CN109897831B - Adeno-associated virus virions with mutant capsids and uses thereof - Google Patents

Abstract

The invention discloses an adeno-associated virus virion with mutant capsid and application thereof. The adeno-associated virus virions with a mutated capsid have a mutated AAV6 capsid protein, which confers enhanced infectivity of retinotropic Muller cells. Wherein the 663 th serine in the amino acid sequence of the mutant AAV6 capsid protein is mutated to leucine relative to the corresponding parental AAV6 capsid protein. The pharmaceutical composition comprises the adeno-associated virus virion and a pharmaceutically acceptable excipient. The invention obtains the specific AAV vector for efficiently transducing Muller cells by site-directed mutagenesis of the amino acids encoding AAV6 capsid, and is suitable for a method for treating retinopathy by transducing Muller cells with exogenous therapeutic genes.

Description

Adeno-associated virus virions with mutant capsids and uses thereof

Technical Field

The invention relates to a recombinant AAV virion, in particular to a method for giving targeted retina Muller cells by single-point amino acid mutation in a selected adeno-associated virus (AAV) sequence and application in treating retinopathy by carrying exogenous therapeutic genes to transduce the Muller cells, belonging to the technical field of genetic engineering.

Background

Retinal diseases are one of the main causes of blindness, and macular degeneration, diabetic retinopathy, glaucoma, hereditary retinopathy and the like are common. Most of the lesions are abnormal due to gene mutation, protein non-operation or over-expression, and then visual cell death and finally blindness are caused. The correct gene can be mediated or the abnormal gene can be knocked out through gene therapy, the normal expression is recovered, and the visual function recovery effect is further achieved.

Adeno-associated virus (AAV), a member of the genus dependovirus of the family parvoviridae, is an icosahedron with a diameter of about 20-26 nm, carrying a linear single-stranded DNA genome of 4.6-4.8 kb. AAV viruses of 13 serotypes and over 120 mutants have been isolated from many mammals, including humans. AAV as a gene vector has advantages in gene therapy applications not possessed by other viral or non-viral vectors: (1) can infect both dividing cells and resting cells; (2) AAV is the only known gene vector which can be integrated to a specific site of a human genome at a fixed point, and the integrated site is safe and reliable; most importantly, although 80-90% of humans are positive for AAV, AAV has not been reported to cause any disease. To date, 33 gene therapy studies of ocular diseases have been conducted in clinical trials worldwide, and 23 clinical trials have been conducted with AAV as a gene therapy vector. American Gene therapy medicine company Spark therapeutics, and a medicament for treating Leber congenital amaurosis (LCA2) caused by mutation of RPE65 gene by using AAV vector carrying RPE65 gene, has been approved to be marketed by the American FDA.

AAV has been shown to infect a variety of retinal cells, including photoreceptor cells, retinal pigment epithelial cells (RPE), Muller cells, retinal ganglion cells, and corneal endothelial cells. Muller cells are the most prominent glial cells in vertebrate retina, which penetrate the entire retina and play an important role in maintaining neuronal integrity, metabolism, homeostasis, and signal transduction. Recent 10 years of research have shown that Muller cells exhibit retinal stem cell potential, both morphologically and functionally. In retinopathy, Muller cells are involved in the entire process, and gliosis reactions with Muller cells are found in various diseases of the retina. Muller cells also regulate the entire process of retinopathy, with alterations in neurotransmitter receptors, glutamate receptors, gated voltage channels, synthesized secreted trophic factors, and self-proliferative differentiation on Muller cell membranes.

However, there are no reports of the efficient targeting of recombinant AAV virions to retinal Muller cells and the gene therapy of retinopathy by administering to Muller cells.

Disclosure of Invention

It is a primary object of the present invention to provide adeno-associated virus (AAV) virions with mutated capsids that overcome the deficiencies of the prior art.

It is also an object of the present invention to provide a mutant AAV6 capsid protein comprising the aforementioned adeno-associated viral virion.

It is also an object of the invention to provide pharmaceutical compositions and their use in medicaments for delivering a gene product to retinal muller cells of an individual.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiments of the present invention provide an adeno-associated viral virion with a mutated capsid, having a single amino acid mutated AAV6 capsid protein, which confers enhanced infectivity of retinotropic muller cells.

Embodiments of the present invention also provide compositions comprising an antibody encoding the aforementioned adeno-associated virus virions, comprising:

a) mutating the AAV6 capsid protein, wherein serine at position 663 in the amino acid sequence is mutated to leucine relative to the corresponding parental AAV6 capsid protein.

b) A heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

The embodiment of the invention also provides a nucleic acid sequence for coding the mutant AAV6 capsid protein, wherein the 663 th serine in the amino acid sequence of the capsid protein is mutated into leucine relative to the corresponding parental AAV 6.

The embodiment of the invention also provides a mutant AAV6 capsid protein, wherein the 663 th serine in the amino acid sequence of the capsid protein relative to the corresponding parental AAV6 is mutated into leucine.

The embodiments also provide recombinant vectors comprising a nucleic acid sequence encoding the mutated AAV6 capsid protein of the aforementioned adeno-associated virus virion.

