Variant AAV capsid protein and recombinant adeno-associated virus rAAV

Abstract

The invention relates to the technical field of biology, in particular to a variant AAV capsid protein and a recombinant adeno-associated virus rAAV. The variant AAV capsid protein provided by the invention comprises: 1) an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 93%, 95% and 98% of identity with an amino acid sequence as shown in SEQ ID NO.1; and 2) an amino acid sequence shown as SEQ ID NO.1, wherein the amino acid sequence comprises amino acid substituted A587D, Q588G, A589N and Q590P, and an amino acid sequence TLAVPFK is inserted after the 588th site and before the 589th site; related products constructed by the variant AAV capsid protein can be used for cross-blood-brain-barrier efficient transduction to realize drug delivery to a central nervous system to treat related diseases of the central nervous system, and meanwhile, the related products have the characteristic of remarkably reducing liver transduction.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a variant AAV capsid protein and a recombinant adeno-associated virus rAAV. Background Art

[0002] Central nervous system diseases include neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, and brain tumors such as glioblastoma and meningioma. These diseases are particularly common in the elderly and are usually caused by inherited gene mutations that lead to abnormal development of the nervous system, neurodegeneration, or impaired neuronal function. Central nervous system diseases impose a serious burden and impact on families and society. Treatment of central nervous system diseases requires whole-brain gene repair or functional protein compensation, and the blood-brain barrier of the brain itself, that is, the blood-brain barrier refers to the barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between plasma and cerebrospinal fluid formed by the choroid plexus. While preventing harmful substances from entering the brain, it is also the main obstacle to drugs entering the central nervous system. How to break through the blood-brain barrier and efficiently and specifically deliver drugs is currently the main problem in nervous system treatment and neuroscience research.

[0003] Delivery of target drugs to the nervous system requires a tool vector. Among the many existing tool vectors, AAV vectors are popular due to their excellent delivery efficiency, stable safety, and relatively low cytotoxicity. There are many AAV serotypes, and different serotypes have different targeted infection characteristics. AAV type 9 (AAV9) is the most widely studied one. Based on AAV9, capsid protein modification is performed to obtain a series of AAV variants that can efficiently cross the blood-brain barrier, but most of them have strong liver tropism. This off-target delivery may cause a strong immune response or cause liver toxicity. In addition, studies have reported that other serotypes of AAV can also cross the blood-brain barrier, such as AAVhu.32 (Yoon et.al., 2020, 143; 2058-2072), but AAVhu.32 is not efficient in crossing the blood-brain barrier. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a variant AAV capsid protein, a recombinant adeno-associated virus rAAV and uses thereof. The recombinant adeno-associated virus rAAV can more efficiently and specifically cross the blood-brain barrier for extensive drug delivery to the central nervous system and has low liver tropism, which can provide a better tool for neuroscience research and brain disease treatment, and has broad application value and market prospects.

[0005] To this end, the present invention provides the following technical solutions:

[0006] The present invention provides a variant AAV capsid protein, comprising:

[0007] 1) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, or 98% identical to the amino acid sequence shown in SEQ ID NO.1; and

[0008] 2) The amino acid sequence shown in SEQ ID NO.1 includes: amino acid substitutions A587D, Q588G, A589N, Q590P, and the amino acid sequence TLAVPFK is inserted after position 588 and before position 589.

[0009] The variant AAV capsid protein is obtained by amino acid substitutions A587D, Q588G, A589N, Q590P on the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence TLAVPFK is inserted after position 588 and before position 589. In some embodiments, the variant AAV capsid protein can also be subjected to at least one amino acid deletion, substitution and / or insertion, such as the inserted 7-mer polypeptide can be replaced with TLAVPFS, FVVGQSY, TLQVPFK, ELAVPFK, DLAVPFK, TMAVPFK, TLAVPPK, TLAVLFK, TLAGPFK, and the above polypeptide scheme is combined with at least one amino acid substitution scheme among amino acid substitutions A587D, Q588G, A589N, Q590P to obtain Different variant AAV capsid proteins, the present invention has found that the insertion polypeptide of the present invention is TLAVPFK and the amino acid substitution A587D, Q588G, A589N, Q590P combination is the best solution, for example, the inserted 7-mer amino acid is changed from TLAVPFK to TLAVPFS, FVVGQSY (and the original amino acids at positions 589 and 590 remain AQ unchanged), other conditions remain unchanged, such as the constructed AAVhu.32-DGN improves central specificity and reduces liver targeting. Figure 2 , Figure 3 AAVhu.32-DGF improves liver specificity and reduces central targeting. Figure 2 , Figure 3 As shown. Based on this method, the performance of the vector can be optimized, such as the targeting and transduction efficiency of delivering exogenous target products to target organs, tissues or cells, and efficiently and specifically crossing the blood-brain barrier. The obtained variant AAV capsid protein can have an amino acid sequence with 80%, 83%, 85%, 88%, 90%, 93%, 95%, 98% or more identity with the natural AAV capsid protein.

