CN116003533A - Capsid protein mutant MutF for improving AAV retina targeting and application thereof - Google Patents

Abstract

The invention discloses capsid protein mutant MutF for improving AAV retina targeting and application thereof. The inventor researches and discovers that the capsid protein mutant MutF obtained by using DENEIRTTNPVATEQYGDVSTNLQRGNR to replace 561-588 amino acids of wild AAV2 virus capsid protein can surprisingly and effectively improve the retina infection capability of AAV virus and fundamentally solve the problem of AAV2 retina targeting.

Description

Capsid protein mutant MutF for improving AAV retina targeting and application thereof

The invention belongs to the technical field of biology, and particularly relates to a capsid protein mutant MutF for improving AAV retina targeting and application thereof.

Background

Adeno-associated virus (AAV) is a delivery vector widely applied to gene therapy, and the principle is that sequences among ITRs of AAV genomes are replaced by target gene sequences through a genetic engineering method, and the target gene sequences are transferred to target cells through cell infection, so that the aim of gene therapy is fulfilled. Based on the characteristics of safety, high efficiency, stability, persistence, specificity, low integration and the like of recombinant AAV, AAV becomes one of main delivery means in the field of gene therapy. In the course of gene therapy applications, living animals often need to inject high doses of high purity AAV viruses, and the high AAV production costs become one of the bottlenecks in the gene therapy process.

Along with the maturation of downstream development techniques for gene therapy, the limitations of upstream AAV production throughput become more and more apparent. To address this problem, existing strategies focus on the following two aspects, one is to optimize the existing AAV production process; and secondly, searching a virus vector with better packaging capability and tissue targeting. More of the AAV production process is through optimization of its external packaging conditions, and no modification of AAV itself is involved. Based on the structural characteristics of AAV viruses, the characteristics of the viruses such as tissue targeting, immunogenicity, packaging yield and the like are mainly determined by the surface capsid proteins.

The relationship between AAV capsid proteins and their targeting is not clear, and AAV capsid proteins with specific targeting or high targeting are mainly found by mutants in the prior art. CN113766934A, CN111770999A, CN105755044A, CN103561774a et al discloses a number of different AAV viruses with a certain retinal targeting.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provides a capsid protein mutant MutF for improving the retina targeting of AAV and application thereof.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided:

an AAV viral capsid protein mutant MutF, wherein amino acids 561-588 of the wild type AAV2 viral capsid protein are replaced with a polypeptide sequence DENEIRTTNPVATEQYGDVSTNLQRGNR as compared to the wild type AAV2 viral capsid protein.

In some examples of AAV viral capsid protein mutants MutF, the amino acid sequences thereof are as set forth in SEQ ID No.: 2.

In a second aspect of the invention, there is provided:

nucleic acid sequence molecules expressing the AAV viral capsid protein mutants MutF according to the first aspect of the invention.

For better expression of the corresponding proteins, the nucleic acid sequence molecules may be sequence optimized for better expression.

In a third aspect of the invention, there is provided:

an expression system capable of expressing an AAV viral capsid protein mutant MutF according to the first aspect of the invention, or comprising a nucleic acid sequence molecule according to the second aspect of the invention.

In some examples of expression systems, the expression system is a recombinant AAV vector that is engineered by insertion or replacement of an AAV vector.

In some examples of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, and mutants thereof.

In some examples of expression systems, the expression system further includes a nucleic acid molecule encoding a functional gene product. The functional gene product may be a protein, mRNA, or the like.

In a fourth aspect of the invention, there is provided:

a composition comprising a nucleic acid sequence molecule according to the second aspect of the invention, or an expression system according to the third aspect of the invention.

In a fifth aspect of the invention, there is provided:

the use of a composition according to the fourth aspect of the invention in the preparation of an eye-targeted gene therapy formulation or transgene formulation.

In some examples of application, the composition targets the retina or lens.

The beneficial effects of the invention are as follows:

the capsid protein mutant MutF of some examples of the invention can effectively improve the retina targeting of AAV virus, and fundamentally solve the problem of the efficiency of AAV2 infecting cells of each layer of retina.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a diagram showing agarose gel electrophoresis identification of mutant plasmids constructed in the test examples of the present invention;

FIG. 2 is a map of pAAV2 packaging plasmid in an example of the present invention;

FIG. 3 is a map of pAAV2-MutF packaging plasmid constructed in the examples of the present invention;

FIG. 4 is a map of pAAV-CAG-EGFP expression plasmids of a viral packaging three plasmid system in an embodiment of the present invention;

FIG. 5 is a pHelper helper plasmid map of a three plasmid viral packaging system according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of the detection of the total amount of virus particles of adeno-associated virus variants prepared according to the inventive protocol and wild-type adeno-associated virus of the control group in the test example of the present invention;