The embodiments of the present invention also provide a pharmaceutical composition comprising the aforementioned adeno-associated virus virion with a mutated capsid, and a pharmaceutically acceptable excipient.

The embodiments of the present invention also provide the use of the aforementioned adeno-associated virus virions or compositions with a mutated capsid in the manufacture of a medicament for delivery of a gene product to retinal muller cells of an individual.

The present embodiments also provide a product for use in a method of treating a retinal disease comprising administering to an individual in need thereof an effective amount of the aforementioned adeno-associated viral virions with a mutated capsid.

Compared with the prior art, the invention has the beneficial effects that:

1) the invention obtains the AAV vector for specifically and efficiently transducing Muller cells by site-directed mutagenesis of the amino acid encoding AAV6 capsid, and is suitable for carrying exogenous therapeutic genes to transduce Muller cells to treat retinopathy;

2) the invention adopts a safer administration mode, the recombinant AAV virions are directly injected into the vitreous cavity, and the virus suspension diffuses into the retina along with the vitreous body. Wild type AAV6 viral vectors transduce ganglion cells efficiently and relatively poorly to other cells of the retina by intravitreal injection. The Muller cells penetrate through the whole retina, and in view of the important function of the Muller cells on maintaining the functions of retinal homeostasis, metabolism and the like, the AAV vector for efficiently and specifically transducing the Muller cells is developed by performing site-directed mutagenesis on the AAV6 virus capsid, and has important significance on gene therapy of retinopathy.

Drawings

FIGS. 1A to 1D are schematic diagrams of maps of recombinant plasmids in example 1 of the present invention, respectively.

FIG. 2 is a schematic diagram of the SDS-PAGE electrophoresis for detecting the purity of the rAAV vector in example 1 of the present invention.

FIG. 3 is a schematic diagram of the in vivo imaging results of different periods after the injection of the rAAV6S663L-CMVEGFP vitreous body in example 1 of the present invention.

FIG. 4 is a schematic diagram showing the results of different time-courses of slide-laying after the injection of rAAV6-S663L-CMV-EGFP vitreous body in example 1 of the present invention.

FIGS. 5A-5B are schematic views of the results of slicing at different stages after injection of the rAAV6-CMV-EGFP and rAAV6-S663L-CMV-EGFP vitreum in example 1 of the present invention, respectively.

FIG. 6 is a schematic diagram showing the result of GS antibody immunofluorescence staining after vitreous injection of rAAV6-S663L-CMV-EGFP, rAAV6-S663L-GFAP-EGFP and rAAV6-CMV-EGFP in example 1 of the present invention.

FIG. 7 is a schematic diagram showing the fluorescence expression results of the simultaneous infection of AAV6-S663L-GFAP-EGFP and AAV6-S663L-CMV-mCherry virus in example 1 of the present invention on retina.

Detailed Description

rAAV becomes the most promising virus vector with the advantages of nonpathogenicity, low immunogenicity, stable expression of target genes and the like, and is widely applied to gene therapy of retinopathy. Therefore, in view of the defects of the prior art, the present inventors have made extensive studies and extensive practices to provide the technical solution of the present invention, which mainly develops an AAV vector for efficiently and specifically transducing muller cells by performing site-specific mutagenesis on the capsid of AAV6 virus and a preparation method thereof, and has important significance for gene therapy of retinopathy.

The following definitions of some terms mentioned in the present invention are explained:

"AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or derivatives thereof. Unless otherwise required, the term includes subtypes and naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV infecting primates, "non-primate AAV" refers to AAV infecting non-primate mammals, "bovine AAV" refers to AAV infecting bovine mammals, and the like.

As used herein, "rAAV vector" refers to an AAV vector comprising a polynucleotide sequence of non-AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and typically two AAV Inverted Terminal Repeats (ITRs). The term rAAV vector encompasses rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single stranded (ssav) or self-complementary (scAAV).

An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically all capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene delivered to a mammalian cell), it is often referred to as a "rAAV vector particle" or simply as a "rAAV vector". Thus, production of rAAV particles necessarily includes production of rAAV vectors, as such vectors are contained within rAAV particles.

"packaging" refers to a series of intracellular events that lead to the assembly and encapsidation of AAV particles.

A "helper virus" of AAV refers to a virus that allows a mammalian cell to replicate and package AAV (e.g., wild-type AAV). A variety of such helper viruses for AAV are known in the art, including adenovirus, herpesvirus, and poxvirus (e.g., vaccinia). Although adenovirus type 5 of subgroup C is most commonly used, adenoviruses encompass many different subgroups. Many adenoviruses of human, non-human mammalian and avian origin are known and available from stores such as the ATCC. Viruses of the herpes family include, for example, Herpes Simplex Virus (HSV) and Epstein-Barr virus (EBV) as well as Cytomegalovirus (CMV) and pseudorabies virus (PRV); also available from depositories such as ATCC.

An "isolated" plasmid, nucleic acid, vector, virus, virosome, host cell or other substance refers to a preparation of a substance that is free of at least some other components that may be present in the substance or similar substance in nature or when originally prepared. Thus, for example, the isolated material can be prepared using purification counts to enrich it from the source mixture. The enrichment may be measured absolutely, for example, weight per volume of solution, or may be measured relative to the presence of a second potentially interfering species in the source mixture. More and more enrichments of embodiments of the disclosure are separated in stages.