[0010] As the best embodiment, the variant AAV capsid protein is a variant of the AAV capsid protein shown in SEQ ID NO.1, in which the amino acid sequence is substituted by A587D, Q588G, A589N, Q590P, and the amino acid sequence TLAVPFK is inserted after the 588th position and before the 589th position. The recombinant adeno-associated virus vector constructed by the variant AAV capsid protein can efficiently transduce gene delivery to the central nervous system, and has significantly reduced liver transduction characteristics. It can be used to more efficiently transduce the central nervous system across the blood-brain barrier, and can provide better tools for neuroscience research and the treatment of whole-brain systemic genetic diseases, neurodegenerative diseases, etc., and has broad application value and market prospects.

[0011] In some embodiments, the AAV capsid protein is present in VP1, VP2 and / or VP3 of the AAV capsid. As the best embodiment, the AAV capsid protein is present in VP1 of the AAV capsid.

[0012] In some embodiments, the AAV capsid protein is chimeric.

[0013] In some embodiments, when delivered systemically to a subject, the variant AAV capsid protein has increased specificity for tissues and / or cells of the central nervous system and / or increased transduction efficiency relative to the native AAVhu.32 capsid protein shown in SEQ ID NO.1 when measured in the central nervous system of the subject.

[0014] In some embodiments, the cell of the central nervous system is selected from at least one of the following cell types: neurons, oligodendrocytes, astrocytes, and cerebral vascular cells;

[0015] In some embodiments, the tissue of the central nervous system is selected from at least one tissue type of the following: cerebrum, thalamus, cortex, striatum, ventral midbrain, spinal cord, and cerebellum.

[0016] In some embodiments, when systemically delivered to a subject, the variant AAV capsid protein has reduced specificity for off-target tissues and / or off-target cells and / or reduced transduction efficiency relative to the native AAVhu.32 capsid protein set forth in SEQ ID NO.1 when measured in off-target tissues and / or off-target cells of the subject;

[0017] Optionally, the off-target tissue includes liver tissue; optionally, the off-target cells include liver cells.

[0018] The present invention provides a biomaterial comprising any one of the following:

[0019] 1) A nucleic acid molecule encoding the variant AAV capsid protein;

[0020] 2) A recombinant vector containing the nucleic acid molecule described in 1);

[0021] 3) A host cell containing the nucleic acid molecule described in 1) or the recombinant vector described in (2).

[0022] The embodiments disclosed in the present invention relate to a recombinant adeno-associated virus rAAV, comprising the variant AAV capsid protein;

[0023] As the best embodiment, it also includes a heterologous nucleotide sequence encoding a target product;

[0024] As the best embodiment, the target product includes nucleic acid, peptide or protein;

[0025] As the best embodiment, the nucleic acid is selected from siRNA or small guide RNA;

[0026] As the best embodiment, the original vector of the recombinant adeno-associated virus rAAV is rAAVhu.32.

[0027] As the best embodiment, the variant AAV capsid protein exists in the recombinant adeno-associated virus rAAV capsid protein VP1.

[0028] The embodiment disclosed in the present invention relates to a pharmaceutical composition containing the recombinant adeno-associated virus rAAV and pharmaceutically acceptable excipients.

[0029] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt are generally chemically and / or physically compatible with the other ingredients making up the dosage form, and physiologically compatible with the recipient thereof.

[0030] "Pharmaceutically acceptable excipients" refer to ingredients other than the active ingredient in a pharmaceutical dosage form that are

[0031] The pharmaceutically acceptable carrier used in the pharmaceutical composition disclosed herein may include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous medium, a non-aqueous medium, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, an anesthetic, a suspending / formulating agent, a multivalent sequestering or chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art, or various combinations thereof.

