们统计前 30秒小鼠在 TQ象限中的搜索时间和经过平台位置 次数作为评价小鼠空间学习和记忆能力的指标。
探索实验结果显示，在治疗组， rAAV/CB-A P 42免疫小鼠的学习和记忆 努力得到明显的改善， 而 rAAV/A P 42免疫小鼠的空间学习和记忆能力与 rAAV/GFP 组和非处理组之间没有明显的差异。 如图 10.A 所示， rAAV/CB-A β 42免疫小鼠 30秒内在 TQ象限中搜索时间的百分比明显高于 AAV-GFP组、非处理组和 rAAV/A 042组。 rAAV/CB-A β 42肌注免疫小鼠 在 TQ象限中搜索时间的百分比为 37.5 %， 而 rAAV/GFP组为 27.2 %， 非 处理组 25.9 %。 AAV-GFP组和非处理组与 rAAV/CB-A β 42肌注免疫组之 间均有明显的差异（p<0.05 ) 。 rAAV-A P 4242肌注免疫小鼠在 TQ象限中 搜索时间的百分比为和平均经过平台的次数分别为 28.9 %，与 rAAV/CB-A β 42肌注免疫组之间也有明显的差异（ρ<0.05 ) 。 而与 rAAV/GFP组和非 处理组之间均没有明显的差异（p〉0.05 ) 。 同样， 在预防组中， 如图 10.B 所示， rAAV/CB-A β 42免疫的小鼠在 TQ象限中搜索时间的百分比也均明 显高于 rAAV/GFP 组和非处理组（ ρ<0·05 ) 。
如图 11.A所示， rAAV/CB-A β 42肌注免疫组在 30秒搜索时间内平均 经过平台位置的次数为（ 1.70士 0.35 )， 明显高于 rAAV/GFP组（ 0.49±0.30 ) ( P<0.01 )和非处理组 ( 0.55士 0.21 ) (Ρ<0·01)。 r AAV/A β 42肌注免疫组在 30秒搜索时间内平均经过平台位置的次数为 0.66±0.25， 与 rAAV/GFP组
和非处理组之间没有明显的差异 (均 Ρ〉0·05)， 也明显地少于 rAAV/CB-A β 42肌注免疫组（PO.01 ) 。 各组非转基因小鼠之间 30秒内在 TQ象限中搜 索时间的百分比和经过平台位置的次数没有明显的差异。
在预防组中， 如图 11.B所示， rAAV/CB-A β 4242免疫的小鼠在探索实 验中经过平台位置的次数也均明显高于 rAAV/GFP 组和非处理组 ( p<0.05 ) 。
( 3 )可见平台水迷宫实验 ( Visible water maze ) 在做完 "probe test" 后， 我们在平台的上方插一个标志杆， 黑白相间， 非常醒目， 小鼠很容发现。 同时在标志杆的上方放一个白色小球， 防止影 像示踪系统对标志杆的识别而引起实验终止。 可见平台水迷宫实验结果显 示， 在治疗组中， 在第四天时 rAAV/CB-A P 42免疫的各组小鼠的平均潜伏 期明显的短于其它各组， 但是 rAAV/CB-A β 42基因重组疫苗的三种免疫途 径之间没有明显的差异。如图 12所示， rAAV/CB-A β 42肌肉注射免疫组在 第四天的平均潜伏期为 15.1±l.ls， 而 rAAV/GFP组、 非处理组和 rAAV/A β 42肌注组分别为 19.4±3.1s、 21±3.6s和 19.9±3.3s，经可重复方差分析发现， rAAV/CB-A β 42肌肉注射免疫组与 AAV-GFP组、非处理组和 rAAV/A β 42 肌注组之间具有显著性差异（Ρ值均小于 0.05 ) 。
在预防组中， 如图 13所示， 在第四天时 rAAV/CB-A β 42免疫的各组小 鼠的平均潜伏期与 rAAV/GFP组和非处理組之间均没有明显的差异， 而且 rAAV/CB-A β 42基因重组疫苗的三种免疫途径之间没有明显的差异。 我们 进一步研究发现 PPDAPP转基因小鼠与非转基因小鼠之间在第 4天实验中 搜索可见平台的平均潜伏期没有明显的差异（Ρ>0.05 ) 。
通过这几种氷迷宫实验检测发现 rAAV/CB-A β 42基因重组疫苗免疫 PDAPP转基因小鼠与其它各组小鼠相比较，其学习和记忆能力明显地得到 改善。
实施例 6
ELISA检测脑组织中 A P淀粉样蛋白的含量 为了进一步证实 rAAV/CB-Ap42基因重组疫苗在降低 PDAPP转基因小 鼠脑组织内 A P蛋白的作用，我们采用了非常敏感的 ELISA方法定量检测 小鼠脑组织内人 Αβ4() 和 Αβ42的含量。此方法可以检测脑组织内总的人 A o和 Αβ42的含量， 包括可溶性和沉淀的。 但这种方法不能检测小鼠 A