As used herein, the terms "treat," "treating," and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or a symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or a side effect attributable to the disease. As used herein, "treatment" includes any treatment of a disease in a mammal (particularly a human) and includes: (a) preventing the occurrence of a disease in a subject who may be susceptible to or at risk of developing the disease but has not yet been diagnosed as diseased; (b) inhibiting the disease, i.e. arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The technical solution, the implementation process and the principle thereof will be further explained with reference to the drawings.

An aspect of the embodiments of the present invention provides an adeno-associated virus virion with a mutant capsid having a single amino acid mutation of the AAV6 capsid protein, which adeno-associated virus virion with a mutant capsid confers enhanced infectivity of a retinophilippiner cell.

Further, wherein the amino acid sequence of the mutant AAV6 capsid protein has a mutation of serine at position 663 to leucine relative to the corresponding parental AAV6 capsid protein.

Further, the virion is of a muller cell.

Yet another aspect of embodiments of the present invention provides a composition comprising an adenovirus-encoding virion as defined above, comprising:

a) mutating the AAV6 capsid protein, wherein serine at position 663 in the amino acid sequence is mutated to leucine relative to the corresponding parental AAV6 capsid protein;

b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

In another aspect of the embodiments of the invention, there is also provided a recombinant vector comprising a nucleic acid sequence encoding the mutated AAV6 capsid protein of the aforementioned adeno-associated virus virion.

Yet another aspect of the embodiments of the invention provides a nucleic acid sequence comprising a nucleic acid sequence encoding a mutant AAV6 capsid protein, wherein serine at position 663 is mutated to leucine with respect to the amino acid sequence of the corresponding parental AAV6 capsid protein.

In another aspect of the embodiments of the present invention, there is provided a mutant AAV6 capsid protein, wherein serine at position 663 in the amino acid sequence relative to the corresponding parental AAV6 capsid protein is mutated to leucine.

In another aspect of the embodiments of the present invention there is also provided a recombinant vector comprising the aforementioned isolated nucleic acid.

Further, the recombinant vector is a plasmid.

In some preferred embodiments, the invention constructs three plasmids (comprising the AAV genome, AAV mutant capsid protein, and replication protein, respectively) according to AAV packaging requirements. The skeletons of the three plasmids are all derived from pFastbacadual plasmid (purchased from invitrogen), namely, a target gene expression cassette is amplified by PCR and cut into a Multiple Cloning Site (MCS) of the pFastbacadual plasmid by enzyme.

The first plasmid involved in the present invention is plasmid pFastbacadual-inCap 6S663L encoding AAV6 mutant capsid protein. The gene coding AAV6Cap protein is subjected to site-directed mutagenesis to change serine (S) at position 663 to leucine (L). Intron sequences were synthesized as described in the literature (Chen H. Intron cleavage-mediated expression of AAV Rep and Cap genes and production of AAV vectors in infection cells. mol Ther,2008,16(5): 924) 930, and were inserted between bases 25 and 26 of Cap6 sequence by fusion PCR method, and the PCR product was digested with BamHI and XbaI into the Multiple Cloning Site (MCS) ligated to pFastbadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadadaduP 6. Furthermore, the gene sequence coding the 663 serine of the Cap6 protein was site-directed mutated to leucine by fusion PCR method using pFastbacadual-inCap 6 plasmid as template. The second round of PCR product was ligated into MSC of vector pFastba cdual by double digestion with BamHI and XbaI to obtain pFastbacadual-inCap 6S 663L.

The second plasmid related in the invention is AAV genome plasmid pFastbacual-ITR-EGFP, which comprises two inverted terminal repeat sequences (ITR) of AAV serotype 2(AAV2) and also comprises an exogenous gene expression cassette (comprising a promoter, an enhancer, an intron and a polyA sequence, wherein the exogenous gene expression cassette comprises a green fluorescent protein gene EGFP and the like) expressed in eukaryotic cells.

The third plasmid related in the invention is a plasmid pFastbinary-inrep for coding AAV replication protein (Rep), which comprises a Rep gene expression frame of AAV2, an intron sequence for enhancing expression and the like (Litamine and the like, an AAV-ITR gene expression microcarrier prepared by insect cells, a report of bioengineering, 2015,31(8), page 1232, and a method of 'constructing pFastbinary-ITR-EGFP plasmid' in 1.2.1).

Further, the AAV vectors involved in the invention are produced in insect cells, and specifically comprise the following steps:

firstly, respectively transforming the three recombinant plasmids pFastbacal-inCap 6S 663L/pFastbacal-ITR-EGFP/pFastbacal dual-inrep into escherichia coli DH10Bac competent cells by a conventional method, screening the competent cells through two blue-white spots, selecting white colonies containing recombinant Bacmid-inCap6S663L/Bacmid-ITR-EGFP/Bacmid-inrep, amplifying the white colonies, and extracting the recombinant Bacmid-inCap6S 663L/Bacmid-ITR-EGFP/Bacmid-inrep.