[0032] The embodiments disclosed in the present invention relate to the use of the recombinant adeno-associated virus rAAV in the preparation of a drug for delivering a target product to the central nervous system;

[0033] As the best embodiment, the target product includes nucleic acid, peptide or protein;

[0034] As the best embodiment, the nucleic acid is selected from siRNA or small guide RNA;

[0035] As the best embodiment, it includes use in preparing drugs for treating central nervous system diseases;

[0036] As the best embodiment, the central nervous system disease includes but is not limited to at least one of neurodegenerative diseases, brain tumor diseases, infectious diseases, cerebrovascular diseases and traumatic brain injury caused by external forces;

[0037] As the best embodiment, the neurodegenerative disease includes but is not limited to at least one of Alzheimer's disease (AD), Parkinson's disease stroke, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis;

[0038] As the best embodiment, the brain tumor disease includes but is not limited to at least one of glioblastoma, meningioma, oligodendroglioma, pituitary tumor and pineal tumor;

[0039] As the best embodiment, the infectious disease includes but is not limited to brain or meningitis;

[0040] As the best embodiment, the cerebrovascular disease includes but is not limited to stroke or subarachnoid hemorrhage.

[0041] The technical solution of the present invention has the following advantages:

[0042] 1. A variant AAV capsid protein provided by the present invention comprises: 1) an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, 98% identity with the amino acid sequence shown in SEQ ID NO.1; and 2) an amino acid sequence having at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, 98% identity with the amino acid sequence shown in SEQ ID NO.1; The amino acid sequence shown in NO.1 includes: amino acid substitutions A587D, Q588G, A589N, Q590P, and the amino acid sequence TLAVPFK is inserted after position 588 and before position 589; relative to the natural AAV capsid protein, the above variant AAV capsid protein has high specificity and / or transduction efficiency for tissues and / or cells of the central nervous system, and can be used for more efficient and specific delivery of a wide range of drugs (including but not limited to gene therapy drugs) to the central nervous system across the blood-brain barrier, while having low specificity and / or transduction efficiency for liver tissue and / or liver cells, indicating that it has low liver tropism, and thus related products constructed using the above variant AAV capsid protein (including but not limited to recombinant adeno-associated virus rAAV or pharmaceutical composition) can be used for efficient transduction across the blood-brain barrier to achieve drug delivery to the central nervous system, treat central nervous system-related diseases, and have significantly reduced liver transduction characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 is the expression profile of the recombinant adeno-associated virus serotype plasmid pAAV2 / hu.32-EBNP in Example 1 of the present invention;

[0045] Figure 2 The imaging results of brain tissue slices after the C57BL / 6 mice were injected with rAAVhu.32-EBNP-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGN-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGF-CMV-EGFP-WPRE-hGH polyA and rAAhu.32-CMV-EGFP-WPRE-hGH polyA virus vectors through the tail vein in Example 3 of the present invention; Figure A is the fluorescence imaging comparison result of the coronal slice of the mouse head; Figure B shows the fluorescence expression results of each brain region; Figure C is the average fluorescence intensity statistics of the mouse brain slice (coronal slice of the head) (**** indicates p < 0.0001);

[0046] Figure 3 These are the results of liver tissue section imaging and average fluorescence density statistics after tail vein injection of rAAVhu.32-EBNP-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGN-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGF-CMV-EGFP-WPRE-hGH polyA and rAAhu.32-CMV-EGFP-WPRE-hGH polyA viral vectors in Example 3 of the present invention in C57BL / 6 mice; Figure A represents liver fluorescence section imaging; Figure B represents the average fluorescence intensity statistics of the liver (**** represents p < 0.0001). DETAILED DESCRIPTION

[0047] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0048] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0049] Terminology Note:

[0050] As used herein, AAV stands for adeno-associated virus.

[0051] Native AAV capsid protein refers to the capsid protein of the natural, unmodified adeno-associated virus. As used herein, the native AAV capsid protein is AAVhu.32.

[0052] Variant AAV capsid protein refers to a natural AAV capsid protein or an artificial AAV capsid protein obtained by modification such as deletion, substitution and / or insertion.

[0053] rAAV stands for recombinant adeno-associated virus.