P 40或 Αβ42， 因此， 在检测非转基因小鼠脑组织提取液时， 无检测信号。 检测结果显示， 在治疗组中， rAAV/CB-Ap42免疫 PDAPP转基因小鼠脑组 织内 A β 4。 or A β 42的含量均明显低于其它各组。 rAAV/CB-Ap42三种免疫 途径之间没有明显的差异， 我们以 rAAV/CB-Ap42肌肉注射组与其它各组 进行统计学分析。 结果显示， rAAV/CB-Ap42肌肉注射组脑组织内 Αβ42的 平均含量 （ 253.6±32.7ng/g 脑组织湿重） 明显低于非处理组 （ 432.8 ±51.3ng/g )、 rAAV/GFP组（ 415·9±61.3 ng/g )和 rAAV/A β 42肌肉注射组
(413.5±56.1ng/g) (p值均小于 0.05) 。 同样， r AA V/CB-A β 42肌肉注射 组脑组织内 A Ρ 40的平均含量 (756.4±112.5 ng/g)也明显低于低于非处理 组（ 954.6 ±156.3ng/g ) 、 rAAV/GFP组（ 960·4±154.9 ng/g )和 rAAV/A β 42肌肉注射组 ( 925.3±147.8 ng/g ) ( ρ值均小于 0.05 ) 。 同样，在预防组中，我们发现 PDAPP转基因小鼠脑组织内 Αβ4。和 A β 42的含量明显低于治疗组。虽然 rAAV/CB-Ap42三种免疫途径之间没有明 显的差异， 但是 rAAV/CB-Ap42免疫 PDAPP转基因小鼠脑组织内 A β 40和 Αβ42的含量均明显低于其它各组。 其中， rAAV/CB-Ap42肌肉注射免疫组 PDAPP转基因小鼠脑组织内 A β 40的含量为 90.1±13.2 ng/g, A β 42的含量 为 40.2±10.3 ng/g, 均明显低于非处理组（ Αβ40为 130.6±17.9 ng/g, Α 42 为 51.9±10.2ng/g ) 和 rAAV/GFP 组 （ 127.8±18.6 ng/ , A β 42 为 55.3±13.5ng/g ) (p值均小于 0.05) 。 实施例 7
PDAPP小鼠脑组织内 P淀粉样蛋白免疫组织化学染色
为了进一步研究 PDAPP转基因小鼠脑组织内 P淀粉样蛋白的沉积情 况， 我们利用 A β特异性单克隆抗体 3D6 (购于美国 Chemicon公司）做免 疫組织化学染色。 如图 14和图 15所示， 在治疗组中， 在非处理组 PDAPP 小鼠脑组织的大脑皮质和海马区域均可以看到有明显的 P淀粉样蛋白沉积 区域， 大量成熟的老年斑。 rAAV/CB-A β 42免疫组小鼠脑组织内 β淀粉样 蛋白沉积区域明显减少， 虽然也可以看到老年斑， 但是老年斑的直径明显 比非处理组小， 看不到大的老年斑。 而 rAAV/GFP组和 rAAV/A β 42组小 鼠脑组织内 Ρ淀粉样蛋白沉积情况与非处理组之间没有发现明显的差异。 我们利用 NIH image J 图像灰度扫描定量分析软件 （ http: rsb.info.nih.gov/nih-image )分析各组 PDAPP小鼠脑组织内 β淀粉样蛋白 沉积区域的百分比。 结果显示， rAAV/CB-A β 42免疫组小鼠脑组织大脑皮 质和海马区域 Ρ淀粉样蛋白的平均值明显低于非处理组、 rAAV/GFP组和 rAAV/A P 42组。 如图 16 和图 17 所示， 在预防组中， 在非处理组和 rAAV/GFP组 PDAPP小鼠脑组织内也可以看到有明显的 β淀粉样蛋白沉 积区域，少量成熟的老年斑，两组之间没有发现明显的差异，而 rAAV/CB-A β 42免疫组小鼠脑组织内 β淀粉样蛋白沉积区域明显减少， 看不到成熟的 老年斑。利用图像灰度扫描定量分析各组 PDAPP小鼠脑组织内 p淀粉样蛋 白沉积区域的百分比。 结果显示， rAAV/CB-A β 42免疫组小鼠大脑皮质和 海马区域 Ρ淀粉样蛋白的平均值明显低于非处理组和 rAAV/GFP组。 实施例 8
PDAPP小鼠脑组织刚果红组织学染色
如图 18和图 19所示， 在治疗组中， 在非处理组和 rAAV/ A β 42免疫 组 PDAPP 小鼠脑组织的大脑皮质和海马区域均可以看到有大量明显的嗜 刚果红神经细胞。 rAAV/CB-A β 42免疫组小鼠脑组织的大脑皮盾和海马区 域嗜刚果红神经细胞数目明显减少。 表明神经细胞受 蛋白的损伤明显
减少。 同样， 在预防组， 如图 20和图 21所示， rAAV/CB-A P 42免疫组小 rAAV/GFP组明显减少 实施例 9
PDAPP小鼠脑组织内星形胶质细胞增生状况的免疫组织化学染色