Then, transfecting the three recombinant Bacmid-inCap6S663L/Bacmid-ITR-EGFP/Bacmid-inrep with an insect cell transfection reagent for 4-5 days, collecting cell supernatant, filtering the cell supernatant by using a 0.22 mu m filter, and obtaining P1 generation recombinant Baculovirus baculovir-inCap 6S 663L/baculovir-ITR-EGFP/baculovir-inrep; the P1 generation recombinant baculovirus is amplified by infecting Sf9 cells twice to obtain the P3 generation recombinant baculovirus. The titer of baculoviruses from P3 generation was determined by plaque assay, with the virus titer (pfu/mL) being 1/dilution x plaque number x 1/volume inoculated per well.

Finally, three recombinant baculoviruses (Baculovir-inCap 6S 663L/Baculovir-ITR-EGFP/Baculovir-inrep) of P3 generation are co-infected with Sf9 cells, and packaged to obtain rAAV6S 663L.

And a method for purifying and concentrating high-concentration recombinant AAV by using a CsCl density gradient centrifugation method, a method for detecting the titer of rAAV6S663L by using fluorescent quantitative PCR, and a method for detecting the purity of rAAV6S663L by using SDS-PAGE.

In another aspect of the embodiments of the present invention there is also provided an isolated, genetically modified host cell comprising the aforementioned isolated nucleic acid.

Yet another aspect of an embodiment of the invention provides a pharmaceutical composition comprising the aforementioned adeno-associated virus virion with a mutated capsid, and a pharmaceutically acceptable excipient.

Further, such excipients, carriers, diluents and buffers include any agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Which may include pharmaceutically acceptable salts such as mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulphate and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been described extensively in a variety of publications, including, for example, a. gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20 th edition, Lippincott, wil iams, & Wilkins; edited by Pharmaceutical document Forms and Drug delivery systems (1999) H.C. Ansel et al, 7 th edition, Lippincott, Williams, & Wilkins; and handbook of Pharmaceutical Excipients (2000) A.H.Kibbe et al, 3 rd edition, Amer.pharmaceutical Assoc.

Yet another aspect of an embodiment of the invention provides the use of the aforementioned adeno-associated virus virions or compositions with a mutated capsid in the manufacture of a medicament for delivery of a gene product to retinal muller cells of an individual.

Yet another aspect of the embodiments of the present invention provides a product for use in a method of treating a retinal disease, the method comprising administering to an individual in need thereof an effective amount of the aforementioned adeno-associated viral virions with a mutated capsid.

Further, the product is administered by intraocular injection.

Still further, the mode of administration of the product is by intravitreal injection.

Furthermore, the administration mode related to the invention is vitreous injection, and the specific steps comprise: 0.01mL/g of 4% chloral hydrate is used for anesthetizing a mouse, mydriasis is performed in both eyes, sodium hyaluronate is used for keeping the surface of the eyes moist, and an antibiotic eye drop are dropped before the surface anesthesia operation. The head position of the mouse is adjusted to ensure that the eyeball keeps the corneal limbus level. A31G needle was used to puncture 1mm behind the limbus and a hamilton33G syringe injected 1 μ L of virus at the puncture site. The needle point vertically enters, slowly pushes in the vitreous cavity, keeps the needle for 1min after the injection is finished, and slowly withdraws the needle head. Ofloxacin eye drops can be used for local anti-inflammation and infection prevention.

In some preferred embodiments, the present invention demonstrates that AAV6S663L mutant targets retinal muller cell properties by various technical schemes:

first, the pattern of EGFP expression as well as the morphology, distribution of infected cells were initially judged by in vivo imaging, retinal plating and sectioning to target infected muller cells.

Secondly, at different times after the AAV6S663L mutant is injected into the vitreous body, living body imaging, retina plating and section observation are carried out to observe the EGFP expression, and the expression mode is unchanged, which indicates that the EGFP expression is stably and specifically infected with Muller cells.

Thirdly, staining the retinal sections with antibodies to Glutamine Synthetase (GS), a protein specific to Muller cells, can coincide with green fluorescent protein expressed by viral transduction, further demonstrating that the Muller cells are infected by the virus in a targeted manner.

Fourthly, constructing a recombinant AAV6S663L vector (AAV6/S663L-GFAP-EGFP) with a target gene driven by a Muller cell-specific promoter (GFAP), wherein the expression pattern of the vector is not obviously different from that of a constitutive promoter (CMV), and when two viruses (AAV6/S663L-CMV-mCherry and AAV6/S663L-GFAP-EGFP) are injected simultaneously, two fluorescences can be overlapped.

In conclusion, the AAV6S663L mutant was injected vitreally, targeting infected retina Muller cells.

In summary, the present invention employs a safer mode of administration by injecting recombinant AAV virions directly into the vitreous cavity, and the viral suspension diffuses with the vitreous into the retina. Wild type AAV6 viral vectors transduce ganglion cells efficiently and relatively poorly to other cells of the retina by intravitreal injection. The Muller cells penetrate the whole retina, and in view of the important function of the Muller cells on maintaining the functions of retinal homeostasis, metabolism and the like, the AAV vector for specifically and efficiently transducing the Muller cells is obtained by site-directed mutagenesis of the amino acids encoding the AAV6 capsid, and is suitable for carrying exogenous therapeutic genes to transduce the Muller cells to treat retinopathy.

