CN109897831A - Adeno-associated virus virion and its application with mutant capsids - Google Patents

Abstract

The invention discloses a kind of adeno-associated virus virion and its application with mutant capsids.The adeno-associated virus virion with mutant capsids has mutation AAV6 capsid protein, and the adeno-associated virus virion with mutant capsids assigns the infectivity of the thermophilic view self-maintenance of enhancing.Wherein relative to corresponding parent AAV6 capsid protein, the 663rd mutant serine is leucine in the amino acid sequence of the mutation AAV6 capsid protein.Described pharmaceutical composition includes the adeno-associated virus virion and pharmaceutically acceptable excipient.The present invention obtains the AAV carrier of specifical and efficient transduction M ü ller cell, the method for treating retinopathy suitable for carrying exogenous therapeutic gene transduction M ü ller cell by the Amino Acid-Induced Site-Directed Mutation to coding AAV6 capsid.

Description

Adeno-associated virus virion and its application with mutant capsids

Technical field

The present invention relates to a kind of recombination AAV virion, and in particular to a kind of in selected adeno-associated virus (AAV) sequence Single-point amino acid mutation is to assign its method for targeting view self-maintenance, and is carrying exogenous therapeutic gene transduction M ü Ller cell treats the application in retinopathy, belongs to gene engineering technology field.

Background technique

Retinal disease be cause blindness the main reason for one of, common are macular degeneration, diabetic retinopathy, Glaucoma, Inherited retinal lesion etc..Wherein, most of lesion do not worked or be overexpressed by gene mutation, albumen cause it is different Often, and then cause visual cell dead, final blinding.Correct gene can be mediated by gene therapy or knocks out aberrant gene, it is extensive Multiple normal expression, and then play the role of restoring visual function.

Adeno-associated virus (Adeno-associated virus, AAV) belongs to a member of Parvoviridae dependovirus, It is the icosahedron that a diameter is about 20~26nm, carries the linear ssdna genome of 4.6~4.8kb.At present Through the AAV virus for being separated to 13 kinds of serotypes and 120 various mutations types in vivo for from many mammals including people.AAV conduct Genophore has the advantages that not available for other viral vectors or non-virus carrier in gene therapy application: (1) can both feel Dividing cell is contaminated, akinete can also be infected；(2) AAV is that unique energy site-directed integration known today is special to human genome The genophore of anchor point, and integration site is safe and reliable；Most of all, although the mankind of 80-90% are positive in AAV, Do not report that AAV causes any disease.So far, the existing 33 eye disease gene therapies research in the whole world enters clinical examination It tests, wherein having 23 clinical test projects is by AAV as gene therapy vector.Gene therapy medicament company, the U.S. It is congenital to carry Leber caused by RPE65 gene therapy RPE65 gene mutation with AAV carrier by SparkTherapeutics The drug of blindness (LCA2), has been approved listing by U.S. FDA.

A variety of retina cells, including photoreceptor cell, retinal pigment epithelium can be infected by having proven to AAV (RPE), M ü ller cell, retinal ganglial cells and endothelial cell.M ü ller cell is in vertebrate retina Most important Deiter's cells, it runs through entire retina, M ü ller cell for maintain the integrality of neuron, metabolism, Homeostasis and signal transduction etc. all have important role.In the past 10 years studies have shown that M ü ller cell either Morphologically still functionally all it is presented with retinal stem cells potential.In retinopathy, M ü ller cell participates in whole A process, and have been found that in the various diseases of retina and reacted with the gliosis of M ü ller cell.Müller Cell also regulates and controls the whole process of retinopathy, neurotransmitter receptor, glutamate receptor, door on M ü ller cell membrane simultaneously Control voltage channel, the trophic factors of synthesized secretion and own amplification differentiation all change.

But view self-maintenance is efficiently targeted about recombination AAV virion there has been no any at present, and be administered Report in terms of M ü ller cell gene therapy retinopathy.

Summary of the invention

Adeno-associated virus (AAV) virion with mutant capsids that the main purpose of the present invention is to provide a kind of, with gram Take deficiency in the prior art.

Another object of the present invention, which also resides in, provides the mutation AAV6 capsid protein comprising aforementioned adeno-associated virus virion.

Another object of the present invention, which also resides in, provides pharmaceutical composition and its view for gene product to be delivered to individual Purposes in the drug of nethike embrane M ü ller cell.

For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:

The adeno-associated virus virion with mutant capsids that the embodiment of the invention provides a kind of, it has single amino acids It is mutated AAV6 capsid protein, the thermophilic retina M ü ller that the adeno-associated virus virion with mutant capsids assigns enhancing is thin The infectivity of born of the same parents.

The embodiment of the invention also provides the composition comprising encoding aforementioned adeno-associated virus virion, it includes:

A) it is mutated AAV6 capsid protein, wherein relative to corresponding parent AAV6 capsid protein, the 663rd in amino acid sequence Mutant serine is leucine.

B) heterologous nucleic acids of the nucleotide sequence comprising encoding gene product (.

The embodiment of the invention also provides a kind of nucleic acid sequences of encoding mutant AAV6 capsid protein, wherein relative to corresponding The 663rd mutant serine is leucine in parent's AAV6 capsid protein amino acid sequence.

The embodiment of the invention also provides a kind of mutation AAV6 capsid proteins, wherein relative to corresponding parent AAV6 capsid egg The 663rd mutant serine is leucine in casamino acid sequence.

The embodiment of the invention also provides the mutation AAV6 capsid proteins comprising encoding aforementioned adeno-associated virus virion The recombinant vector of nucleic acid sequence.

The embodiment of the invention also provides a kind of pharmaceutical compositions, include the adeno-associated virus above-mentioned with mutant capsids Virion and pharmaceutically acceptable excipient.

