CN117964716A - Adeno-associated virus mutant and application thereof - Google Patents
Adeno-associated virus mutant and application thereof Download PDFInfo
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- CN117964716A CN117964716A CN202410201060.3A CN202410201060A CN117964716A CN 117964716 A CN117964716 A CN 117964716A CN 202410201060 A CN202410201060 A CN 202410201060A CN 117964716 A CN117964716 A CN 117964716A
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- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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Abstract
The invention belongs to the technical field of biology, and discloses an adeno-associated virus mutant and application thereof. The invention provides an adeno-associated virus capsid protein mutant, the amino acid sequence of which is shown as SEQ ID No. 11. The invention carries out the strategy of polypeptide combinatorial library design based on RGD motif, and cooperates with the directional screening method, effective AAV variants can be discovered through a single screening process, and the screening precision is high. The AAV mutant with muscle specificity is obtained, has good effect in muscle tissues, and has low hepatic tropism and good specificity. The invention provides more useful and selectable carrier tools for gene therapy of muscle diseases, and has huge clinical value and commercial application scene.
Description
The present invention is a divisional application of Chinese patent application No. 202310898798.5 based on the invention name "adeno-associated virus mutant and its application" filed on the basis of month 21 of 2023, 07.
Technical Field
The invention relates to the technical field of biological medicine, in particular to an adeno-associated virus mutant with muscle targeting and application thereof.
Background
Adeno-associated virus (AAV) is a type of non-enveloped small virus that encapsulates a linear single-stranded DNA genome, belonging to the genus lentivirus (Parvoviridae) dependent virus (Dependovirus), which requires helper virus (usually adenovirus) to participate in replication. AAV genomes are single-stranded DNA fragments contained in non-enveloped viral capsids and can be divided into three functional regions: two open reading frames (Rep gene, cap gene) and an Inverted Terminal Repeat (ITR). The recombinant adeno-associated virus vector (rAAV) is derived from a non-pathogenic wild adeno-associated virus, and is widely applied to gene therapy and vaccine research as a gene transfer vector due to the advantages of wide host range, non-pathogenicity, low immunogenicity, long-term stable expression of exogenous genes, good diffusion performance, stable physical properties and the like. rAAV is used in medical research for research (including in vivo and in vitro experiments) of gene therapy of various diseases, such as gene function research, construction of disease models, preparation of gene knockout mice, and the like.
In recent years, gene therapy has become a novel approach to the treatment of muscle meat diseases. Taking Dunaliella Muscular Dystrophy (DMD) as an example, it is a rare fatal neuromuscular genetic disease, DMD is caused by alterations or mutations in the gene encoding the muscular dystrophy protein. Symptoms of DMD are often present in infants and infants, and patients experience developmental delays, such as walking, climbing stairs, or difficulty standing from a seated position. SRP-9001 (AAVrh74. MHCK7. Micro-Dystrophin) is a Sarepta/Roche gene therapy candidate, and utilizes a MHCK7 promoter in combination with an AAVrh74 vector to deliver a miniature anti-dystrophin transgene capable of producing a smaller but functional dystrophin protein (known as a micro-dystrophin), a functional protein that is deficient in DMD patients. 22, 2023, 6, sarepta Therapeutics announced that its AAV gene therapy Delandistrogene moxeparvovec (SRP-9001) was FDA approved for use in the treatment of Duchenne Muscular Dystrophy (DMD), under the trade name Elevidys. The medicine has good curative effect and obviously improves the muscle function of patients. However, AAV therapy also suffers from drawbacks. For example, targeting, poor specificity, overdosing can cause a response to the immune system, leading to side effects. Furthermore, high doses also mean higher production difficulties and higher cost. Therefore, development of drugs with higher targeting to reduce drug dosage or better specificity to avoid adverse reactions is a main aim of AAV serotype engineering.
In view of the foregoing, although AAV is one of the most widely used and safe gene therapy vectors at present, there is still a need for further improvement in terms of lower dose muscle targeting in vivo, and the like. Thus, serotype types with better therapeutic efficacy, reduced therapeutic dose, reduced side effects and cost of use were developed to meet patient needs and drive AAV-based gene therapy approaches toward large-scale, social applications. This has great significance in improving the benefit of gene therapy and serving a wide range of patients.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an adeno-associated virus mutant and application thereof, wherein the adeno-associated virus mutant has muscle targeting, high specificity and good safety.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
In a first aspect, the present invention provides an adeno-associated virus capsid protein mutant having a heterologous peptide inserted therein; the amino acid sequence of the heterologous peptide is shown as any one of SEQ ID No. 1-4.
The adeno-associated virus capsid protein mutant has good targeting to different muscle tissues (quadriceps femoris, biceps brachii, abdominal muscle and the like), compared with a control AAV9, the muscle targeting is improved by about 10 times at most, and the liver tropism is lower than that of the control AAV9 by 2-3 times, so that the specificity is good.