FIG. 7 is a graph of fluorescence OCT of the 3W living fundus of wild-type and MutF-infected mice according to an embodiment of the present invention;

FIG. 8 is a diagram of a retinal tissue section of wild type and MutF infected mice of an embodiment of the present invention at 3W;

FIG. 9 is a sequence alignment of wild-type Cap561-588 and MutF sequences according to an embodiment of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1

This example provides an adeno-associated virus variant (AAV 2-MutF) prepared by the steps of:

TABLE 1 AAV2-WT and its mutant Cap561-588 amino acid sequence information

Sequence name	Sequence information	Numbering device
			AAV2-WT-Cap561-588	DEEEIRTTNPVATEQYGSVSTNLQRGNR	6
AAV2-MutF	DENEIRTTNPVATEQYGDVSTNLQRGNR	3
			AAV2-Mut051	HSEDEIKTTNPVATEGYGEVATNLQRGPQQ	7
AAV2-Mut052	AVEGEIKTTNPVATEGYGEVATNLQRGPNG	8
			AAV2-Mut053	DEEEIRTTNPVATEQSCGSVSTNLVQYRGNR	9
AAV2-Mut054	NAAEIKTTNPVATEQYGEVASPQAAQAWKQVTDL	10
			AAV2-Mut055	HDEEEIAATNPVSTEHYGESATNLQSGPAG	11
AAV2-Mut056	AENEIATTNPVATEQYGEASENLQRGGR	12
			AAV2-Mut057	DEEEIRTTNPVATEVYGEASTNLQHGGR	13
AAV2-Mut058	QEEEIATTNPVATEQYGEVSENLQRGNR	14
			AAV2-Mut059	DEEEIRTTNPVSTEVYGEASTNLQHGGR	15
AAV2-Mut060	DEEEIRTTNPVATEQYGEVSENLQRGER	16
			AAV2-Mut061	DEEEIRTTNPVATEQYGEVATNLQRGNR	17
AAV2-Mut062	AENEIATTNPVATEQYGEVSENLQRGNR	18
			AAV2-Mut063	DEEEIRTTNPVATEQYGGVSENLQRGNR	19
AAV2-Mut064	DEQEIRTTNPVATEQYGEVSTNLQRGNM	20
			AAV2-Mut065	DEEEIVTTNPVATEQYGEVSENLQRGNR	21
AAV2-Mut066	DEEEIAATNPVATEQYGSVSELLQTGNR	22
			AAV2-Mut067	DEEEVRTTNPVATEQYGSVSTTLQHGNR	23
AAV2-Mut068	DEEEIRTTNPVSTEQYGEVSTNLQRGGR	24
			AAV2-Mut069	DEEEIATTNPVATEQYGDVSTNLQRGNR	25
AAV2-Mut070	DEQEIATTNPVCTEQYGEVSENLQRGNT	26

1. Plasmid construction

The sequence obtained by synthesis is cloned to pAAV2 plasmid by the prior method through the nucleic acid sequences corresponding to amino acid sequences (shown in table 1) such as MutF, mut051-Mut070, etc., of gene synthesis (biological engineering Co., ltd.), and Cap2 561-588 amino acid is replaced by mutation sequence, so that pAAV-mutation plasmid vector is constructed; the ligation product is transformed into escherichia coli competent Stbl3, single colony extracted plasmids are selected for sequencing verification and enzyme digestion verification, and agarose gel electrophoresis identification is carried out, and the result is shown in figure 1, so that the mutant plasmids are successfully constructed.

2. AAV2-WT and AAV2-MutF mutant virus packages

AAV2-WT plasmid and AAV 2-mutant plasmid are transfected into HEK293T cells by adopting a virus packaging three-plasmid system, the plasmid maps are respectively shown in figures 2-5, and the method comprises the following steps:

a cell culture

(1) Taking out HEK293T cell from liquid nitrogen tank, rapidly placing into 37deg.C water bath, slightly shaking to rapidly defrost the cell, centrifuging at 200 Xg for 5min, discarding supernatant, re-suspending into 10ml DMEM complete medium, placing into 10cm culture dish, and adding 5% CO at 37deg.C ₂ Medium culture

(2) After 48-72 hours of culture, the cell confluence reaches about 80%, and the cells can be subjected to subculture;

(3) The culture supernatant was aspirated, washed 1 time with 5ml PBS buffer, digested 3-5min at 37℃with 1.5ml pancreatin digest, and stopped with 4.5ml DMEM complete medium;

(4) Blowing cells to form a cell suspension according to 1:4-1:5, and inoculating into T300 bottle for culturing.