The embodiment of the invention also provides the adeno-associated virus virion or composition above-mentioned with mutant capsids to be used for Purposes gene product being delivered in the drug of the view self-maintenance of individual.

The embodiment of the invention also provides a kind of product applied to retinal disease treatment method, the method includes to Individual in need applies the adeno-associated virus virion a effective amount of, above-mentioned with mutant capsids.

Compared with prior art, beneficial effects of the present invention at least that:

1) present invention obtains specifical and efficient transduction M ü ller cell by the Amino Acid-Induced Site-Directed Mutation to coding AAV6 capsid AAV carrier, treat retinopathy suitable for carrying exogenous therapeutic gene transduction M ü ller cell；

2) recombination AAV virion is directly injected into vitreous chamber, virus using a kind of safer administration mode by the present invention Suspension diffuses into retina with vitreum.Wild type AAV6 viral vectors, which transduces to gangliocyte by intravitreal, imitates Rate is high, and to retina, other cell transduction rates are relatively low.M ü ller cell runs through entire retina, and regards in view of it to maintenance The important function of the functions such as nethike embrane stable state and metabolism develops a kind of efficient spy by carrying out rite-directed mutagenesis to AAV6 viral capsid The AAV carrier of different transduction M ü ller cell, has great importance for the gene therapy of retinopathy.

Detailed description of the invention

Figure 1A-Fig. 1 D is recombinant plasmid map schematic diagram in the embodiment of the present invention 1 respectively.

Fig. 2 is the purity schematic diagram of SDS-PAGE electrophoresis detection rAAV carrier in the embodiment of the present invention 1.

Fig. 3 is different times living imaging knot after rAAV6S663L-CMVEGFP intravitreal injection in the embodiment of the present invention 1 Fruit schematic diagram.

Fig. 4 is different times tile result after rAAV6-S663L-CMV-EGFP intravitreal injection in the embodiment of the present invention 1 Schematic diagram.

Fig. 5 A- Fig. 5 B is rAAV6-CMV-EGFP, rAAV6-S663L-CMV-EGFP glass in the embodiment of the present invention 1 respectively Different times are sliced result schematic diagram after body injection.

Fig. 6 is rAAV6-S663L-CMV-EGFP, rAAV6-S663L-GFAP-EGFP, rAAV6- in the embodiment of the present invention 1 GS antibody mediated immunity fluorescent staining result schematic diagram after CMV-EGFP intravitreal injection.

Fig. 7 is AAV6-S663L-GFAP-EGFP and AAV6-S663L-CMV-mCherry virus in the embodiment of the present invention 1 Retina luciferase expression result schematic diagram is infected simultaneously.

Specific embodiment

RAAV becomes most promising virus with the advantages that non-pathogenic, low immunogenicity, stable expression target gene and carries Body is widely used in the gene therapy of retinopathy.Therefore, in view of many defects of the prior art, inventor's warp It studies for a long period of time and largely practices, be able to propose technical solution of the present invention, mainly by determining AAV6 viral capsid Point mutation develops a kind of AAV carrier and preparation method thereof of efficient specialized transduction M ü ller cell, for the base of retinopathy Because treatment has great importance.

The definition of some terms referred to below to the present invention is made explanations:

" AAV " is the abbreviation of adeno-associated virus, and can be used for referring to virus itself or derivatives thereof.Unless otherwise needs When, the term includes hypotype and naturally occurring and recombinant forms.Abbreviation " rAAV " refers to recombinant adeno-associated virus, also referred to as recombinates AAV carrier (or " rAAV carrier ").Term " AAV " include 1 type AAV (AAV-1), 2 type AAV (AAV-2), 3 type AAV (AAV-3), 4 type AAV (AAV-4), 5 type AAV (AAV-5), 6 type AAV (AAV-6), 7 type AAV (AAV-7), 8 type AAV (AAV-8), fowl AAV, Ox AAV, dog AAV, horse AAV, primate AAV, non-human primate AAV and sheep AAV." primate AAV " refers to infection primate AAV, " non-human primate AAV " refer to the AAV of infection non-primate mammal, and " ox AAV " refers to the AAV etc. of infected cattle mammal.

As used herein, " rAAV carrier " refers to the polynucleotide sequence comprising the origin non-AAV (that is, with heterologous more of AAV Nucleotide), the usually AAV carrier of the target sequence of genetic transformation of cell.In general, heterologous polynucleotide two sides have at least One, and usually there are two AAV inverted terminal repeat (ITR).Term rAAV carrier covers rAAV carrier granular and rAAV Vector plasmid.RAAV carrier can be single-stranded (ssAAV) or self-complementary (scAAV).

" AAV virus " or " AAV virion " or " rAAV carrier granular " refer to by least one AAV capsid protein (usually For all capsid proteins of wild type AAV) and encapsidate polynucleotides rAAV carrier constitute virion.If the particle Comprising heterologous polynucleotide, (polynucleotides i.e. in addition to wild type AAV genome, such as be delivered to mammalian cell turn base Cause), it is usually referred to as " rAAV carrier granular " or is referred to as " rAAV carrier ".Therefore, the generation of rAAV particle must include The generation of rAAV carrier, because containing this carrier in rAAV particle.

" packaging " guidance causes a series of events intracellular of AAV particle assembly and encapsidate.

" helper virus " of AAV, which refers to, allows mammalian cell to replicate and pack the virus of AAV (such as wild type AAV). It is known in the art a variety of such helper viruses of AAV, including adenovirus, herpesviral and poxvirus (such as cowpox).Though 5 type adenovirus of right C subclass are the most frequently used, but adenovirus covers many different subclass.Known people, non-human mammal and fowl It many adenovirus in class source and can be obtained from repository such as ATCC.The virus of bleb family includes (for example) herpe simplex Viral (HSV) and Epstein-Barr virus (Epstein-Barr viruses) (EBV) and cytomegalovirus (CMV) and virtual genus Dog disease virus (PRV)；It can also be obtained from repository such as ATCC.