As a preferred embodiment of the adeno-associated virus capsid protein mutant of the invention, the insertion site of the heterologous peptide is located between amino acids 583 and 591 of the adeno-associated virus capsid protein.
As a preferred embodiment of the adeno-associated virus capsid protein mutant of the present invention, the amino acid sequence thereof is as shown in any one of SEQ ID No.9 to 12.
In a second aspect, the invention provides a nucleic acid encoding an adeno-associated virus capsid protein mutant, the nucleotide sequence of which comprises the heterologous peptide nucleotide sequence as shown in SEQ ID nos. 5-8.
As a preferred embodiment of the nucleic acid encoding the adeno-associated virus capsid protein mutant of the present invention, the nucleotide sequence is as shown in any one of SEQ ID No.13 to SEQ ID No. 16.
In a third aspect, the invention provides a recombinant adeno-associated virus comprising said adeno-associated virus capsid protein mutant.
The recombinant adeno-associated virus vector obtained by constructing the adeno-associated virus capsid protein mutant has higher specificity, better safety and wide application range
As a preferred embodiment of the recombinant adeno-associated virus of the invention, a heterologous gene of interest is also included.
As a preferred embodiment of the recombinant adeno-associated virus of the invention, the heterologous gene of interest encodes any one of the gene products interference RNA, aptamer, endonuclease, guide RNA.
In a fourth aspect, the invention provides the use of said adeno-associated virus capsid protein mutant, said recombinant adeno-associated virus in the manufacture of a medicament or formulation for delivering a gene product into a cell or tissue of a subject.
As a preferred embodiment of the use according to the invention, the cells are muscle cells; the tissue is muscle tissue.
In a fifth aspect, the invention provides the use of said adeno-associated virus capsid protein mutant, said recombinant adeno-associated virus in the manufacture of a drug delivery vehicle for the prevention and/or treatment of muscle disorders.
In a sixth aspect, the invention provides the use of the adeno-associated virus capsid protein mutant and the recombinant adeno-associated virus in the manufacture of a medicament for the treatment of muscle and meat disorders.
As a preferred embodiment of the use according to the invention, the muscular diseases include, but are not limited to, any one of Duchenne Muscular Dystrophy (DMD), becker Muscular Dystrophy (BMD), X-linked myotubular myopathy (XLMTM), limb Girdle Muscular Dystrophy (LGMD), myotonic muscular dystrophy, facial shoulder muscular dystrophy.
Compared with the prior art, the invention has the beneficial effects that:
The invention adopts a method based on specific motif to construct a small AAV virus library, and can discover effective AAV variants through a single screening process, thereby having high screening precision and needing no repeated screening and verification for multiple rounds. The invention obtains 4 AAV9 mutants with muscle specificity, and verifies from mRNA and protein expression level that the AAV9 mutants have good effect in muscle tissues such as quadriceps femoris, biceps brachii, abdominal muscle and the like (compared with a control AAV9, the AAV9 has improved muscle targeting by about 10 times at most), low hepatic tropism (lower than that of the control AAV9 by 2-3 times), and good specificity. Provides more useful and selectable carrier tools for gene therapy of muscle diseases, benefits to vast patients, and has great clinical value and commercial application scenes.
Drawings
FIG. 1 is an analysis of the muscle (quadriceps femoris) targeting of C57 mice by different serotypes (3 weeks); in the figure, A is the mRNA relative expression level of quadriceps femoris, and B is the protein expression level of quadriceps femoris;
FIG. 2 is an analysis of muscle (biceps brachii) targeting of different serotypes to C57 mice (3 weeks); in the figure, A is the mRNA relative expression level of biceps brachii, and B is the protein expression level of biceps brachii;
FIG. 3 is an analysis of muscle (abdominal) targeting of C57 mice by different serotypes (3 weeks); in the figure, A is the relative mRNA expression level of the abdominal muscle, and B is the protein expression level of the abdominal muscle;
FIG. 4 is a liver tropism analysis (3 weeks) of C57 mice for different serotypes; in the figure, A is the relative expression level of mRNA in liver, and B is the expression level of protein in liver.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: screening of novel mutants
(1) Construction of AAV9 capsid protein mutant library backbone plasmid
AAV9 library backbone vectors comprise MHCK promoter, intron, mutated AAV9CAP sequence [ the sequence after T582 of AAV9CAP sequence was removed, the T582 nucleic acid sequence ACA was mutated to ACT, thus constituting with the polyA pre-sequence the cleavage site BsrG I (TGTACA) for subsequent backbone enzyme tangentially-cutting ] and polyA. The sequences are synthesized by a gene synthesis mode and inserted between ITRs of AAV vector plasmids to form AAV9 library skeleton vectors.
(2) Construction of mutant Rep-CAP vectors
By introducing a stop codon into the N-terminal of VP1, VP2 and VP3 proteins of CAP sequence in AAV9, the Rep-CAP vector can normally express Rep protein and AAP protein, but can not express VP1, VP2 and VP3 proteins of CAP, thereby avoiding pollution of CAP sequence in parental AAV 9. The sequences were synthesized by gene synthesis and inserted into CAP sequences replacing the AAV9 Rep-CAP vector.