The technical solutions of the present invention are further explained below with reference to some preferred embodiments and the accompanying drawings, but the experimental conditions and the setting parameters should not be construed as limitations of the basic technical solutions of the present invention. And the scope of the present invention is not limited to the following examples.

Example 1

The embodiment relates to an AAV variant for efficiently and specifically transducing Muller cells, and the specific construction method comprises the following steps:

(1) preparation of recombinant plasmid (see FIGS. 1A-1D):

preparation of pFastbacadual-inCap 6S663L

According to the literature (Chen H. Intron cracking-mediated expression of AAV Rep and Cap genes and production of AAV vectors in infection cells. mol Ther,2008,16(5): 924) insert it between bases 25 and 26 of Cap6 sequence by fusion PCR method, PCR product was digested with BamHI and XbaI into the Multiple Cloning Site (MCS) of pFastbacadial plasmid to obtain pFastbacadial-inCap 6. Furthermore, the gene sequence coding the 663 serine of the Cap6 protein was site-directed mutated to leucine by fusion PCR method using pFastbacadual-inCap 6 plasmid as template. Performing site-directed mutagenesis by a fusion PCR method, wherein two rounds of PCR reactions are required, wherein in the first round of PCR, an inCap gene in pFastbacdual-inCap6 plasmid is used as a template, the first round of PCR relates to two reaction systems, and a 3 'primer of a first sequence and a 5' primer of a second sequence have a complementary region; then all products of the first round of PCR are added into a reaction system as a template, and a 5 'primer of the first section of sequence and a 3' primer of the last section of sequence are used as primers to carry out second amplification to form a fusion sequence. And (3) purifying the fusion PCR product by using a DNA purification kit, then carrying out enzyme digestion by using BamHI and XbaI, carrying out enzyme digestion on the vector pFastbacal-inCap 6 by using BamHI and XbaI, connecting the vector pFastbacal-inCap 6 by using T4DNA Ligase, transforming escherichia coli Top10 competent cells, selecting a single clone colony for amplification, extracting a plasmid, and carrying out enzyme digestion identification to obtain pFastbacal-inCap 6S 663L. This plasmid also contains other elements necessary for the expression of the capsid protein in eukaryotic cells, including the promoter Polyhedrin promoter (P)_PH) And SV40 poly conjugation signal sequence.

Preparation of pFastbacanal-ITR-EGFP, pFastbacanal-ITR-GFAP-EGFP, pFastbacanal-ITR-CMV-mCherry

The pFastbacdual-ITR-EGFP plasmid contains two ITRs of AAV2 and a CMV promoter, a beta-globin intron, a coding EGFP gene and an hGH polyA sequence, and is constructed and stored in a laboratory (reference: Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cells, bioengineering, 2015,31(8), page 1232, method "pFastbacdual-ITR-EGFP plasmid" in 1.2.1).

The method comprises the following steps of constructing the pFastbacadial-ITR-GFAP-EGFP by taking the pFastbacadial-ITR-EGFP as a framework, and specifically comprising the following steps: the GFAP promoter sequence is synthesized by Suzhou Jinweizhi biotechnology limited, MluI enzyme cutting sites are arranged at the upstream, EcoRI enzyme cutting sites are arranged at the downstream, the MluI and the EcoRI are used for respectively cutting pFastbdual-ITR-CMV-EGFP and GFAP fragments, the fragments are recovered and purified, and the fragments are connected by T4DNA ligase, so that the pFastbdual-ITR-GFAP-EGFP is obtained.

The method comprises the following steps of constructing pFastbacadial-ITR-CMV-mCherry by taking pFastbacadial-ITR-EGFP as a framework, and specifically comprising the following steps: the mCherry sequence is obtained by amplifying plasmid pAAV-CaMKIIa-hRhR 2-mChery, the upstream of the mCherry sequence is provided with a BamHI enzyme cutting site, the downstream of the mChery sequence is provided with an XhoI enzyme cutting site, the pFastbacal-ITR-EGFP and the mChery fragment are respectively cut by BamHI and XhoI, the pFastbacal-ITR-CMV-mChery fragment is recovered and purified, and the pFastbacal-ITR-CMV-mChery fragment is obtained by connecting the pFastbacal-ITR-CMV.

Preparation of pFastbacadual-inrep plasmid

The pFastbacdual-inrep plasmid contains AAV2rep gene, intron sequence, the expression of which is regulated by p10 promoter and HSV tk polyA element on pFastbacdual plasmid vector, and is constructed and stored in laboratory (reference: Litamine et al, AAV-ITR gene expression microcarrier prepared by insect cell, report of bioengineering, 2015,31(8), p 1232, 1.2.2 method "construct pFastbinary-inrep plasmid").

(2) Preparation of recombinant Bacmid

The three recombinant plasmids in the last step are respectively used for preparing recombinant Bacmid, and the specific method is as follows:

1) 100 μ L of DH10Bac was slowly thawed on ice.

2) Add 50ng plasmid DNA and mix gently.

3) Standing on ice for 30min, heat shocking at 42 deg.C for 90s, immediately transferring to ice and standing for 2 min.