" separation " plasmid, nucleic acid, carrier, virus, virion, host cell or other materials refer to not in the substance Or the like naturally occurring or when initially preparation the preparation of the substances of at least some other components that is also likely to be present.Therefore, For example, purifying can be used to count preparative separation substance so that it is enriched with from the mixture of source.Enriched substance can be utterly measured, such as The weight of every bulk solution, or can be measured relative to the second potential interference substance present in the mixture of source.Gradually separating for several times The more and more enriched substances of disclosure embodiment.

As used herein, pharmacology needed for the fingers such as term " treatment " obtain and/or physiological role.It is described to have acted on Fully or partially prevent in terms of disease or its symptom to be preventative and/or to disease and/or be attributable to the pair of the disease It can be therapeutic in terms of the partially or completely healing of effect.As used herein, " treatment " includes to mammal (especially people Class) disease any treatment, and to include: (a) prevention disease easily ill or may have risk but be not yet diagnosed as suffering from Occur in the subject of disease；(b) inhibit disease, that is, prevent its development；(c) alleviate disease, that is, cause disease regression.

The technical solution, its implementation process and principle etc. will be further explained in conjunction with attached drawing as follows.

A kind of adeno-associated virus virion with mutant capsids that the one aspect of the embodiment of the present invention provides, it has Single amino acids are mutated AAV6 capsid protein, and the adeno-associated virus virion with mutant capsids assigns the thermophilic view of enhancing The infectivity of film M ü ller cell.

Further, wherein relative to corresponding parent AAV6 capsid protein, the amino acid of the mutation AAV6 capsid protein The 663rd mutant serine is leucine in sequence.

Further, the virion is thermophilic M ü ller cell.

The other side of the embodiment of the present invention additionally provides the composition comprising encoding aforementioned adeno-associated virus virion, It includes:

A) it is mutated AAV6 capsid protein, wherein relative to corresponding parent AAV6 capsid protein, the 663rd in amino acid sequence Mutant serine is leucine；

B) heterologous nucleic acids of the nucleotide sequence comprising encoding gene product (.

The other side of the embodiment of the present invention additionally provides the mutation comprising encoding aforementioned adeno-associated virus virion The recombinant vector of the nucleic acid sequence of AAV6 capsid protein.

The other side of the embodiment of the present invention additionally provides the nucleic acid sequence comprising encoding mutant AAV6 capsid protein, In relative to the 663rd mutant serine in corresponding parent AAV6 capsid protein amino acid sequence be leucine.

The other side of the embodiment of the present invention additionally provides a kind of mutation AAV6 capsid protein, wherein relative to corresponding parent The 663rd mutant serine is leucine in this AAV6 capsid protein amino acid sequence.

The other side of the embodiment of the present invention additionally provides the recombinant vector comprising aforementioned separation nucleic acid.

Further, the recombinant vector is plasmid.

In some preferred embodiments, the present invention packs necessary condition three plasmids of building according to AAV and (separately includes AAV Genome, AAV mutant capsid protein and replication protein).The skeleton of above three plasmid all derives from pFastbacdual matter Grain (is purchased from invitrogen company), that is, connects destination gene expression box into pFastbacdual by PCR amplification, digestion In the multiple cloning sites (MCS) of plasmid.

First plasmid involved in the present invention, for the plasmid pFastbacdual- for encoding AAV6 mutant capsid protein inCap6S663L.Gene by encoding AAV6Cap albumen carries out rite-directed mutagenesis, its 663rd serine (S) is made to become bright Propylhomoserin (L).According to document (Chen H.Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells.Mol Ther,2008,16(5): 924-930) the synthesis intron sequence is inserted by fusion DNA vaccine method between the bit base of Cap6 sequence 25 and 26, PCR product is connected in the multiple cloning sites (MCS) of pFastbacdual plasmid with BamHI and XbaI enzyme cutting, is obtained pFastbacdual-inCap6.It further, will by fusion DNA vaccine method using pFastbacdual-inCap6 plasmid as template The gene order for encoding the 663rd serine of Cap6 albumen carries out rite-directed mutagenesis so that it becomes leucine.PCR product is taken turns by second It is connected by BamHI and XbaI double digestion in the MSC of carrier pFastba cdual, obtains pFastbacdual-inCap6 S663L。

Second plasmid involved in the present invention is AAV geneome plasmid pFastbacdual-ITR-EGFP, includes AAV Two inverted terminal repeats (ITR) of serotype 2 (AAV2), also include the exogenous gene expression expressed in eukaryocyte Box (including promoter, enhancer, introne, polyA sequence and exogenous gene expression frame include green fluorescence protein gene EGFP etc.).

Third plasmid involved in the present invention, for the plasmid pFastbacdual- for encoding AAV replication protein (Rep) Inrep, the Rep gene expression frame including AAV2, (Li Taiming etc., the insect cell preparation such as intron sequence of Enhanced expressing AAV-ITR gene expression microcarrier, bioengineering journal, 2015,31 (8), page 1232, " the building in 1.2.1 method PFastBacdual-ITR-EGFP plasmid ").

Further, the AAV carrier involved in the present invention produces acquisition in insect cell, specifically includes following step It is rapid:

Firstly, by above three recombinant plasmid pFastbacdual-inCap6S663L/pFastbacdual-ITR- EGFP/pFastbac dual-inrep conventional method converts Escherichia coli DH10Bac competent cell respectively, passes through two-wheeled Blue hickie screening, the bacterium colony comprising restructuring rod granule are white, and the bacterium colony not recombinated is blue, select white colony amplification, Extract restructuring rod granule Bacmid-inCap6S663L/Bacmid-ITR-EGFP/Bacmid-inrep.