[0054] "Identity" with respect to a sequence refers to the extent to which the two sequences have the same nucleotide residues or amino acid residues at the same position when the two sequences are aligned to achieve the maximum level of identity, expressed as a percentage. Wherein, the "identity" between the sequences of two nucleic acid molecules or two amino acid sequences can be determined using a known computer algorithm, such as at least one of the GCG program package, BLASTN or FASTA, and other commercial or publicly available programs may also be, for example, the DNAStar-"MegAlign" program.

[0055] As used herein, the term "subject" refers to a mammal, including humans, veterinary or farm animals, livestock animals or pets, and animals commonly used in clinical studies. In one embodiment, the subject of these methods and compositions is a human. Other suitable subjects include, but are not limited to, rodents, rats, canines, felines, swine, bovines, ovines, non-human primates, and other animals. As used herein, the term "subject" is used interchangeably with "patient".

[0056] As used herein, "systemic delivery" refers to delivery resulting in a wide range of biodistribution of the AAV capsid proteins described herein (including variant AAV capsid proteins) and / or recombinant adeno-associated viruses constructed thereof in an organism, for example, due to movement from one location to another through systemic circulation. Systemic delivery means exposing a useful, preferably therapeutic amount of AAV capsid proteins (including variant AAV capsid proteins) and / or recombinant adeno-associated viruses constructed thereof to most parts of the body. Systemic delivery of the AAV capsid proteins (including variant AAV capsid proteins) and / or recombinant adeno-associated viruses constructed thereof provided herein is preferably achieved by intravenous delivery, but may also be achieved by other delivery routes (including oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular and other parent routes).

[0057] The construction method of the laboratory-constructed plasmid pAAV2 / hu.32 is as follows:

[0058] The capsid protein (SEQ ID NO.1) of AAVhu.32 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using the plasmid with Addgene No. 112862 as a template, the gene encoding the capsid protein (SEQ ID NO.1) of AAVhu.32 (cap gene, SEQ ID NO.2, synthesized by Sangon Biotech (Shanghai) Co., Ltd.) was inserted into the 112862 plasmid by enzyme ligation. The specific steps are as follows:

[0059] (1) Enzyme digestion of plasmid 112862 and target gene respectively, enzyme digestion system:

[0060] Table 1. Enzyme digestion system

[0061] Reagents concentration volume SwaI enzyme (commercially available) 10000units / ml 1ul AgeI enzyme (commercially available) 20000units / ml 1ul NEB 3.1 buffer (commercially available) / 5ul 112862 / hu.32-Cap 500ng / ul 6ul Ultrapure water / 37ul

[0062] Enzyme digestion conditions: 37℃ water bath reaction for 3 hours

[0063] (2) The 112862 plasmid and the target gene after enzyme digestion were connected. The connection system is as follows:

[0064] Table 2. Connection system

[0065] Reagents concentration volume 2×Universal Ligation Mix (commercially available) / 5ul F-112862 50ng / ul 2.5ul F-hu.32-Cap 50ng / ul 2.5ul

[0066] Connection conditions: 25℃ for 5 minutes for quick connection

[0067] (3) Obtaining pAAV2 / hu.32

[0068] pAAV2 / hu.32 was extracted and purified from the reaction solution after ligation, and then sequenced. If the sequencing was correct, it was pAAV2 / hu.32, and its plasmid map was the same as Figure 1 The present invention is similar to Figure A, except that EBNP is replaced by the gene encoding the capsid protein (SEQ ID NO.1) of AAVhu.32.

[0069] The plasmid pAAV-CMV-EGFP-WPRE-hGH polyA loaded with core elements was purchased from Brinkase Biotechnology Co., Ltd. or referenced in patent document CN117230117A.

[0070] The adenovirus element helper plasmid pAd-Helper was purchased from Addgene (No.: 112867).

[0071] SYBR Green qPCR kit was purchased from Bio-Rad.

[0072] Tissue embedding medium was purchased from Sakura Co., Ltd., Japan.

[0073] 2×TransStart FastPfu Fly PCR SuperMix is ​​a commercially available product.

[0074] PEI transfection reagent is a commercially available product.