(3) Construction of random polypeptide vector libraries comprising RGD motifs
Primer design: the insertion and modification sequence between the N583 and A591 sequences of AAV9 CAP, the original sequence being N583-HQSAQAQ-A591, and the insertion and substitution of the random tetrapeptide sequence comprising RGD at different positions therein forms the following 18 combinations: N583-HQSAQ (RGD+ random tetrapeptide) AQ-A591, N583-HQSAQ (RGD+ random tetrapeptide) Q-A591, N583-HQSAQ (RGD+ random tetrapeptide) -A591, N583-HQSA (RGD+ random tetrapeptide) AQ-A591, N583-HQSA (RGD+ random tetrapeptide) Q-A591, N583-HQSA (RGD+ random tetrapeptide) -A591, N583-HQS (RGD+ random tetrapeptide) AQ-A591, N583-HQS (RGD+ random tetrapeptide) Q-A591, N583-HQS (RGD+ random tetrapeptide) -A591, N583-HQ (RGD+ random tetrapeptide) AQ-A591, N583-HQ (RGD+ random tetrapeptide) Q-A591, N583-HQ (RGD+ random tetrapeptide) -A591, N583-H (RGD+ random tetrapeptide) AQ-A591, N583-H (RGD+ random tetrapeptide) Q-A591, N583-H (RGD+ random tetrapeptide) -A591, N583- (RGD+ random tetrapeptide) AQ-A591, N583- (RGD+ random tetrapeptide) Q-A591. Wherein (rgd+random tetrapeptide) itself also has 5 RGD position combinations: RGDXXXX, XRGDXXX, XXRGDXX, XXXRGDX, XXXXRGD. Thus, 90 sequence primers are formed in total, the primers are used as an upstream primer and a downstream primer to form a primer pair, a target fragment library is amplified under the condition that an AAV9 CAP vector is used as a template, and homologous arms are arranged at two ends of the fragment library, so that the fragment library can be recombined with AAV9 library skeleton plasmids after enzyme digestion in a homologous manner to form a vector library.
The specific operation steps are as follows: the AAV9 CAP-containing vector was used as a template and PCR amplification was performed using the primers described above to obtain fragments containing random sequences. Gel electrophoresis and gel recovery are carried out on the fragments to obtain purified nucleic acid fragments; connecting the nucleic acid fragments into AAV9 library skeleton vectors (subjected to BsrG I digestion and glue recovery purification) through a Gibson homologous recombination connection mode, purifying the connected vectors through a PCR product purification kit, and then digesting the connected vectors with Plasmid-SAFE DNASE enzyme to remove fragments which are not connected; finally purifying by a PCR product purification kit to obtain the constructed AAV9 random polypeptide carrier library containing the RGD motif.
(4) Construction of AAV9 mutant Virus library
The mutant Rep-Cap plasmid, AAV9 mutant plasmid library and pHelper plasmid are co-transferred into HEK-293T cells, iodixanol gradient ultra-high speed centrifugation is adopted to purify adeno-associated virus, the virus titer is measured to be proper titer at 10 12GC/mL～1013 GC/mL, and the AAV9 mutant virus library is obtained and placed at the temperature of minus 80 ℃ for standby.
(5) Screening of AAV9 mutants
(5.1) Animal injection and dissection
The animal experiment uses C57 male mice of 6-8 weeks old, the mice are grouped according to different dosages, each group is respectively injected with 2E10GC and 3E9GC virus libraries according to each mouse, related viruses are prepared according to the experiment group, animal dissection and organ material (muscle, heart, liver and the like at each part) are carried out after 21 days of injection, and liquid nitrogen quick freezing is carried out immediately after sample material taking and the obtained sample material is used for the subsequent RNA extraction experiment.
(5.2) Total RNA extraction and RT-PCR
Grinding of the sample: pre-cooling the grinder 10min in advance and setting grinding parameters. The animal tissue sample stored in the refrigerator at-80 ℃ is taken out, about 50-100 mg of tissue is cut into Huang Douli pieces in a sterile culture dish, and then transferred into a 1.5mL RNase-free EP tube. According to each 50-100 mg tissue: 1mL TransZol Up, adding a proper amount of TransZol Up, then adding two clean sterile 3mm grinding steel balls, and winding a sealing film. The sample was placed in a 24-well grind adapter and trimmed, the screw was tightened, and the cap closing button was pressed. And starting a grinding program, taking out the sample after the operation of the instrument is finished, and observing the grinding granularity of the sample, wherein the subsequent extraction operation can be performed if no massive tissue residue exists. The milled sample was centrifuged at 12,000Xg for 2min at 4℃and the supernatant was pipetted into a new 1.5mL RNase-free EP tube with corresponding labeling.