Then, with insect cell transfection reagent, respectively by above-mentioned three kinds of restructuring rod granule Bacmid-inCap6S663L/ Bacmid-ITR-EGFP/Bacmid-inrep transfection insect cell Sf9 after 4~5 days, collects cell conditioned medium through 0.22 μm of filter After filtering, P1 is obtained for recombinant baculovirus Baculovirus-inCap6S663L/Baculovirus-ITR-EGFP/ Baculovirus-inrep；P1 is obtained into the rod-shaped disease of P3 generation recombination by the amplification of two subinfection Sf9 cells for recombinant baculovirus Poison.Titre using plaque assay measurement P3 for baculoviral, virus titer (pfu/mL)=1/ extension rate × plaque number × 1/ every hole is inoculated with volume.

Finally, by three kinds of recombinant baculovirus (Baculovirus-inCap6S663L/Baculovirus- in P3 generation ITR-EGFP/Baculovirus-inrep) co-infection Sf9 cell, packaging obtain rAAV6S663L.

And purified with the method for CsCl density gradient centrifugation be concentrated out high concentration recombination AAV virus method, fluorescence The method of quantitative PCR detection rAAV6S663L titre, the method that SDS-PAGE detects rAAV6S663L purity.

The other side of the embodiment of the present invention additionally provides a kind of repairing through separation, heredity comprising separation nucleic acid above-mentioned The host cell of decorations.

The other side of the embodiment of the present invention additionally provides a kind of pharmaceutical composition, has mutant capsids comprising above-mentioned Adeno-associated virus virion and pharmaceutically acceptable excipient.

Further, such excipient, carrier, diluent and buffer include that can apply and any medicine of toxicity without exception Agent.Pharmaceutically acceptable excipient includes but is not limited to liquid, such as water, salt water, glycerol and ethyl alcohol.It wherein may include pharmacy Upper acceptable salt, such as mineral acid salt such as hydrochloride, hydrobromate, phosphate, sulfate etc.；For example with the salt of organic acid Acetate, propionate, malonate, benzoate etc..In addition, auxiliary substance may be present in such medium, such as soak Agent or emulsifier, pH buffer substance etc..Be known in the art diversified pharmaceutically acceptable excipient without It is discussed in detail herein.Pharmaceutically acceptable excipient, including (example are at large described in a variety of publications As) A.Gennaro (2000) " Remington:The Science and Practice of Pharmacy, " the 20th edition, Lippincott,Will iams,&Wilkins；Pharmaceutical Dosage Forms and Drug The editor such as DeliverySystems (1999) H.C.Ansel, the 7th edition, Lippincott, Williams , &Wilkins；With The editor such as Handbookof Pharmaceutical Excipients (2000) A.H.Kibbe, the 3rd edition, Amer.Pharmaceutical Assoc。

The other side of the embodiment of the present invention additionally provides the adeno-associated virus virion above-mentioned with mutant capsids Or composition is used to for gene product being delivered to the purposes in the drug of the view self-maintenance of individual.

The other side of the embodiment of the present invention additionally provides a kind of product applied to retinal disease treatment method, institute The method of stating includes applying the adeno-associated virus virion a effective amount of, above-mentioned with mutant capsids to individual in need.

Further, the method for application of the product is to pass through intraocular injection.

Further, the method for application of the product is to pass through intravitreal injection.

Further, the administration mode involved in the present invention is intravitreal injection, and specific steps include: 4% hydration chlorine Aldehyde 0.01mL/g anesthetized mice, eyes mydriasis keep ocular wet using sodium hyaluronate, and antibiotic ophthalmic liquid medicine, table anaesthetic are preoperative Eye drip.Adjustment mouse head position makes eyeball keep corneal limbus horizontal position.It is punctured at 1mm after corneal limbus using 31G syringe needle, Hamilton33G syringe 1 μ L of injecting virus at puncture.Needle point is vertically into row vitreous chamber is slowly injected, and injection finishes Let the acupuncture needle remain at a certain point afterwards 1min, slowly extracts syringe needle out.Ofloxacin eyedrops, local anti-inflammatory, prevention infection.

In some preferred embodiments, the present invention proves that AAV6S663L mutant targets view by multiple technologies scheme Film M ü ller cell characteristics:

First, pass through the mode of living imaging, inner nuclear layer retina and sections observation EGFP expression and the cell shape of infection Its targeting infection M ü ller cell of state, distribution preliminary judgement.

Different time after second, AAV6S663L mutant intravitreal injection, carry out living imaging, inner nuclear layer retina and Sections observation EGFP expression, expression pattern is constant, illustrates its stable specific infection M ü ller cell.

Third, it is anti-with M ü ller cell-specific proteins glutamine synthelase (Glutamine Synthetase, GS) Body dyes retinal slice, and the green fluorescent protein that can be expressed with viral transduction is overlapped, and further proves viral target To infection M ü ller cell.

4th, building target gene is carried by the recombination AAV6 S663L that M ü ller cell specificity promotor (GFAP) starts Body (AAV6/S663L-GFAP-EGFP), expression pattern and constitutive promoter (CMV) no significant difference, and two are injected simultaneously When kind virus (AAV6/S663L-CMV-mCherry and AAV6/S663L-GFAP-EGFP), two kinds of fluorescence can be overlapped.

To sum up, AAV6S663L mutant intravitreal injection, targeting infection view self-maintenance.