[0075] The gene sequences or amino acid sequences involved in the following examples were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0076] The sequences involved in the following embodiments are specifically shown in the following table:

[0077] Table 3. Sequence

[0078]

[0079]

[0080] Example 1 Construction of recombinant adeno-associated virus AAVhu.32-EBNP serotype plasmid pAAV2 / hu.32-EBNP

[0081] This embodiment provides a recombinant adeno-associated virus serotype packaging plasmid pAAV2 / hu.32-EBNP, comprising the following steps:

[0082] The recombinant adeno-associated virus AAVhu.32-EBNP is obtained by mutating and inserting a nucleotide sequence encoding a peptide segment based on the Cap gene (SEQ ID NO.2) encoding the capsid protein VP1 (SEQ ID NO.1) in the original adeno-associated virus AAVhu.32. The amino acid sequence of the capsid protein VP1 of AAVhu.32-EBNP is shown in SEQ ID NO.3, and the Cap gene sequence of AAVhu.32-EBNP is shown in SEQ ID NO.4. Compared with the capsid protein VP1 of AAVhu.32, the capsid protein VP1 of AAVhu.32-EBNP has 5 position changes, including 4 site mutations of amino acid substitutions A587D, Q588G, A589N, and Q590P, and inserting a 7-amino acid peptide segment TLAVPFK after the 588th site and before the 589th site of the original VP1. The Cap gene of the variant AAV capsid protein after mutation and insertion of the peptide segment is shown in SEQ ID NO.4.

[0083] To construct AAVhu.32-EBNP, primers Mu-hu.32-EBNP-F (SEQ ID NO.5) and Mu-hu.32-EBNP-R (SEQ ID NO.6) were synthesized respectively. The recombinant adeno-associated virus serotype packaging plasmid pAAV2 / hu.32-EBNP was obtained by PCR reaction using the Fast Mutagenesis System kit (TransGen Biotech) with the laboratory constructed plasmid pAAV2 / hu.32 as a template. The details are as follows:

[0084] The PCR reaction system is: 2×TransStart FastPfu Fly PCR SuperMix, 25μl; 10μM forward primer, 1μl; 10μM reverse primer, 1μl; template pAAV2 / hu.32, 2μl; ddH2O, 31μl. The reaction procedure is: 98℃, 5min; {98℃, 30s; 60℃, 30s; 72℃, 5min; 72℃, 10min} 36 cycles; 16℃, 30min. The PCR product needs to add 1ul template digestion enzyme DMT, and digest the template at 37℃ for 1 hour. 5ul of the digested product is used to transform the competent Stbl3. After 16 hours, a single colony is picked for activation and inoculated into 15 ml LB liquid culture medium. After culture and amplification, the plasmid is extracted and sequenced. The map of the constructed recombinant adeno-associated virus serotype packaging plasmid pAAV2 / hu.32-EBNP expression vector is shown in the figure Figure 1 All PCR primers, gene synthesis and sequencing in the present invention were completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0085] Example 2 Recombinant adeno-associated virus AAVhu.32-EBNP

[0086] The virus packaging method refers to AAV13 Enables Precise Targeting of Local Neural Populations, Han et al.; Int. J. Mol. Sci, 2022, 23 (21), 12806; doi: 10.3390 / ijms232112806. The three-plasmid packaging system is used to package adeno-associated virus. The plasmid pAAV-CMV-EGFP-WPRE-hGH polyA (the coding gene of EGFP fluorescent protein is selected in this example) loaded with the core element (target gene), the adenovirus element auxiliary plasmid pAd-Helper and the pAAV2 / hu.32-EBNP serotype plasmid are co-transfected into HEK-293T cells in a ratio of 1:1:1 of the number of plasmid molecules. Among them, the plasmid pAAV-CMV-EGFP-WPRE-hGH polyA loaded with core elements is used to load the target gene, wherein the position of the coding gene of the EGFP fluorescent protein is the position for delivering the target gene. In this embodiment, the coding gene of the EGFP fluorescent protein is selected, and it can also be conventionally replaced with other target genes.