Extraction of total RNA of a sample: reference is made specifically to TransZol Up Plus RNA Kit (Beijing full gold, cat# ER 501) specification. Every time 1mL TranZol up is used, 0.2: 0.2mL RNA Extraction Agent is added, and the mixture is vigorously vibrated for 5min;12,000Xg, centrifuged at 4℃for 10min. At this time, the sample was divided into three layers, the colorless aqueous phase was transferred to a new 1.5mL RNase-free EP tube, and an equal volume of absolute ethanol (precipitation may occur at this time) was added, and mixed by gently inverting; adding the obtained solution and the precipitate into a centrifugal column, centrifuging at room temperature for 30s at 12,000Xg, and discarding the filtrate; adding 500 mu L of CB9, centrifuging at 12,000Xg for 30s at room temperature, and discarding the filtrate; repeating the previous steps for one time; 500. Mu. LWB9 was added and centrifuged at 12,000Xg for 30s at room temperature, and the filtrate was discarded; repeating the previous steps for one time; centrifuging for 2min at room temperature of 12,000Xg to thoroughly remove residual ethanol; putting the centrifugal column into a 1.5mL RNase-free EP tube, adding 30-50 mu L (depending on the tissue size) of RNase-FREE WATER in the center of the centrifugal column, and standing for 1min at room temperature; centrifuging at room temperature for 1min at 12,000Xg, eluting RNA;
Sample nucleic acid concentration determination by detecting RNA concentration using a micro-nucleic acid quantitative instrument detector, recording the concentration, OD260/280, OD260/230, and storing RNA at-80 ℃.
RT-PCR: RNA samples were extracted and first strand cDNA synthesis was performed using PRIMESCRIPTTM IV A st strand cDNA Synthesis Mix (Takara, 6215A). Then using NEB Q5 for 2 rounds of PCR amplification (first round using outside primer amplification; second round using gel recovered first round product as template, with NGS primer amplification), gel recovered corresponding band size PCR product to send company for NGS sequencing.
NGS sequencing, data analysis and selection of candidate vectors: sequencing data analysis is carried out after sequencing, sequences which are ranked in front of the occurrence frequency and are repeatedly appeared in a plurality of samples are selected as candidates, and subsequent construction and verification work of AAV mutants is carried out.
Example 2: construction of AAV capsid protein mutants and production of viruses
(1) Construction of mutant serotype vector and plasmid extraction
AAV9 Rep-CAP plasmid is digested with Smi I and BshT I, gel electrophoresis is carried out, and about 5000bp fragment band is cut off for gel recovery, so that digested skeleton fragment is obtained.
According to the Cap sequence of the mutant 1, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ221-1 by using a CAP-f+YJ221-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ221-2 by using a YJ221-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing the Rep-CAP plasmid of the mutant 1 by mixing the framework fragments, YJ221-1 and YJ221-2 according to the following steps and proportions;
according to the Cap sequence of the mutant 2, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ222-1 by using a CAP-f+YJ222-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ222-2 by using a YJ222-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 2 by mixing a framework fragment, YJ222-1 and YJ222-2 according to the following steps and proportions;
according to the Cap sequence of the mutant 3, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ223-1 by using a CAP-f+YJ223-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ223-2 by using a YJ223-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 3 by mixing a framework fragment, YJ223-1 and YJ223-2 according to the following steps and proportions;
According to the Cap sequence of the mutant 4, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ224-1 by using a CAP-f+YJ224-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ224-2 by using a YJ224-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 4 by mixing a framework fragment, YJ224-1 and YJ224-2 according to the following steps and proportions;
According to the Cap sequence of the mutant 5, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ225-1 by using a CAP-f+YJ225-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ225-2 by using a YJ225-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 5 by mixing a framework fragment, YJ225-1 and YJ225-2 according to the following steps and proportions;
according to the Cap sequence of the mutant 6, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ226-1 by using a CAP-f+YJ226-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ226-2 by using a YJ226-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 6 by mixing a framework fragment, YJ226-1 and YJ226-2 according to the following steps and proportions;
according to the Cap sequence of the mutant 7, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ227-1 by using a CAP-f+YJ227-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ227-2 by using a YJ227-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 7 by mixing a framework fragment, YJ227-1 and YJ227-2 according to the following steps and proportions;
According to the Cap sequence of the mutant 8, the following primers are designed, and the specific steps are as follows: amplifying and gel-recovering a target product YJ228-1 by using a CAP-f+YJ228-R primer with a Rep-CAP plasmid of AAV9 as a template, amplifying and gel-recovering a target product YJ228-2 by using a YJ228-F+cap-R primer with a Rep-CAP plasmid of AAV9 as a template, and recombining and constructing a Rep-CAP plasmid of a mutant 8 by mixing a framework fragment, YJ228-1 and YJ228-2 according to the following steps and proportions;
the primers involved in the construction of the Rep-CAP vector for the AAV capsid mutants described above are shown in Table 1:
TABLE 1 primer sequences
Taking 1 clean 200 mu L PCR tube as a mark and placing the mark on an ice box, and cutting the enzyme-cleaved skeleton and each target fragment according to the skeleton: preparing a reaction solution with the fragment molar ratio of 1:3, and carrying out recombination connection in a PCR instrument at 50 ℃ for 30 min. Thawing 50 mu L of competent cells on ice, mixing 10 mu L of the ligation product with DH5 alpha competent cells, and standing on ice for 20-30 minutes; heat shock at 42 ℃ for 45 seconds; rapidly placing on ice for 2 minutes, adding 400 mu L of recovery SOC culture medium (without antibiotics), and culturing at 37 ℃ and 200rpm for 1 hour; the mixture was spread on an Amp-resistant plate (50. Mu.g/mL) and incubated at 37℃for 14 hours. Monoclonal bacteria were selected and grown in 4mL of liquid LB medium (Amp+ resistant) for 14 hours at 37 ℃.