In conclusion the present invention is directly injected into vitreum using a kind of safer administration mode, by recombination AAV virion Chamber, viral suspension diffuse into retina with vitreum.Wild type AAV6 viral vectors is thin to neuromere by intravitreal Dysuria with lower abdominal colic is led high-efficient, and to retina, other cell transduction rates are relatively low.M ü ller cell runs through entire retina, and in view of it The important function for maintaining the functions such as retina stable state and metabolism is obtained by the Amino Acid-Induced Site-Directed Mutation to coding AAV6 capsid The AAV carrier for obtaining specifical and efficient transduction M ü ller cell, is treated suitable for carrying exogenous therapeutic gene transduction M ü ller cell Retinopathy.

Below in conjunction with several preferred embodiments and attached drawing the technical solution of the present invention is further explained explanation, but its In experiment condition and setup parameter be not construed as the limitation to basic technical scheme of the present invention.And protection scope of the present invention It is not limited to the following embodiments.

Embodiment 1

The present embodiment is related to a kind of AAV variant of efficient specialized transduction M ü ller cell, and specific construction method includes such as Lower step:

(1) preparation (please referring to Figure 1A-Fig. 1 D) of recombinant plasmid:

The preparation of A.pFastbacdual-inCap6S663L

According to document (Chen H.Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells.Mol Ther,2008,16(5): 924-930) the synthesis intron sequence is inserted by fusion DNA vaccine method between the bit base of Cap6 sequence 25 and 26, PCR product is connected in the multiple cloning sites (MCS) of pFastbacdual plasmid with BamHI and XbaI enzyme cutting, is obtained pFastbacdual-inCap6.It further, will by fusion DNA vaccine method using pFastbacdual-inCap6 plasmid as template The gene order for encoding the 663rd serine of Cap6 albumen carries out rite-directed mutagenesis so that it becomes leucine.Pass through fusion DNA vaccine method Rite-directed mutagenesis is carried out, two-wheeled PCR reaction need to be carried out, wherein when first round PCR, in pFastbacdual-inCap6 plasmid For inCap gene as template, first round PCR is related to two reaction systems, wherein the 3' primer and second segment sequence of first segment sequence The 5' primer of column has one section of complementary region；Then all products of first round PCR are added in a reaction system as template, the The 5' primer of one section of sequence and the 3' primer of final stage sequence do primer, carry out second and expand, form fusion sequence.It will melt PCR product is closed to carry out by DNA purification kit after purification, with BamHI and XbaI enzyme cutting, carrier pFastbacdual- InCap6 also uses BamHI and XbaI enzyme cutting, and the two is connected by T4DNA Ligase, and conversion Escherichia coli Top10 competence is thin Born of the same parents, the amplification of picking monoclonal colonies are extracted plasmid and are identified by digestion, obtain pFastbacdual-inCap6S663L.This matter Grain further includes other elements needed for expressing capsid protein in eukaryocyte, including promoter Polyhedrin promoter (P_PH), SV40 polyade nylation signal sequence etc..

B.pFastbacdual-ITR-EGFP、pFastbacdual-ITR-GFAP-EGFP、pFastbacdual-ITR- The preparation of CMV-mCherry

PFastbacdual-ITR-EGFP plasmid includes that two ITR of AAV2 and CMV promoter, beta-globin are included Son, coding EGFP gene, hGH polyA sequence save (bibliography: Li Taiming etc., insect cell preparation by laboratory building AAV-ITR gene expression microcarrier, bioengineering journal, 2015,31 (8), page 1232, " the building in 1.2.1 method PFastBacdual-ITR-EGFP plasmid ").

Using pFastbacdual-ITR-EGFP as framework construction pFastbacdual-ITR-GFAP-EGFP, specific steps As follows: GFAP promoter sequence is synthesized by Suzhou Jin Weizhi Biotechnology Co., Ltd, and upstream has MluI restriction enzyme site, downstream With EcoRI restriction enzyme site, digestion pFastbacdual-ITR-CMV-EGFP and GFAP segment is distinguished using MluI and EcoRI, Recovery purifying, T4DNA ligase connect the two to arrive pFastbacdual-ITR-GFAP-EGFP.

It is specific to walk using pFastbacdual-ITR-EGFP as framework construction pFastbacdual-ITR-CMV-mCherry Rapid as follows: mCherry sequence is obtained from plasmid pAAV-CaMKIIa-hChR2-mCherry amplification, and upstream has BamHI digestion Site, downstream have XhoI restriction enzyme site, using BamHI and XhoI difference digestion pFastbacdual-ITR-EGFP and MCherry segment, recovery purifying, T4DNA ligase connect the two to arrive pFastbacdual-ITR-CMV- mCherry。

The preparation of C.pFastbacdual-inrep plasmid

PFastbacdual-inrep plasmid includes AAV2rep gene, intron sequence, is expressed by pFastbacdual P10 promoter and the regulation of HSV tk polyA element on plasmid vector, are constructed by laboratory and save (bibliography: Li Taiming Deng, insect cell preparation AAV-ITR gene expression microcarrier, bioengineering journal, 2015,31 (8), page 1232,1.2.2 method In " building pFastBacdual-inrep plasmid ").

(2) preparation of Bacmid is recombinated

Three recombinant plasmids of previous step are distinguished into preparation and reorganization Bacmid, the specific method is as follows:

1) slowly melt 100 μ L DH10Bac competence on ice.

2) 50ng Plasmid DNA is added, mixes gently.

3) 30min is placed on ice, and 42 DEG C of heat shock 90s are transferred to place 2min on ice immediately.

4) 900 μ L SOC culture mediums are added, 37 DEG C of 225rpm shake 4h.