B cell transfection

(1) Culturing the cells for 48 hours, wherein the cell confluence reaches 80% -90%, and the cells can be transfected;

(2) Per bottle of cells, molar ratio 1 in 1mL DMEM medium: 1:1, sequentially adding 70 mug of packaging plasmid, pHelper and pAAV-CAG-EGFP, and uniformly mixing to obtain a plasmid diluent;

(3) Taking 1ml of DMEM culture medium, adding 180ug PEI, and mixing uniformly to obtain PEI diluent;

(4) Pouring PEI diluent into plasmid diluent, quickly and uniformly mixing, and standing at room temperature for 25 minutes to form a transfection complex; adding the transfection complex into 80ml of DMEM culture medium, and mixing evenly with light shaking;

(5) Taking out the T300 cell culture flask, discarding the culture supernatant, and adding a culture medium containing the transfection complex;

(6) At 37℃5% CO ₂ Is cultured.

C Virus harvesting

(1) The cells are cultured for 72 hours after transfection, and virus harvesting is started;

(2) Collecting culture supernatant in a centrifugal bottle, adding 50% PEG8000 solution (containing 0.5mol/L NaCl) of 0.245mL per mL supernatant, mixing thoroughly, and standing at 2-8deg.C overnight;

(3) Adding 5ml of 0.5% TritonX-100 cell lysis buffer solution into the culture flask, treating at 37deg.C for 1-2 hr, adding 0.55ml of 5mol/L NaCl, mixing thoroughly, centrifuging at 4deg.C for 15min at 3000g, collecting supernatant, and temporarily storing at 2-8deg.C;

(4) The supernatant obtained after concentration of PEG8000 was centrifuged at 4℃and 3000g for 15min, the supernatant was removed, 2ml of 0.5% Triton X-100 cell lysis buffer was added, the mixture was treated at 37℃for 1-2h, then 0.22ml of 5mol/L NaCl was added, and after thorough mixing, 3000g was centrifuged for 15min, and the supernatant was collected. The 2 supernatants were incorporated into a centrifuge tube.

D virus purification

1) Taking an Ultra-Clear centrifuge tube, sequentially adding 0.5mL of 60% Iodixanol,2mL of 40% Iodixanol,1.5mL of 25% Iodixanol and 1.5mL of 15% Iodixanol at the bottom, collecting virus suspension, and balancing by using cell lysate;

2) 10 ℃, 230000g, 8-up and 9-down ultracentrifugation for 18h;

3) Taking a 100KD ultrafiltration tube, and infiltrating the filter membrane with 2mL PBS buffer solution for standby;

4) Carefully extracting a 40% -60% -Iodixanol intermediate liquid layer of the ultra-fast centrifuge tube by using a pipetting gun, avoiding sucking protein, and transferring to an ultra-fast tube;

5) Adding a proper amount of PBS buffer solution, blowing uniformly, centrifuging for 3-5min at 4500g, and repeating the steps for 5-7 times until Iodixanol is removed;

6) Adding 1mL of PBS buffer solution, blowing 40-50 parts of the PBS buffer solution into virus suspension, and transferring the virus suspension into an EP tube;

7) The virus suspension in the EP tube was filtered through a 0.22 μm filter by a 5mL syringe, and 20. Mu.L of the virus solution was collected as a test sample, and then split-packed with 100. Mu.L of the virus solution per tube to obtain an adeno-associated virus variant.

Comparative example 1

This comparative example provides a wild-type adeno-associated virus (AAV 2-WT) which differs from the preparation of example 1 only in that the sequence of the adeno-associated virus capsid protein employs a wild-type AAV2 capsid protein sequence.

Test case

1. Virus titer detection

(1) Virus pretreatment

1) 10. Mu.L of the virus samples of example 1 and comparative example were taken, respectively, and ddH was used ₂ O dilution 10-fold to 100ul;

2) Respectively adding omnipotent nuclease to 50U/ml, incubating for 30min at 37 ℃, then adding protease K5 mu L, incubating for 30min at 55 ℃, and then incubating for 15min at 95 ℃;

3) 10. Mu.L of the treated virus dilution was taken and subjected to ddH ₂ O is diluted to 10000 times in a 10-time gradient for standby.

(2) Preparation of standard for standard curve

Taking 1×10 ⁹ Plasmid standard of copies/. Mu.L with ddH ₂ O was diluted 10-fold to 1X 10 ⁸ copies/µL、1×10 ⁷ copies/µL、1×10 ⁶ copies/µL、1×10 ⁵ copies/µL、1×10 ⁴ copies/µL。

(3) qPCR system preparation

1) A0.2 mL PCR tube was used to prepare the following reaction system, 3 replicates for each viral dilution. 2 XqPCR Mix 10. Mu.L; forward and reverse primers were each 0.2. Mu.L (100 nM); 5. Mu.L of amplified template (virus dilution or plasmid standard, etc.); adding ddH ₂ O to 20ul.