Centrifuging the bacterial liquid for 1 minute at 12000rpm, and pouring out the supernatant culture medium; adding 250 mu L of buffer P1/RNaseA mixed solution, and high-speed vortex to re-suspend bacteria; adding 250 mu L buffer P2, and reversing the above steps for 8 to 10 times; adding 350 mu L buffer P3, immediately reversing and uniformly mixing for 8-10 times to thoroughly neutralize the solution; centrifuging at 13000rpm for 10min, and collecting supernatant; centrifuging 12000 for 1 minute, pouring out the waste liquid, adding 500 mu L PW1, centrifuging 12000 for 1 minute, and pouring out the waste liquid; 600 μl of PW2 was added, 12000 centrifuged for 1 min, and the supernatant was decanted; 600 μl of PW2 was added, 12000 centrifuged for 1 min, and the supernatant was decanted; idle at 12000rpm for 2 minutes; adding 30-50 mu L of preheated eluent at 55 ℃, standing for 2 minutes, and centrifuging at 12000rpm for 1 minute. Concentration detection was performed using a micro nucleic acid quantitative instrument.
The obtained plasmid is subjected to concentration detection, 10 mu L of positive plasmid identified by enzyme digestion is taken and sequenced, and the positive plasmid is stored at-20 ℃. Sequencing results showed that the obtained plasmid was able to encode the variant capsid protein VP1. Finally, relevant Helper plasmids were extracted according to the amount of virus required for the later test, and each group of Rep-Cap plasmids (AAV 9 wild type and AAV9 mutants 1 to 8) plasmids and GOI plasmids (ssAAV. CAG. Fluc-2a-eGFP. WPRE. SV40 pA).
(2) Packaging and purification of mutant serotype viruses
Rep-Cap plasmids of each group (AAV 9 wild type and AAV9 mutants 1-8) are obtained, GOI plasmids expressing firefly luciferase (Fluc) and green fluorescent protein (EGFP) are co-transferred into HEK-293T cells in proper quantity, AAV viruses are purified by iodixanol gradient ultra-high speed centrifugation, and the virus titer is measured to be proper titer between 1E+11GC/mL and 1E+13GC/mL and is placed at the temperature of minus 80 ℃ for standby.
Example 3 comparative testing of various indicators of mutant serotypes
(1) Injection and dissection of animals
The animal experiment uses C57 male mice of 6-8 weeks old, relevant viruses are prepared according to designed experimental groups and control groups, each group is injected with 2E11GC virus according to each mouse, animal dissection and material drawing of each organ are carried out after 3 weeks of injection, liquid nitrogen quick freezing is carried out immediately after sample material drawing, and the liquid nitrogen quick freezing is respectively used for subsequent experiments such as RNA extraction, WB detection and the like.
(2) Detection of mRNA expression level of target Gene
(2.1) Total RNA extraction and reverse transcription:
Grinding of the sample: pre-cooling the grinder 10min in advance and setting grinding parameters. The animal tissue sample stored in the refrigerator at-80 ℃ is taken out, about 50-100 mg of tissue is cut into Huang Douli pieces in a sterile culture dish, and then transferred into a 1.5mL RNase-free EP tube. According to each 50-100 mg tissue: 1mL TransZol Up, adding a proper amount of TransZol Up, then adding two clean sterile 3mm grinding steel balls, and winding a sealing film. The sample was placed in a 24-well grind adapter and trimmed, the screw was tightened, and the cap closing button was pressed. And starting a grinding program, taking out the sample after the operation of the instrument is finished, and observing the grinding granularity of the sample, wherein the subsequent extraction operation can be performed if no massive tissue residue exists. The milled sample was centrifuged at 12,000Xg for 2min at 4℃and the supernatant was pipetted into a new 1.5mL RNase-free EP tube with corresponding labeling.