4) Add 900. mu.L of SOC medium and shake at 37 ℃ and 225rpm for 4 h.

5) 40. mu.L of 2% (20mg/mL) Blue-gal and 7. mu.L of 20% (200mg/mL) IPTG were added dropwise to the center of a pre-prepared 90mm agar plate containing 50. mu.g/mL kanamycin (Kan), 7. mu.g/mL gentamicin (Gen), 10. mu.g/mL tetracycline (Tet). The plates were spread over the entire surface using a sterile spreader and incubated at room temperature until all liquid disappeared.

6) Using SOC culture mediumCells were diluted 10-fold in gradient (10)^-1，10^-2，10^-3) 100 μ L of each gradient was applied to LB plates.

7) After 48h at 37 ℃ 10 white colonies were picked and dipped on fresh LB agar plates (resistant as above) overnight at 37 ℃. The confirmed white spots were picked and inoculated into LB liquid medium (containing 50. mu.g/mL kanamycin (Kan), 7. mu.g/mL gentamicin (Gen), 10. mu.g/mL tetracycline (Tet)).

8) The mixture was left overnight at 4 ℃ to allow the blue color to develop sufficiently during this period.

9) Sf9 can be transfected after the correct PCR identification can be carried out for white spots.

10) Extracting and separating recombinant bacmid DNA by using an OMEGA kit, and measuring the concentration of bacmid by the experimental method according to the kit specification, subpackaging and freezing at-20 ℃ to avoid repeated freezing and thawing.

11) And (3) PCR identification of Bacmid, wherein the used primers are respectively as follows: an upstream primer 5'-CCC AGT CAC GAC GTT GTA AAA CG-3' and a downstream primer 5'-GCT CTA GAT TAC TTG TAC AGC TCG TCC AT-3'.

12) Taking out the Bacmid strain with correct identification, and inoculating the Bacmid strain to 3mL LB (Kan +, Gen +, Tet +) for 12h according to the proportion of 1: 300. Then inoculating 150mL LB (Kan +, Gen +, Tet +) shake bacteria for 16h according to the proportion of 1:100, and extracting Bacmid in large quantity according to the instruction of a large-scale/large-scale plasmid extraction kit so as to prepare baculovirus by transfecting cells.

(3) Preparation of baculovirus

Culture of Sf9 cells. Sf9 cells were plated one day in advance in six well plates at 50% well density using complete medium, 95% viability. The recombinant Bacmid DNA is subjected to warm bath in a 70 ℃ water bath for 20min, and then 12000g of the recombinant Bacmid DNA is centrifuged for 10min to obtain supernatant.

② cell plating.

Ensuring the cell density to be 1.5-2.5 × 10⁶cells/mL (medium without antibiotics). Add 2mL basal (Grace) medium without additives (no antibiotics and serum) to 6-well plates. Inoculation 8X 10⁵cells/mL Sf9 in step 1 (without changing medium and washing cells) and allow cells to adhere for 15min at room temperature.

Preparing transfection reagent.

a) Transfection reagent II was mixed well, 8. mu.L to 92. mu.L basal medium without additives (without antibiotics and serum) was added, and vortexed.

b) And (3) taking 5 mu L of bacmid DNA (500 ng/mu L, ensuring that the quantity of bacmid is 2-3 mu g) to 95 mu L of basal medium without additives (without antibiotics and serum), and gently mixing the bacmid DNA and the basal medium.

c) Mixing the above two solutions, and incubating at room temperature for 30 min.

And fourthly, dripping the DNA-Lipid mixture into the hole paved with the cells, and incubating the cells for 5 hours at the temperature of 27 ℃.

Fifthly, the culture medium in the plate is removed and 2mL of complete culture medium is replaced.

Sixthly, incubating for 72 hours at the temperature of 27 ℃ and observing the sign of virus infection.

Isolation P1:

after confirming that the cells are in the late stage of infection (usually 4-5 days after transfection), 2mL of virus-containing medium per well was collected into a sterile 15mL centrifuge tube and centrifuged at 1000g for 5min to remove cell debris.

The supernatant was filtered through a 0.22 μm filter into a sterile 15mL centrifuge tube and stored at 4 ℃ in the dark. If long-term storage is desired, subpackaging and freezing at-80 ℃.

And (3) virus amplification:

taking 10mL suspension culture cells with MOI of 0.05-0.1 and density of 2 multiplied by 10⁶cells/mL; or cells in 6-well plates at a density of 2X 10⁶cells/well, calculate the required P1 volume.

(ii) Sf9 cells plated on six-well plates, 2X 10⁶cells/well. The plate was left at room temperature for 1 hour to adhere and observed under a microscope.

② adding proper amount of P1 into each hole, culturing for 48-72 h at 27 ℃.

③ 2mL of the virus-containing culture medium was collected per well in a sterile 15mL centrifuge tube and centrifuged at 1000g for 5 min.

Transferring the supernatant to a sterile 15mL centrifuge tube, wherein the virus supernatant is P2. Storing at 4 deg.C in dark place, and freezing at-80 deg.C if long-term storage is desired.