5) contain 50 μ g/mL kanamycins (Kan), 7 μ g/mL gentamicins (Gen), 10 μ g/mL tetracyclines (Tet) it is pre- The Blue-gal and 7 μ L 20% (200mg/mL) IPTG of 40 μ L 2% (20mg/mL) is added dropwise in the 90mm agar plate center of system.Make It is allowed to be scattered in culture plate whole surface with sterile spreader, in incubation at room temperature until whole liquid disappear.

6) 10 times of gradient dilution cells (10 of SOC culture medium are used^-1, 10^-2, 10^-3), each gradient takes 100 μ L to apply LB plate.

7) 37 DEG C of placement 48h, 10 white colonies of picking dip on new LB agar plate (ibid resistance), 37 DEG C of mistakes Night.The hickie of picking confirmation, is inoculated into LB liquid medium (containing 50 μ g/mL kanamycins (Kan), 7 μ g/mL gentamicins (Gen), 10 μ g/mL tetracycline (Tet)).

8) it stands overnight for 4 DEG C, makes blue sufficiently colour developing during this period.

9) hickie can be carried out that Sf9 can be transfected after PCR identification is correct.

10) using the bacmid dna of OMEGA kit extracting separation recombination, experimental method is referring to kit specification, measurement Freeze after being dispensed after rod granule concentration in -20 DEG C, avoids multigelation.

11) PCR identifies that Bacmid, the primer are respectively as follows: 5 '-CCC AGT CAC GAC GTT GTA of upstream primer AAA CG-3 ', 5 '-GCT CTA GAT TAC TTG TAC AGC TCG TCC AT-3 ' of downstream primer.

12) it takes out and identifies correct Bacmid strain, be inoculated in 3mL LB (Kan+, Gen+, Tet according to the ratio of 1:300 +) shake bacterium 12h.Then be inoculated in 150mL LB (Kan+, Gen+, Tet+) according to the ratio of 1:100 and shake bacterium 16h, referring to it is large-scale/ A large amount of plasmid extraction kit specifications largely extract Bacmid, in case transfection cell prepares baculoviral.

(3) preparation of baculoviral

1. Sf9 cell culture.Sf9 cell mentions the previous day and spreads six orifice plates, 50% hole density, using complete medium, 95% survival degree.Bacmid DNA warm bath 20min inside 70 DEG C of water-baths will be recombinated, 12000g centrifugation 10min takes supernatant.

2. plating cells.

Ensure cell density 1.5~2.5 × 10⁶(culture medium is free of antibiotic) is operated when cells/mL.Add 2mL Additive-free basis (Grace) culture medium (without antibiotic and serum) is into 6 orifice plates.Inoculation 8 × 10⁵Cells/mL step Sf9 (do not replace culture medium and wash cell) in 1, allows the adherent 15min of cell room temperature.

3. preparing transfection reagent.

A) mixing transfection reagent II, 8 μ L of addition to 92 μ L additive-free basal medium (without antibiotic and serum), It is vortexed and mixes.

B) take 5 μ L bacmid dnas (500ng/ μ L guarantees that the amount of rod granule is 2~3 μ g) to the additive-free basis culture of 95 μ L Base (without antibiotic and serum), mixes gently.

C) above-mentioned two solution is mixed, is incubated at room temperature 30min.

4. above-mentioned DNA-Lipid mixture is added dropwise into the hole for being covered with cell, 27 DEG C of incubated cell 5h.

5. removing culture medium in plate, 2mL complete medium is changed.

6. observing virus infection sign in 27 DEG C of incubation 72h.

Separate P1:

Confirmation cell is in after the later infections stage (4~5 days after usually transfecting), and training of the 2mL containing virus is collected in every hole Base is supported into sterile 15mL centrifuge tube, 1000g is centrifuged 5min and removes cell fragment.

Supernatant is filled into sterile 15mL centrifuge tube through 0.22 μm of filter, and 4 DEG C are kept in dark place.If thinking long-term preservation, Packing is frozen in -80 DEG C.

Amplification virus:

Taking MOI is the cell of 0.05~0.1,10mL suspension culture, and density is 2 × 10⁶cells/mL；Or in 6 orifice plates Cell, density be 2 × 10⁶The hole cells/ calculates required P1 volume.

1. Sf9 cell six orifice plates of paving, 2 × 10⁶The hole cells/.Being placed at room temperature for 1h makes its attaching, microscopically observation.

2. suitable P1,27 DEG C of culture 48h~72h is added in every hole.

3. culture of the 2mL containing virus is collected based in sterile 15mL centrifuge tube in every hole, 1000g is centrifuged 5min.

4. shifting supernatant into sterile 15mL centrifuge tube, which is P2.4 DEG C are kept in dark place, if thinking to protect for a long time It deposits, packing is frozen in -80 DEG C.

5. can expand to obtain P3 according to the above method, (the P1 virus titer generally yielded is 1 × 10⁶~1 × 10⁷Between, P2 drop Degree is 1 × 10⁷~1 × 10⁸Between).

Virus titer is measured with plaque assay.Detailed experimental steps are as follows:

1. the hole 2mL/ cell (5 × 105cells/mL) is planted into 6 orifice plates, being incubated at room temperature 1h keeps its adherent, after incubation microscopy its Adherent degree.

2. 4%agarose gel is put into 70 DEG C of water-baths to melt, 2 × Grace and 100mL aseptic bottle is put into 40 The preheating of DEG C water-bath.

3. baculoviral is carried out gradient dilution with serum-free basal medium: 10^-1~10^-8。

4. abandoning supernatant in 6 orifice plates, the virus diluted is rapidly joined, the hole 1mL/ (multiple holes) are incubated at room temperature 1h.

5. configuring top-layer agar, 20mL high-temperature inactivation FBS to 2 × Grace 100mL, 2 × Grace of 25mL is added (to contain FBS) 4% Ago-Gel of+12.5mL sterile water+12.5mL, until the 100mL aseptic bottle of preheating, mixes gently, be put into 37 DEG C of water-baths Pot is spare.