Extraction of total RNA of a sample: reference is made specifically to TransZol Up Plus RNA Kit (Beijing full gold, cat# ER 501) specification. Every time 1mL TransZol up is used, 0.2: 0.2mL RNA Extraction Agent is added, and the mixture is vigorously vibrated for 5min;12,000Xg, centrifuged at 4℃for 10min. At this time, the sample was divided into three layers, the colorless aqueous phase was transferred to a new 1.5mL RNase-free EP tube, and an equal volume of absolute ethanol (precipitation may occur at this time) was added, and mixed by gently inverting; adding the obtained solution and the precipitate into a centrifugal column, centrifuging at room temperature for 30s at 12,000Xg, and discarding the filtrate; adding 500 mu L of CB9, centrifuging at 12,000Xg for 30s at room temperature, and discarding the filtrate; repeating the previous steps for one time; 500. Mu. LWB9 was added and centrifuged at 12,000Xg for 30s at room temperature, and the filtrate was discarded; repeating the previous steps for one time; centrifuging for 2min at room temperature of 12,000Xg to thoroughly remove residual ethanol; putting the centrifugal column into a 1.5mL RNase-free EP tube, adding 30-50 mu L (depending on the tissue size) of RNase-FREE WATER in the center of the centrifugal column, and standing for 1min at room temperature; centrifuging at room temperature for 1min at 12,000Xg, eluting RNA;
Sample nucleic acid concentration determination by detecting RNA concentration using a micro-nucleic acid quantitative instrument detector, recording the concentration, OD260/280, OD260/230, and storing RNA at-80 ℃.
Reverse transcription: use of RNA samples from each groupAll-in-One First-STRAND CDNA SYNTHESIS SuperMix for qPCR (One-Step gDNA Removal) (Beijing full gold, cat# AE 341-03), specific steps of which are referred to the specification.
(2.2) Quantitative PCR (qPCR) experiments:
the qPCR system configuration was performed using each set of cDNAs as templates according to the specification of 2 XSYBR GREEN QPCR MASTER Mix (Bimake, cat# B21203), see tables 2-4:
TABLE 2qPCR System
| Reagent(s) | Usage amount |
| 2×SYBR Green qPCR Master Mix | 10μL |
| CDNA template | 1.5μL |
| Upstream primer (10. Mu.M) | 1μL |
| Downstream primer (10. Mu.M) | 1μL |
| ROX Reference Dye | 0.4μL |
| Deionized water | Up to 20μL |
TABLE 3 primer sequences
| Primer name | Primer sequence (5 '- > 3') |
| Fluc2-qPCR-F1 | AACCAGCGCCATTCTGATCA |
| Fluc2-qPCR-R1 | TCGGGGTTGTTAACGTAGCC |
| GAPDH-F2 | CAGGAGAGTGTTTCCTCGTCC |
| GAPDH-R2 | TTCCCATTCTCGGCCTTGAC |
Table 4 qPCR program settings
(2.3) Data analysis
Based on the Ct value of each group, the relative expression amount is calculated according to equation 2 -ΔΔct.
(3) Detection of expression level of target protein by WB
Sample pretreatment:
Cutting the tissue into small fragments, weighing and recording the weight, placing the fragments into a 1.5mL or 2mL centrifuge tube, marking the tube, cooling at-80 ℃ for standby, and precooling a cryogrinder; lysates of RIPA (bi yun, P0013B) were dissolved (PMSF was added to a final PMSF concentration of 1mM during the minutes prior to use);
The complete lysate is added according to the proportion of adding 150-250 mu L of lysate into each 20mg of tissue, then two sterilized zirconia grinding beads are added, and the samples (brain, spinal cord and other tissue samples: temperature-20 ℃, frequency 70Hz, time for shaking 50s for 10s, circulation 3-4 times, muscle, liver and other samples: temperature-20 ℃, frequency 70Hz, time for shaking 50s for 10s, 5-7 times) are directly ground in the lysate. After the sample is ground, centrifuging the sample in a refrigerated centrifuge at 4 ℃ and 12,000Xg for 5-10 min, and transferring the supernatant to a new sterilized EP tube for preservation at-20 ℃ or-80 ℃;
protein concentration determination:
after protein concentration was measured by the method in the modified BCA protein concentration measurement kit (Protect, cat. No. C503051), an appropriate amount of the protein homogenate sample was taken according to the required amount, and mixed with a corresponding amount of 5 XSDS-PAGE protein loading buffer, and the mixture was boiled in water for 10 minutes, cooled, centrifuged at a low speed for a moment, and then loaded.