P3 can be obtained by amplification according to the above method (normally the obtained P1 virus titer is 1X 10)⁶～1×10⁷P2 titre between 1X 10⁷～1×10⁸In between).

Viral titers were determined by plaque assay. The detailed experimental procedure is as follows:

firstly, 2 mL/well of cells (5 multiplied by 105cells/mL) are planted into a 6-well plate, the cells are incubated for 1h at room temperature to adhere to the wall, and the degree of adherence is examined under a microscope after incubation.

② 4 percent agarose gel is put into a 70 ℃ water bath kettle to be melted, 2 XGrace and a 100mL sterile bottle are put into a 40 ℃ water bath kettle to be preheated.

③ using a serum-free basic culture medium to carry out gradient dilution on the baculovirus: 10^-1～10^-8。

And fourthly, abandoning the supernatant in the 6-well plate, quickly adding the diluted virus, and incubating for 1 hour at room temperature per 1 mL/well (multiple wells).

Fifthly, preparing upper agar, adding 20mL of high-temperature inactivated FBS to 2 XGrace 100mL, adding 25mL of 2 XGrace (containing FBS) +12.5mL of sterile water and 12.5mL of 4% agarose gel to a preheated 100mL sterile bottle, gently mixing, and placing in a 37 ℃ water bath for later use.

Sixthly, removing the supernatant in the 6-hole plate, quickly adding 2mL of upper agar to prevent the bacterial layer from drying, and standing for 10-20 min to solidify. The 6-well plate was placed in an incubator at 27 ℃ and cultured for 5 days.

Preparing 1mg/mL neutral red solution, and performing sterile filtration in a basic complete culture medium.

Eighty percent (1.5) mL of the solution, 16.5mL of the basic complete medium, and 6mL of 4% agar were prepared as the neutral red upper agar.

Ninthly, 4 days after the virus infection, 1mL of neutral red upper agar is added.

And (5) putting the red (R) into an incubator continuously, observing plaques after 4-5 days, counting the number of the plaques, and obtaining the virus titer.

Note: viral titer (pfu/mL) 1/dilution factor × plaque number × 1/inoculation volume per well

(4) Preparation and purification of rAAV vector

The purity schematic diagram of the rAAV vector detected by SDS-PAGE in the embodiment can be seen in figure 2, wherein A represents rAAV6-CMV-EGFP, B represents rAAV6-S663L-CMV-EGFP, and C represents rAAV 6-S663L-GFAP-EGFP.

(5) Mouse vitreous injection method

The specific operation steps are as follows:

4% chloral hydrate 0.01ml/g anesthetizes the mice, mydriasis of both eyes, uses sodium hyaluronate to keep the ocular surface moist, and uses antibiotic eye drops and topical anesthetic to drop the eye before operation. The head position of the mouse is adjusted to ensure that the eyeball keeps the corneal limbus level. A31G needle was used to puncture 1mm behind the limbus and a Hamilton (Hamilton)33G syringe injected 1. mu.l of virus at the puncture site. The needle point enters vertically, then inclines and pushes slowly, the needle is left for 0.5-1min after pushing the needle, and the needle is taken out quickly.

(6) Living body imaging method

After injection of viral vectors 1-5, mice were anesthetized with weekly intraperitoneal injections of chloral hydrate. The compound tropicamide eye drops are used for dilation of pupils. To prevent dehydration of the cornea, sodium hyaluronate eye drops were used for moisturizing. Fundus imaging was performed using a line-scanning confocal fundus imaging system, with 488nm solid-state laser exciting GFP. After imaging, the sodium hyaluronate eye drops are used for moisturizing eyes.

In this embodiment, fig. 3 is a schematic diagram of in vivo imaging results of different periods after injection of rAAV6S663L-CMVEGFP vitreous body, and the expression pattern of EGFP after the retina is infected with rAAV6S663L-CMVEGFP is observed by in vivo imaging and mainly distributed around blood vessels, and the main cells around the blood vessels are muller cells.

(7) Retinal plating extraction and confocal microscope detection of virus expression in retina

Two weeks after intravitreal injection, mice were sacrificed by cervical dislocation and the eyeball was directly placed in 4% Paraformaldehyde (PFA) and fixed at 4 ℃ for 2 h. After fixation, the eyeball is placed into PBS for washing, placed into a culture dish, the optic nerve is cut off, the cornea is opened, and the cornea is cut off. The sclera and choroid complex were torn open to expose the cup-shaped clear retina, the lens removed, and the residual vitreous removed. The retina was transferred to new PBS and 4 valves were symmetrically cut open. PBS was aspirated and blotted dry with paper. Adjusting the shape of the retina, transferring the retina to a glass slide, covering the glass slide, observing under a confocal microscope, selecting a microscope multiple of 20 multiplied by 10, and observing the cell layer structure.

The rAAV6-S663L-CMV-EGFP is mainly targeted to the peripheral Muller cells after infecting retina through retinal plating, and the wild type rAAV6-CMV-EGFP is mainly targeted to ganglion cells.

In this example, the results of different time slices after the injection of rAAV6-S663L-CMV-EGFP vitreous body are shown in the schematic diagram in FIG. 4, and the main target of peripheral Muller cells after the retina is infected by rAAV6-S663L-CMV-EGFP is observed through the retinal slices, while the main target of the wild-type rAAV6-CMV-EGFP is ganglion cells.