6. abandoning supernatant in 6 orifice plates, quickly add 2mL top-layer agar, dry with fungi-proofing layer, standing 10~20min makes its solidification. 6 orifice plates are put into 27 DEG C of incubators, are cultivated 5 days.

7. preparing 1mg/mL neutral red solution, in basic complete medium, it is sterile filtered.

8. the above-mentioned solution of 1.5mL, the basis 16.5mL complete medium, the agar of 6mL 4% are configured to dimethyl diaminophenazine chloride upper layer Agar.

9. adding 1mL dimethyl diaminophenazine chloride top-layer agar after virus infection 4 days.

10. continuing to be put into incubator, it is observable plaque after 4~5 days, counts the quantity of plaque, acquire viral drop Degree.

Note: the every hole in virus titer (pfu/mL)=1/ extension rate × plaque number × 1/ is inoculated with volume

(4) preparation and purifying of rAAV carrier

The purity schematic diagram of SDS-PAGE electrophoresis detection rAAV carrier sees Fig. 2 in the present embodiment, wherein A is represented RAAV6-CMV-EGFP, B represent rAAV6-S663L-CMV-EGFP, and C represents rAAV6-S663L-GFAP-EGFP.

(5) mouse intravitreal injection method

Specific steps are as follows:

4% chloraldurate 0.01ml/g anesthetized mice, eyes mydriasis keep ocular wet, antibiotic using sodium hyaluronate Eyedrops, the preoperative eye drip of table anaesthetic.Adjustment mouse head position makes eyeball keep corneal limbus horizontal position.Using 31G syringe needle in corneal limbus It is punctured at 1mm afterwards, Hamilton (Hamilton) 33G syringe 1 μ l of injecting virus at puncture.Needle point is vertically into then Inclination, is slowly injected, let the acupuncture needle remain at a certain point after pushing pin 0.5-1min, rapidly needle out.

(6) living imaging method

After injecting virus carrier 1-5, intraperitoneal injection of anesthesia mouse is carried out with chloraldurate weekly.It is dripped with Tropicamide and Phenylephrine The expansion of ocular fluid progress pupil.Corneal dehydration in order to prevent, with sodium hyaluronate eye drops moisturizing.It is scanned using line and is copolymerized burnt eyeground Imaging system carries out fundus imaging, and the solid-state laser of 488nm excites GFP.Eyes are carried out with sodium hyaluronate eye drops again after imaging Moisturizing.

Different times living imaging result schematic diagram is asked after rAAV6S663L-CMVEGFP intravitreal injection in the present embodiment Refering to Fig. 3, rAAV6S663L-CMVEGFP is observed by living imaging and infects postretinal EGFP expression pattern, it is main to be distributed Around blood vessel, and circumvascular main cell is M ü ller cell.

(7) inner nuclear layer retina is extracted and is expressed in retina with Laser Scanning Confocal Microscope detection virus

Intravitreal injection after two weeks, by mouse break neck put to death, take eyeball to be directly placed into 4% paraformaldehyde (Paraformaldehyde, PFA), 4 DEG C of fixed 2h.Eyeball is put into PBS after fixation and is rinsed, is placed in culture dish, is cut off Optic nerve is open at cornea, cornea is wiped out.Sclera and train of thought film composite are torn along opening, exposes cup shaped transparent view Film removes crystalline lens, removes residual glass body.Retina is transferred in new PBS, 4 valves are symmetrically cut off.PBS is sucked, is inhaled with paper It is dry.Retina shape is adjusted, shifts retina to glass slide, covered is observed under Laser Scanning Confocal Microscope, and selection is aobvious Cell deck structure can be observed in micro mirror multiple 20 × 10.

It is observed after rAAV6-S663L-CMV-EGFP infects retina by inner nuclear layer retina and is predominantly targeting M ü around blood vessel Ller cell, and wild type rAAV6-CMV-EGFP is predominantly targeting gangliocyte.

Different times tile result schematic diagram please join after rAAV6-S663L-CMV-EGFP intravitreal injection in the present embodiment Fig. 4 is read, is observed after rAAV6-S663L-CMV-EGFP infects retina by inner nuclear layer retina and is predominantly targeting M ü around blood vessel Ller cell, and wild type rAAV6-CMV-EGFP is predominantly targeting gangliocyte.

(8) frozen section and DAPI are dyed

The mouse neck that breaks is put to death, and is taken out eyeball, is immersed 4%PFA, 4 DEG C of fixed 2h, is open at cornea, immersion 4%PFA, and 4 DEG C Overnight.PFA, 20% 4 DEG C of sucrose dehydration 12h are eliminated, 30% 4 DEG C of sucrose is dehydrated more than for 24 hours.Take out the immersion of 30% sucrose solution Eye clamps corneal moieties, removes cornea, crystalline lens, blots sucrose with paper.The pre-cooling such as tweezers, paper, knife.A small amount of OCT is instilled, it will Eye is disposed vertically in plastic lid, removes bubble removing, is put into slicer freezing, is vertically established after keeping freezing, continues to instill OCT embedding Firmly entire eye.One circle OCT of drop, sample are placed on specimen disc on the specimen disc of pre-cooling, the fixed entire sample of OCT, pocket knife finishing Size.Specimen disc insertion sample head, adjustment knife rest and anti-roll bending, coarse adjustment photo fix, after cutting out tissue, 14 μm of fine cut.After cutting out Slice sticker in glass slide, be packed into slide holding frame.0.01M PBS impregnates glass slide, and 2h is dried in ventilation.