WB (Western Blot) detection:
SDS-PAGE electrophoresis: the proper loading amount is determined according to the protein concentration and the expression level, the loading amount of the tissue homogenate protein is less than 20 mu L/hole, the loading amount of the tissue homogenate protein is about 20-50 mu g, and the specific operation flow of electrophoresis is as follows: pulling out the comb on the prefabricated gel, mounting the gel on the electrophoresis tank, adding electrophoresis buffer solution into the inner tank and the outer tank, adding newly prepared buffer solution into the inner tank, detecting leakage, and adding electrophoresis buffer solution into the outer tank if no leakage exists; and (3) taking a proper amount of treated protein samples for loading, and carrying out 100V constant-pressure electrophoresis on a space energy electrophoresis device by taking pre-dyed standard proteins as references, wherein the electrophoresis time is 100min until bromophenol blue reaches the bottom of the gel. Closing the power supply, carefully removing the prefabricated rubber plate, taking down the gel, and placing the gel in a film transfer buffer solution to wait for subsequent operation;
b. Transferring: 6 filter papers and 1 PVDF membrane were cut according to the glue area. Soaking PVDF membrane in methanol for 5-10sec, transferring to membrane transfer buffer solution for 5min, and pre-wetting filter paper in the membrane transfer buffer solution; and (3) installing and transferring the device: negative electrode (blackboard) -sponge-3 layer wetted filter paper-gel-PVDF film-3 layer wetted filter paper-sponge-positive electrode (transparent plate). Each layer of bubbles are driven away to avoid affecting the transfer effect, the support is clamped, and the support is placed into the electrotransport groove; transferring the film for 100min by using a constant-pressure ice bath with the voltage of 100V; judging whether the membrane transfer is successful or not according to whether the pre-dyed protein molecular weight standard strip is completely transferred to the PVDF membrane or not; soaking the transferred PVDF film in PBST solution, washing for 5min at room temperature, and cutting the PVDF film according to the requirement, wherein the PVDF film is not dried in the film cutting process;
c. Blocking and antibody incubation: incubating the PVDF membrane with a blocking solution (5% nonfat milk powder) at room temperature for 2h or overnight at 4deg.C; transferring the sealed PVDF membrane into primary antibody hybridization solution (Luciferase Rabbit Polyclonal antibody (Proteintech, 27986-1-AP) according to 1:2000;GADPH Rabbit Polyclonal antibody (Proteintech, 10494-1-AP) according to 1:2000;Rabbit GFP tag Polyclonal antibody (Proteintech, 50430-2-AP) according to 1:2000, respectively adding into 4ml QuickBlock TM Western primary antibody dilution solution (Biyun Tian, P0256) to prepare primary antibody hybridization solution), incubating at room temperature for 1h or overnight at 4 ℃, and then washing the membrane with PBST for 3×5min; transferring the washed PVDF membrane into secondary antibody hybridization solution (HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (Proteintech, SA 00001-2), adding into 4mL QuickBlock TM Western secondary antibody dilution solution (Biyundian, P0258) according to a ratio of 1:5000, namely preparing into secondary antibody hybridization solution), incubating for 1H at room temperature, washing the membrane by PBST, and 3×5min;
d. color development: mixing the A solution and the B solution of the ECL chemiluminescence kit in equal volume, and after shaking and mixing uniformly, dripping the luminescent liquid on the PVDF film to ensure that the PVDF film is covered with the luminescent liquid, adjusting different exposure time to ensure that protein strips are clear, and photographing by an instrument.
The different screening mutants had different degrees of enhancement of targeting ability to muscle for mutants 1,2,4, and 7 as compared to the parental AAV9 control, as shown in figures 1,2, and 3. Wherein the mRNA levels of mutants 1 and 2 were 8.34-fold and 10.35-fold higher than that of AAV9 in quadriceps femoris, and the mRNA levels of mutants 4 and 7 were slightly lower than those of AAV9 by 6.52-fold and 5.55-fold, respectively. Protein expression level of quadriceps femoris is significantly improved by mutants 1,2 and 7. The protein expression results of biceps brachii are substantially similar to quadriceps femoris, and mutants 1,2 and 7 are also most pronounced. And the highest mRNA levels were mutants 2 and 7, 2.73-fold and 2.26-fold compared to AAV9 control. mRNA expression levels at the abdominal muscle, also significantly increased as compared to mutants 1,2,4 and 7, reached 2.64-fold, 3.19-fold, 2.29-fold and 3.94-fold, respectively, of AAV9 controls, with protein levels substantially identical. From a combination of the above 3 muscle tissue performances, it was determined that mutants 1,2,4 and 7 had good targeting to the muscle, from which we also found that, although candidate mutants all contained the RGD motif, the targeting was varied around the amino acid species near the RGD motif and the influence on the AAV capsid protein structure. Optimization of adjacent amino acids is thus also an essential process for targeting sequences for which motifs already exist.
To better assess the specificity of the mutants in vivo, the liver tropism of the mutants was analyzed. As a result, as shown in FIG. 4, most of the mutants were lower in mRNA level and protein level than the AAV9 control group, and the mRNA levels of mutants 1,2, 4 and 7 were 0.32-fold, 0.55-fold, 0.4-fold and 0.51-fold, respectively, as compared with the control group. The mutant of the invention has very important effects on the follow-up curative effect, dose reduction, toxic and side effects and the like, and has the advantages of improving the targeting property of muscles, reducing hepatic tropism and obviously improving the specificity.