(8) Frozen sections and DAPI staining

Mice were sacrificed by cervical dislocation, the eyeballs were removed, immersed in 4% PFA, fixed at 4 ℃ for 2h, the cornea was opened, immersed in 4% PFA, and left overnight at 4 ℃. Except PFA, 20 percent of sucrose is dehydrated for 12 hours at 4 ℃, and 30 percent of sucrose is dehydrated for more than 24 hours at 4 ℃. The eyes soaked in the 30% sucrose solution were removed, the corneal portion was clamped, the cornea and the crystalline lens were removed, and sucrose was blotted with paper. Precooling with tweezers, paper, knives, etc. And (3) dripping a small amount of OCT, vertically placing the eye in a plastic cover, removing bubbles, freezing in a microtome, keeping the frozen eye vertically standing, and continuously dripping the OCT to embed the whole eye. Dripping a circle of OCT on a precooled specimen tray, putting a sample on the specimen tray, fixing the whole sample by the OCT, and trimming the size by a knife. The specimen disc is embedded into the specimen head, the tool rest and the anti-rolling plate are adjusted, the trimming sheet is roughly adjusted, and after a tissue to be cut is cut, the tissue is finely cut by 14 mu m. The cut sections were attached to a slide glass and mounted in a slide holder. The slides were soaked in 0.01M PBS and allowed to air dry for 2h in the open air.

Taking out the slicing frame, placing into a repair box containing 0.01M PBS, wiping to dry the periphery, enclosing the periphery of the slice with an immunohistochemical pen, dripping oil to dry, and cleaning with 0.01M PBS. DAPI (Sigma) was added (1: 1000PBS) at room temperature for 20 min. 0.01M PBS 3 times washing. Sections were kept wet and observed by inverted fluorescence microscopy.

In this example, the results of different sections of rAAV6-CMV-EGFP and rAAV6-S663L-CMV-EGFP after vitreous injection are shown in FIGS. 5A-5B. The observation of rAAV6-S663L-CMV-EGFP infection on the retina through the retina section shows the morphology of Muller cells penetrating the whole retina, and the main infection site of the wild type rAAV6-CMV-EGFP is ganglion cells.

(9) Tissue section immunofluorescence staining:

1) the slide holder was removed and placed in a repair box containing 0.01M PBS or rinsed with a Pasteur pipette.

2) Taking out and wiping only the periphery, and enclosing the periphery by an immunohistochemical pen.

3) After a few minutes the oil was dried and washed by adding 0.01 PBS.

4) Adding sealing liquid containing 3% goat serum, and sealing for more than 60 min.

5) Most of the serum blocking solution was aspirated and washed once with 0.01M PBST.

6) Blocking solution diluted primary anti-GS monoclonal antibody (1: 100) 16-18 h/overnight at 4 ℃.

7)0.01M PBST washing 4 times, each time for 5 minutes.

8) A diluted secondary goat anti-mouse IgG (Alexa Flour 594) (1: 100) incubate at room temperature for 2 h.

9)0.01M PBST washing 4 times, each time for 5 minutes.

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

11)0.01M PBS 3 times, the last PBS was not discarded to keep the sections moist.

(10) Specific promoter fluorescent expression:

constructing a recombinant AAV6S663L vector (AAV6-S663L-GFAP-EGFP) of which the target gene is started by a Muller cell specific promoter (GFAP), theoretically, the expression of the EGFP protein can only be started in the Muller cell, and the AAV6-S663L-GFAP-EGFP can express green fluorescence in the Muller cell according to the section staining result, which indicates that the AAV6S663L can infect the Muller cell. FIG. 6 shows GS antibody immunofluorescence staining results of the glass body injection of rAAV6-S663L-CMV-EGFP, rAAV6-S663L-GFAP-EGFP and rAAV6-CMV-EGFP in the present example.

The result of retinal fluorescence expression is schematically shown in FIG. 7, in which AAV6-S663L-GFAP-EGFP and AAV6-S663L-CMV-mCherry virus are infected simultaneously with the mixed virus AAV6-S663L-GFAP-EGFP carrying a specific promoter and a green fluorescent protein and the virus AAV6-S663L-CMV-mCherry carrying a constitutive promoter and a mChery red fluorescent protein, and FIG. 7 shows that the two viruses are expressed and overlapped in the retina, which indicates that AAV6S663L targets Muller cells.

As can be seen from the above examples, the present invention obtains AAV vector capable of specifically and efficiently transducing Muller cells by site-directed mutagenesis of the amino acids encoding AAV6 capsid, and is suitable for transduction of Muller cells with exogenous therapeutic genes to treat retinopathy.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

It will be appreciated by persons skilled in the art that the above-described embodiments of the present invention are not intended to limit the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims (1)

1. Use of a recombinant AAV virion for the preparation of a medicament for the targeted treatment of retinal Muller cell pathology, wherein the recombinant AAV virion is an adeno-associated virus type 6, wherein serine at position 663 in the amino acid sequence of the capsid protein is mutated to leucine, and the recombinant AAV virion has an enhanced infectivity of retinal Muller cells.

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