Slide holding frame is taken out, is put into the reparation box for containing 0.01M PBS, is dried surrounding, enclosed with immunohistochemistry pen and be sliced four Week is cleaned after oil droplet is dry with 0.01M PBS.It is added DAPI (Sigma company) (1:1000PBS), room temperature 20min.0.01M PBS is cleaned 3 times.Keep slice wet, inverted fluorescence microscope observation.

Different times are sliced after rAAV6-CMV-EGFP, rAAV6-S663L-CMV-EGFP intravitreal injection in the present embodiment Result schematic diagram please refers to Fig. 5 A- Fig. 5 B.Observing rAAV6-S663L-CMV-EGFP infection retina by retinal slice is Through the M ü ller cellular morphology of entire retina, and wild type rAAV6-CMV-EGFP main infection position is that neuromere is thin Born of the same parents.

(9) histotomy immunofluorescence dyeing:

1) slide holding frame is taken out, is put into the reparation box for containing 0.01M PBS or pasteur pipet cleans.

2) it takes out and only dries surrounding, enclose surrounding with immunohistochemistry pen.

3) oil droplet is dry after a few minutes, and 0.01PBS cleaning is added.

4) it is added and contains 3% lowlenthal serum confining liquid, close 60min or more.

5) most of serum block is absorbed, then is washed one time with 0.01M PBST.

6) the diluted primary antibody anti-GS monoclonal antibody (1:100) of confining liquid is added, 4 DEG C of 16~18h/ are overnight.

7) 0.01M PBST is cleaned 4 times, every time 5 minutes.

8) diluted secondary antibody goat anti-mouse IgG (Alexa Flour 594) (1:100) is added, is incubated at room temperature 2h.

9) 0.01M PBST is cleaned 4 times, every time 5 minutes.

10) DAPI (1:1000PBS) is added, is incubated at room temperature 20min.

11) 0.01M PBS is cleaned 3 times, and it is wet that last time PBS does not discard holding slice.

(10) specificity promoter luciferase expression:

The recombination AAV6S663L carrier that building target gene is started by M ü ller cell specificity promotor (GFAP) (AAV6-S663L-GFAP-EGFP), theoretically, can only in M ü ller cell initial EGFP protein expression, and by slice dye Color result sees that AAV6-S663L-GFAP-EGFP can express green fluorescence in M ü ller cell, illustrates that AAV6S663L can Infect M ü ller cell.Fig. 6 show rAAV6-S663L-CMV-EGFP, rAAV6-S663L-GFAP-EGFP in the present embodiment, GS antibody mediated immunity fluorescent staining result schematic diagram after rAAV6-CMV-EGFP intravitreal injection.

Respectively with viral AAV6-S663L-GFAP-EGFP and the carrying for carrying specificity promoter and green fluorescent protein The viral AAV6-S663L-CMV-mCherry of constitutive promoter and mCherry red fluorescent protein mixing after simultaneously vitreum Injection, Fig. 7 show AAV6-S663L-GFAP-EGFP and AAV6-S663L-CMV-mCherry virus while infecting retina Luciferase expression result schematic diagram, Fig. 7 show that two kinds of viruses express overlapping in retina, illustrate that AAV6S663L targets M ü ller Cell.

As seen from the above embodiment, the present invention is obtained special high by the Amino Acid-Induced Site-Directed Mutation to coding AAV6 capsid The AAV carrier of effect transduction M ü ller cell, treats retinopathy suitable for carrying exogenous therapeutic gene transduction M ü ller cell Become.

Finally, it is to be noted that, the terms "include", "comprise" or its any other variant be intended to it is non-exclusive Property include so that include a series of elements process, method, article or equipment not only include those elements, but also Further include other elements that are not explicitly listed, or further include for this process, method, article or equipment it is intrinsic Element.

It will be appreciated by those skilled in the art that the above described specific embodiments of the present invention, are not constituted to the present invention The restriction of protection scope.Any any other various changes and modifications in accordance with the technical idea of the present invention, should all Comprising within the scope of the invention as claimed.

Claims (10)

1. a kind of adeno-associated virus virion with mutant capsids, which is characterized in that there are single amino acids to be mutated AAV6 for it Capsid protein, the adeno-associated virus virion with mutant capsids assign the infection of the thermophilic view self-maintenance of enhancing Property.

2. the composition comprising adeno-associated virus virion described in coding claim 1, it includes:

A) it is mutated AAV6 capsid protein, wherein relative to corresponding parent AAV6 capsid protein, the 663rd silk ammonia in amino acid sequence Acid mutation is leucine；

B) heterologous nucleic acids of the nucleotide sequence comprising encoding gene product (.

3. a kind of nucleic acid sequence of encoding mutant AAV6 capsid protein, wherein relative to corresponding parent AAV6 capsid protein amino acid The 663rd mutant serine is leucine in sequence.

4. a kind of mutation AAV6 capsid protein, wherein relative to the 663rd in corresponding parent AAV6 capsid protein amino acid sequence Mutant serine is leucine.

5. the recombination of the nucleic acid sequence of the mutation AAV6 capsid protein comprising adeno-associated virus virion described in coding claim 1 Carrier.

6. a kind of pharmaceutical composition includes the adeno-associated virus virion and pharmacy described in claim 1 with mutant capsids Upper acceptable excipient.

7. the adeno-associated virus virion with mutant capsids or combination as claimed in claim 6 as described in claim 1 Object is used to for gene product being delivered to the purposes in the drug of the view self-maintenance of individual.

8. a kind of product applied to retinal disease treatment method, the method includes applying effective quantity to individual in need The adeno-associated virus virion with mutant capsids as described in claim 1.

9. product according to claim 8, it is characterised in that: the method for application of the product is to pass through intraocular injection.

10. product according to claim 9, it is characterised in that: the method for application of the product is by infusing in vitreum It penetrates.