The amino acid sequences of the AAV capsid protein mutants 1,2,4 and 7 screened by the invention are shown in SEQ ID No. 9-SEQ ID No.12 in sequence, and the nucleotide sequences are shown in SEQ ID No. 13-SEQ ID No.16 in sequence; the amino acid sequence of the targeting peptide in VP1 is shown in SEQ ID No. 1-SEQ ID No.4, and the nucleotide sequence is shown in SEQ ID No. 5-SEQ ID No. 8. The method comprises the following steps:
SEQ ID No.1 (targeting peptide 1 amino acid sequence): QMHRGDA;
SEQ ID No.2 (targeting peptide 2 amino acid sequence): RRGDLVG;
SEQ ID No.3 (targeting peptide 4 amino acid sequence): SVRGDAS;
SEQ ID No.4 (targeting peptide 7 amino acid sequence): DLRSRGD;
SEQ ID No.5 (targeting peptide 1 nucleic acid sequence (5 '- > 3')): CAGATGCATAGAGGAGACGCT;
SEQ ID No.6 (targeting peptide 2 nucleic acid sequence (5 '- > 3')): CGTAGAGGAGACTTGGTTGGG;
SEQ ID No.7 (targeting peptide 4 nucleic acid sequence (5 '- > 3')): TCGGTGAGAGGAGACGCGTCG;
SEQ ID No.8 (targeting peptide 7 nucleic acid sequence (5 '- > 3')): GATCTTCGTAGTAGAGGAGAC;
SEQ ID No.9 (mutant 1VP1 amino acid sequence):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQMHRGDAAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.10 (mutant 2VP1 amino acid sequence):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSARRGDLVGAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.11 (mutant 4VP1 amino acid sequence):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSSVRGDASAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.12 (mutant 7VP1 amino acid sequence):
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQDLRSRGDAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL*
SEQ ID No.13 (mutant 1VP1 nucleic acid sequence (5 '- > 3')):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGATGCATAGAGGAGACGCTGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.14 (mutant 2VP1 nucleic acid sequence (5 '- > 3')):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGAGTGCCCGTAGAGGAGACTTGGTTGGGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.15 (mutant 4VP1 nucleic acid sequence (5 '- > 3')):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGAGTTCGGTGAGAGGAGACGCGTCGGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA;
SEQ ID No.16 (mutant 7VP1 nucleic acid sequence (5 '- > 3')):
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCG
CGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGAC
AACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACA
AGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACC
AGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCA
GGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCC
AAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAA
AGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATC
GGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTC
CCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAA
TGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTA
GTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAG
CACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGC
ACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTT
TGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACA
ACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGT
TACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTC
ACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGC
CGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGC
CAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAAC
GGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTC
ACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCA
AAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCA
GCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGT
CTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGG
CTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGG
AGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAG
ACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACC
CGGTAGCAACGGAGTCCTATGGACAAGTGGCCACTAACCACCAGGATCTTCGTAGTAGAGG
AGACGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGAC
AGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTC
ACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAA
AACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCAT
CACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAAC
AGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTG
AATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA。
by combining the results, the invention constructs a small AAV library by adopting a method based on a specific motif, and can discover effective AAV variants through a single screening process, thereby having high screening precision and needing no repeated screening and verification for multiple rounds. The strategy of designing the polypeptide combinatorial library based on RGD motif is matched with a directional screening method, 4 AAV9 mutants with muscle specificity are obtained, and the effects of the AAV9 mutants in muscle tissues such as quadriceps femoris, biceps brachii, abdominal muscle and the like are verified from mRNA and protein expression levels (compared with a control AAV9, the muscle targeting is improved by about 10 times at most), and the specificity is low (lower than that of the control AAV9 by 2-3 times). Provides more useful and selectable carrier tools for gene therapy of muscle diseases, benefits to vast patients, and has great clinical value and commercial application scenes.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. An adeno-associated virus capsid protein mutant, which is characterized in that the amino acid sequence is shown in SEQ ID No. 11.
2. A nucleic acid for coding a capsid protein mutant of an adeno-associated virus, which is characterized in that the nucleotide sequence is shown in SEQ ID No. 15.
3. A recombinant adeno-associated virus comprising the adeno-associated virus capsid protein mutant of claim 1.
4. The recombinant adeno-associated virus of claim 3, further comprising a heterologous gene of interest.
5. The recombinant adeno-associated virus according to claim 4, wherein the heterologous gene of interest encodes any one of interfering RNA, aptamer, endonuclease, guide RNA gene product.
6. Use of the adeno-associated virus capsid protein mutant of claim 1, the recombinant adeno-associated virus of any one of claims 3-5 in the manufacture of a medicament or formulation for delivering a gene product into a cell or tissue of a subject.
7. The use according to claim 6, wherein the cells are muscle cells; the tissue is muscle tissue.
8. Use of an adeno-associated virus capsid protein mutant according to claim 1, a recombinant adeno-associated virus according to any one of claims 3 to 5 for the manufacture of a drug delivery means for the prevention and/or treatment of muscle disorders.
9. Use of the adeno-associated virus capsid protein mutant of claim 1, the recombinant adeno-associated virus of any one of claims 3-5 for the preparation of a medicament for the treatment of muscle disorders.
10. The use according to claim 8 or 9, wherein the muscular disease includes, but is not limited to, any one of duchenne muscular dystrophy, becker muscular dystrophy, X-linked myotubular myopathy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, facial shoulder brachial muscular dystrophy.
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