[0087] Transfection specific conditions: 18 hours before transfection, the suspended HEK293T cells were transferred to a 10 cm culture dish for culture. Each 10 cm culture dish was inoculated with 7 × 10 cells. 62% FBS and 1% double antibodies (penicillin and streptomycin) were added to the cell solution. The transfection operation was completed by transient co-transfection of three plasmids within 24 hours of plating the cells. The three plasmids included: pAAV-CMV-EGFP-WPRE-pA, adenovirus auxiliary plasmid pAdDeltaF6 (Addgene, 112867) and constructed packaging plasmids pAAVhu.32 and pAAVhu.32-EBNP. The transfection process was as follows: First, the amount of three plasmids and PEI was calculated: The mass of transfected plasmid (ng) was calculated according to the plasmid size (bp) 1:1, and the amount of PEI (1μg / μL) was twice the total amount of plasmid; 2 portions of serum-free and anti-antibody-free DMEM medium were prepared according to 500μL per dish, and added to 2 centrifuge tubes, one tube was added with plasmid (about 30μg), and the other tube was added with PEI (final concentration 6μg / mL), and the mixture was shaken and allowed to stand for 5min; then the two tubes were mixed and allowed to stand for 15min; the incubated composite solution was evenly added to the culture dish with cells. The culture was carried out in an incubator at 37°C and 5% carbon dioxide concentration.

[0088] The supernatant and cell pellet were collected 72 hours after transfection, concentrated and purified by iodixanol gradient centrifugation, and the recombinant adeno-associated virus titer was detected by SYBR Green qPCR. The titer of the rAAVhu.32-EBNP-CMV-EGFP-WPRE-hGH polyA virus was 1.0×10 13 VG / mL, the titer of rAAVhu.32-CMV-EGFP-WPRE-hGHpolyA virus is 1.0×10 13 VG / mL.

[0089] Example 3 Application of recombinant adeno-associated virus

[0090] This experiment investigated the effects of recombinant adeno-associated virus (rAAV) constructed with different variants of AAV capsid protein on efficient transduction across the blood-brain barrier into the central nervous system and liver transduction.

[0091] Table 4. Serotype plasmids of different variant AAV capsid proteins

[0092]

[0093]

[0094] The serotype plasmids in the above table were prepared according to the method of Example 1, except that primers were conventionally designed according to the mutation position, and the coding gene of EBNP in the plasmid map was replaced with the coding gene of the corresponding variant AVV capsid protein, such as the primers of AAVhu.32-DGN were Mu-hu.32-DGN-F (SEQ ID NO.7) and Mu-hu.32-DGN-R (SEQ ID NO.8). The primers of AAVhu.32-DGF were Mu-hu.32-DGF-F (SEQ ID NO.9) and Mu-hu.32-DGF-R (SEQ ID NO.10). The constructed AAVhu.32-DGN and AAVhu.32-DGF serotype plasmid maps are shown in FIG. Figure 1 As shown in , the difference lies in the inserted target gene.

[0095] The serotype plasmids of the different variant AAV capsid proteins obtained above were used according to the method in Example 2, except that pAAVhu.32-EBNP was replaced with the serotype plasmids of the corresponding variant AAV capsid proteins, and finally the corresponding recombinant adeno-associated viruses rAAVhu.32-DGN-CMV-EGFP-WPRE-hGH and rAAVhu.32-DGF-CMV-EGFP-WPRE-hGH were obtained, and the titers were adjusted to 1.0×10 13 VG / mL.

[0096] The rAAVhu.32-EBNP-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGN-CMV-EGFP-WPRE-hGH polyA, rAAVhu.32-DGF-CMV-EGFP-WPRE-hGH recombinant adeno-associated virus (as the experimental group) and rAAVhu.32-CMV-EGFP-WPRE-hGH polyA (as the control group) prepared in Example 2 were respectively injected into 8-10 week old C57BL / 6 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) through the tail vein, with a virus titer of 1.0×10 13 VG / mL, the tail vein injection dose for each mouse was 20uL, and the brain and liver tissues were perfused after 3 weeks. There were 3 experimental mice corresponding to each recombinant adeno-associated virus. The mouse brain tissue and liver tissue were fixed with DEPC-treated 4% paraformaldehyde fixative (PFA solution) for 4 hours and then dehydrated with DEPC-treated 30% sucrose-PBS solution for 48 hours. The dehydrated mouse brain and liver were fully embedded with tissue embedding agent and cut into 40μm thick slices with a freezing slicer. Paste and use a slide scanner for microscopic imaging. The data statistical method is: one-way analysis of variance. The test results are as follows:

[0097] Table 5. Statistics of mean fluorescence intensity of brain slices of mice in each group (AU)

[0098]

[0099] Table 6. Statistics of mean fluorescence intensity of liver in each group (AU)

[0100]

[0101] Exemplary liveness detection results are shown in Figure 2 and Figure 3 .