如图 22所示，在治疗组中， 非处理组和 rAAV/A P 42免疫组组 PDAPP 小鼠脑组织大脑皮质均可见到大量明显的 GFAP阳性星形胶质细胞增生， 而 rAAV/CB-A β 42免疫组 PDAPP小鼠脑组织内仅有非常弱 GFAP阳性星 形胶质细胞增生。 表明脑组织内活化的反应性星形细胞数目减少， 脑组织 内炎症反应程度降低。 同样， 在预防组， 如图 23 所示， rAAV/CB-A β 42 免疫组小鼠脑组织的大脑皮质 GFAP阳性星形胶质细胞数目比非处理组和 rAAV/GFP组明显减少。 实施例 10
CB-A β 42和 A β 42蛋白在原核细胞中的表达与纯化
首先，通过聚合 反应 ( PCR )扩增 CB-A Ρ 42和 Α β 42DNA片段（具 体步骤同实施例 1 ) ， 并将它们分别装入 pET30a ( Novagen公司产品）和 pET42a( Novagen公司产品）原核表达载体中。然后，转化到大肠杆菌 BL21 ( DE3 )表达菌株中， 通过 IPTG诱导， 可以得到融合蛋白。 将融合蛋白 通过 His Trap 纯化柱加以纯化， 然后， 用凝血酶 Xa因子分别将 CB-A β 42蛋白和 Α β 42肽片段切割下来， 并加以纯化， 冻干后， -20 保存。 实施例 11. CB-A P 42蛋白和 Α β 42肽免疫原性的检测
将实施例 10获得的冻干的 CB-A β 42蛋白和 Α Ρ 42肽分别溶于磷酸盐緩 沖液（PBS ) 中， 在无佐剂的情况下， 皮下免疫 Balb/c小鼠， 结杲显示， 在免疫一个月后， CB-A β 42蛋白免疫组可以产生高滴度的抗 Ρ淀粉样蛋白
抗体（抗体滴度为 1: 10,000 ~ 20,000 ) ， 而 A β42肽免疫组仅产生非常低 的抗 β淀粉样蛋白抗体（抗体滴度为 1: 200 ~ 500) 。 由此表明 CB-AP42 蛋白也能够作为预防和治疗阿尔茨海默病的药物。
Claims
1. 一种用于治疗和预防阿尔茨海默病的基因重组疫苗， 该疫苗含有 编码霍乱毒素 B亚单位和 Αβ肽片段的融合蛋白的重组 DNA分子。
2. 如权利要求 1 所述的基因重组疫苗， 其中还含有与该重组 DNA 分子有效连接的并能够在真核细胞中表达融合蛋白的启动子， 以及位于所 述重组 DNA分子的 5，端编码 Ν-末端信号肽的前导序列。
3. 如权利要求 1所述的基因重组疫苗， 其中所述 Αβ肽片段选自 A β39、 Αβ40、 Αβ41、 Α β 42和 Α β 43。
4. 一种重组 DNA分子，其含有编码霍乱毒素 B亚单位和 Αβ舦片段 的融合蛋白的 DNA分子， 以及与该 DNA分子有效连接的并能够在真核细 胞中表达融合蛋白的启动子， 和位于所述 DNA分子的 5，端编码 Ν-末端信 号肽的前导序列。
5. 一种重组 DNA分子， 其含有如下的 DNA序列；
( 1 ) SEQIDNO.1 所示的 DNA序列；
( 2 ) 在严格条件下与 SEQIDNO.1 所示的 DNA序列杂交的 DNA 序列。
6. 如权利要求 5所述的重组 DNA分子， 其中编码霍乱毒素 B亚单位 和 Αβ肽片段的融合蛋白的核苷 列如图 4所示， 所述启动子为 CMV启 动子， 所述前导序列为霍乱毒素 Β亚单位的前导序列。
7. 一种融合蛋白， 其含有如下的氨基^ 列：
( 1 ) SEQIDNO. 2 所示的氨基 列；
( 2 ) 通过一个或多个 ^酸残基的替换、缺失或添加而衍生自 SEQ ID NO. 2 所示的氨基酸序列并具有与其相同功能的氨基酸序列。
8. 一种融合蛋白， 其从 N端至 C端由霍乱毒素 B亚单位、 接头 Αβ 肽片段和 Αβ肽片段组成， 所述 Αβ肽片段选自 Αβ39、 Αβ40、 Αβ41、 Αβ 42和 A Ρ 43， 所述接头的氨基酸序列为甘氨酸-脯氨酸 -甘氨酸-脯氨酸。
9. 如权利要求 8所述的融合蛋白， 其具有 SEQ ID NO. 2所示序列。
10. 一种重组病毒载体，其包含权利要求 4-6任一项的重组 DNA分子， 所迷病毒栽体选自^目关病毒、 腺病毒、 疱疹病毒、 逆转录病毒、 痘苗病 毒、 禽痘病毒、 新培斯病毒或鸡痘病毒。
11.如权利要求 10所述的重组病毒载体， 其为包含权利要求 6-8任一 项的重组 DNA分子的腺相关病毒。
12. 一种药物组合物，其包含权利要求 6-8任一项的重组 DNA分子及 合适的基因转运载体.
13.一种药物组合物， 其包含权利要求 7-9任一项的融合蛋白及药物 学可接受的载体。