Amplification primers:

forward primer-F5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO. 4);

reverse primer-R5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO. 5).

2) PCR amplification

Pre-denaturation: 95 ℃ for 2 min;

40 x cycle: 95 ℃ for 15s;60 ℃ for 30s.

3) qPCR data processing: viral titer = dilution x viral gene array copy number.

As shown in FIG. 6, it can be seen from the graph that AAV2-WT virus packaging three-plasmid system (comprising three plasmids of pAAV2, pAAV-CAG-EGFP and pHelper) and AAV 2-mutant virus packaging three-plasmid system (comprising three plasmids of pAAV2-MutF, pAAV-CAG-EGFP and pHelper) were transfected into HEK293T cells in equal amounts, respectively, wherein the total amount of AAV2-MutF virus particles was 56% of AAV2-WT, and the total amount of the remaining 24 mutant virus particles was 40.5% -1.5% of AAV2-WT, and AAV2-MutF was selected for in vivo infection ability test in mice.

2. Test of retinal infection Effect in mice

(1) Samples of example 1 and comparative example were diluted to 2E+9vg/μl, respectively, and the vitreous cavity was injected into C57BL-6J mice, and 1.5 μl (total virus amount of 3E+9vg) was injected into each eye, and 3 mice were injected with each virus.

(2) After virus injection for 3W, the mice were anesthetized, fluorescent fundus imaging was performed with a micro-n IV small animal retina imaging system, and the photographing parameters were: gain (dB) -10dB, frame Rate (fps) -2fps, exposure-Auto

(3) Taking eyeballs of the mice, embedding paraffin after fixation, slicing, staining with DAPI, and observing and photographing with a fluorescence microscope.

The results of the fluorescence OCT on the fly after 3W virus injection are shown in fig. 7, from which it can be seen that AAV2-MutF virus is not less expressed in the retina than AAV2-WT virus. The results of DAPI staining of paraffin sections of the eyeballs are shown in FIG. 8, in which AAV2-MutF virus was injected into the vitreous cavity to infect cells of each layer of retina from RGC to PRC, whereas AAV2-WT only infects cells of RGC layer. The infection capacity and penetrability of the adeno-associated virus variant AAV2-MutF prepared by the scheme in the retina are obviously improved.

The sequence alignment of the sequence MutF of the adeno-associated virus variant capsid protein and the wild-type AAV2 capsid protein (SEQ ID NO.: 1) is shown in FIG. 9. The sequence of the adeno-associated virus variant capsid protein differs from the wild-type AAV2 capsid protein in that the wild-type capsid protein sequence is replaced by 2 amino acids N563, D578. The amino acid sequence of the wild-type adeno-associated virus capsid protein Cap2 is shown in SEQ ID NO.1, and the amino acid sequence of the adeno-associated virus variant capsid protein Cap2 is shown in SEQ ID NO. 2.

Amino acid sequence of MutF mutant portion: DE (DE)NEIRTTNPVATEQYGDVSTNLQRGNR (SEQ ID NO. 3).

In conclusion, the adeno-associated virus variant prepared by the scheme of the invention can obviously enhance the retinal infection capacity and penetrability of AAV2 viruses compared with a wild type.

Amino acid sequence of wild-type adeno-associated virus capsid protein Cap2 SEQ ID No.1: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (protein ID: YP_ 680426.1)

Amino acid sequence of adeno-associated virus variant capsid protein Cap2, SEQ ID No.2: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDENEIRTTNPVATEQYGDVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

Claims (10)

1. An AAV viral capsid protein mutant MutF, wherein amino acids 561-588 of the wild type AAV2 viral capsid protein are replaced with a polypeptide sequence DENEIRTTNPVATEQYGDVSTNLQRGNR compared to the wild type AAV2 viral capsid protein.

2. The AAV viral capsid protein mutant MutF according to claim 1, wherein the amino acid sequence is as set forth in SEQ ID No.: 2.

3. A nucleic acid sequence molecule that expresses the AAV viral capsid protein mutant MutF of claim 1 or 2.

4. An expression system capable of expressing the AAV viral capsid protein mutant MutF of claim 1 or 2, or comprising the nucleic acid sequence molecule of claim 3.

5. The expression system of claim 4, wherein the expression system is a recombinant AAV vector obtained by insertion or replacement engineering of an AAV vector.

6. The expression system according to claim 5, wherein the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10, and mutants thereof.

7. The expression system of any one of claims 4-6, further comprising a nucleic acid molecule encoding a functional gene product.

8. A composition comprising the nucleic acid sequence molecule of claim 3, or the expression system of any one of claims 4-7.

9. Use of the composition of claim 8 for the preparation of an eye-targeting gene therapy formulation or transgene formulation.

10. The use of claim 9, wherein the composition targets the retina or lens.

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