[0102] From the above results, it can be seen that in the whole brain area, only the recombinant adeno-associated virus of rAAVhu.32-EBNP showed stronger fluorescence and more positive cells than the control group (p < 0.0001). In addition, in the liver, compared with the control group, the fluorescence expression intensity mediated by the recombinant adeno-associated virus of rAAVhu.32-EBNP and rAAVhu.32-DGN was significantly reduced, and the reduction was greater than that of the recombinant adeno-associated virus of the rAAVhu.32-DGF serotype plasmid, reducing off-target infection.

[0103] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A variant AAV capsid protein, characterized in that include: 1) an amino acid sequence that is at least 80%, 83%, 85%, 88%, 90%, 93%, 95%, 98% or more identical to the amino acid sequence shown in SEQ ID NO.1; as well as 2) The amino acid sequence shown in SEQ ID NO.1 includes: amino acid substitutions A587D, Q588G, A589N, Q590P, and the amino acid sequence TLAVPFK is inserted after position 588 and before position 589.

2. The variant AAV capsid protein according to claim 1, characterized in that The AAV capsid protein is present in VP1, VP2 and / or VP3 of the AAV capsid.

3. The variant AAV capsid protein according to claim 1 or 2, characterized in that The AAV capsid protein is chimeric.

4. The variant AAV capsid protein according to any one of claims 1 to 3, characterized in that When delivered systemically to a subject, the variant AAV capsid protein has increased specificity for tissues and / or cells of the central nervous system and / or increased transduction efficiency relative to the native AAVhu.32 capsid protein shown in SEQ ID NO.1 when measured in the central nervous system of the subject.

5. The variant AAV capsid protein according to claim 4, characterized in that The cells of the central nervous system are selected from at least one of the following cell types: neurons, oligodendrocytes, astrocytes and brain vascular cells; And / or, the tissue of the central nervous system is selected from at least one of the following tissue types: cerebrum, thalamus, cortex, striatum, ventral midbrain, spinal cord and cerebellum.

6. The variant AAV capsid protein according to any one of claims 1 to 5, characterized in that When systemically delivered to a subject, the variant AAV capsid protein has reduced specificity for off-target tissues and / or off-target cells and / or reduced transduction efficiency relative to the native AAVhu.32 capsid protein set forth in SEQ ID NO.1 when measured in off-target tissues and / or off-target cells of the subject; Optionally, the off-target tissue includes liver tissue; optionally, the off-target cells include liver cells.

7. Biological materials, including any of the following: 1) A nucleic acid molecule encoding the variant AAV capsid protein according to any one of claims 1 to 6; 2) A recombinant vector containing the nucleic acid molecule described in 1); 3) A host cell containing the nucleic acid molecule described in 1) or the recombinant vector described in (2).

8. A recombinant adeno-associated virus rAAV, characterized in that: Containing the variant AAV capsid protein according to any one of claims 1 to 6; Optionally, it also includes a heterologous nucleotide sequence encoding a target product; Optionally, the target product includes a nucleic acid, a peptide or a protein; Optionally, the nucleic acid is selected from siRNA or small guide RNA; Optionally, the original vector of the recombinant adeno-associated virus rAAV is rAAVhu.32; Optionally, the variant AAV capsid protein is present in the recombinant adeno-associated virus rAAV capsid protein VP1.

9. A pharmaceutical composition, characterized in that Contains the recombinant adeno-associated virus rAAV as described in claim 8 and pharmaceutically acceptable excipients.

10. Use of the recombinant adeno-associated virus rAAV according to claim 8 in the preparation of a medicament for delivering a target product to the central nervous system; Optionally, the target product includes a nucleic acid, a peptide or a protein; Optionally, the nucleic acid is selected from siRNA or small guide RNA; Optionally, including use in the preparation of a medicament for treating a central nervous system disease; Optionally, the central nervous system disease includes but is not limited to at least one of neurodegenerative diseases, brain tumor diseases, infectious diseases, cerebrovascular diseases and traumatic brain injury caused by external forces; Optionally, the neurodegenerative disease includes but is not limited to at least one of Alzheimer's disease (AD), Parkinson's disease stroke, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis; Optionally, the brain tumor disease includes but is not limited to at least one of glioblastoma, meningioma, oligodendroglioma, pituitary tumor and pineal tumor; Optionally, the infectious disease includes but is not limited to brain or meningitis; Optionally, the cerebrovascular disease includes but is not limited to stroke or subarachnoid hemorrhage.

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