14.权利要求 6-8任一项的重组 DNA分子或者权利要求 7-9任一项的 融合蛋白在制备治疗和预防阿尔茨海默病的药物中的应用。
15. 一种预防和 /或治疗阿尔茨海默病的方法， 该方法包括给需要该 治疗的患者服用权利要求 1 - 3的基因重组疫苗或者权利要求 7-9任一项 的融合蛋白。
-24- 替换页（細 弟 6条）
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| WO2004111250A1 (ja) * | 2003-06-13 | 2004-12-23 | Japan As Represented By President Of National Center For Geriatrics And Gerontology | アルツハイマー病の治療のための組換えアデノ随伴ウィルスベクター |
| WO2005014041A3 (en) * | 2003-07-24 | 2005-05-06 | Novartis Ag | Use of an amyloid beta dna vaccine for the treatment and/or prevention of amyloid diseases |
| WO2010050585A1 (ja) * | 2008-10-31 | 2010-05-06 | ディナベック株式会社 | アルツハイマー病治療用ベクター |
| US8497072B2 (en) | 2005-11-30 | 2013-07-30 | Abbott Laboratories | Amyloid-beta globulomer antibodies |
| US8691224B2 (en) | 2005-11-30 | 2014-04-08 | Abbvie Inc. | Anti-Aβ globulomer 5F7 antibodies |
| US8877190B2 (en) | 2006-11-30 | 2014-11-04 | Abbvie Inc. | Aβ conformer selective anti-Aβ globulomer monoclonal antibodies |
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| US8987419B2 (en) | 2010-04-15 | 2015-03-24 | AbbVie Deutschland GmbH & Co. KG | Amyloid-beta binding proteins |
| US9062101B2 (en) | 2010-08-14 | 2015-06-23 | AbbVie Deutschland GmbH & Co. KG | Amyloid-beta binding proteins |
| US9176150B2 (en) | 2003-01-31 | 2015-11-03 | AbbVie Deutschland GmbH & Co. KG | Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof |
| CN116327904A (zh) * | 2022-08-02 | 2023-06-27 | 深圳源兴基因技术有限公司 | 一种用于阿尔茨海默病的治疗和预防的重组腺相关病毒基因疫苗及其用途 |
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| CN101392027B (zh) * | 2008-07-03 | 2012-04-25 | 浙江大学 | 一种治疗阿尔茨海默病的融合蛋白及其制备方法 |
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Also Published As
| Publication number | Publication date |
|---|---|
| AU2003289631A1 (en) | 2004-06-23 |
| CN1504240A (zh) | 2004-06-16 |
| CN100450551C (zh) | 2009-01-14 |
| AU2003289631A8 (en) | 2004-06-23 |
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