CN116555195A

CN116555195A - Adeno-associated virus capable of passing through blood brain barrier and relieving acute hypoxia brain injury and application thereof

Info

Publication number: CN116555195A
Application number: CN202310578716.9A
Authority: CN
Inventors: 常玮; 薛军辉; 李亚娟; 满明昊; 刘峰舟; 刘美杰
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2023-05-22
Filing date: 2023-05-22
Publication date: 2023-08-08

Abstract

The invention discloses an adeno-associated virus capable of passing through a blood brain barrier and relieving acute hypoxia brain injury and application thereof, belonging to the technical field of virus genetic engineering. Inserting a coding gene (the sequence of which is shown as SEQ ID NO. 1) of MICU1 as a target gene into a pHBAAV-SM22A-3flag-T2A-ZsGreen vector to obtain an adeno-associated virus plasmid; after cotransfection of the pAAV-RC vector plasmid, the pHelper vector plasmid and the adeno-associated virus plasmid into 293T cells, adeno-associated virus that passes the blood brain barrier and alleviates acute hypoxia brain injury was obtained. The experiment of acute hypoxia brain injury shows that the adeno-associated virus provided by the invention can infect brain tissues through the blood brain barrier of the mice, obviously improve the brain injury condition caused by the acute hypoxia exposure of the mice, and obviously reduce the apoptosis cell number of the brain tissues of the mice.

Description

Adeno-associated virus capable of passing through blood brain barrier and relieving acute hypoxia brain injury and application thereof

Technical Field

The invention belongs to the technical field of virus genetic engineering, and particularly relates to an adeno-associated virus capable of passing through a blood-brain barrier and relieving acute hypoxia brain injury and application thereof.

Background

Adeno-associated virus (AAV), a genus of the family picoviridae, has a viral particle size of about 20-26 nm and requires helper virus (typically adenovirus or herpes virus) for its complete life cycle. It encodes cap and rep genes in inverted repeats (ITRs) at both ends. Wherein the cap gene encodes a viral capsid protein and the rep gene is involved in viral replication and integration. AAV has multiple serotypes, and different serotypes have different affinities for different tissues, so that the AAV is suitable for various in vivo infection experiments. AAV has become the most promising gene therapy tool at present due to its advantages of good safety and long expression time. AAV packaging systems are classified into 3 plasmids, including shuttle plasmids into which foreign genes can be inserted, pAAV-RC encoding rep and cap protein encoding genes, and pHelper plasmids replacing adenoviruses on which adeno-associated viruses depend. AAV virus particles carrying exogenous inserted genes can be formed after 3 plasmids are co-transfected with tool cells AAV-293.

With the recent trend of the number of people entering the plateau, the human body damage caused by the peculiar low-pressure anoxic environment of the plateau is also more and more concerned. Exposure of the human body to high altitude areas for hours or days can lead to a range of clinical syndromes. Among them, brain damage caused by acute hypoxia is receiving increasing attention as one of the most common acute altitude reactions. The present study suggests that various mechanisms are involved in the development of acute hypoxia brain injury, in which Blood-brain barrier (BBB) disruption plays an important role in cerebral vasomotor dysfunction. For plateau hypoxic brain injury, cerebrovascular dysfunction plays an important role therein. Vascular smooth muscle cells are one of the main components constituting the vascular wall, and their great growth acceleration potential becomes a key factor for the occurrence and development of a number of vascular diseases, so that a method capable of alleviating brain damage caused by acute hypoxia is required.

MICU1, as a calcium ion sensing protein on the inner mitochondrial membrane, can regulate mitochondrial calcium uptake by sensing the concentration of calcium ions. Since calcium homeostasis is important in maintaining normal physiological processes in cells, the regulation of mitochondrial calcium by MICU1 greatly affects the processes of metabolism, growth, and apoptosis in cells.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the present invention aims to provide an adeno-associated virus which can pass through the blood brain barrier and alleviate acute hypoxia brain injury and application thereof, and alleviate brain injury caused by acute hypoxia exposure.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

the invention discloses an adeno-associated virus plasmid, which is transformed by taking AAVPHP.B plasmid as a vector and taking a coding gene of MICU1 as a target gene;

wherein the coding gene sequence of the MICU1 is shown as SEQ ID NO. 1.

Preferably, the ITR of the AAVPHP.B plasmid is inserted with an SM22A promoter, a flag tag, a T2A connecting sequence, a fluorescent protein, a WPRE post-transcriptional regulatory element and a polyA sequence in sequence.

Further preferably, the AAVPHP.B plasmid is a pHBAAV-SM22A-3flag-T2A-ZsGreen vector.

Further preferably, the construction method of the transgenic vector plasmid comprises the following steps: the pHBAAV-SM22A-3flag-T2A-ZsGreen vector is digested with BamHI and MluI, and the digested products are recovered to obtain a linearization expression vector; and recombining and transforming the target gene and the linearized expression vector to obtain the adeno-associated virus plasmid.

The invention also discloses an adeno-associated virus which can pass through the blood-brain barrier and relieve acute hypoxia brain injury, and the adeno-associated virus is obtained by taking the adeno-associated virus plasmid as a core plasmid.

Preferably, the construction method of the adeno-associated virus comprises the following steps: after cotransfection of 293T cells with pAAV-RC vector plasmid, pHelper vector plasmid and adeno-associated virus plasmid according to any one of claims 1-4, adeno-associated virus that passes the blood brain barrier and alleviates acute hypoxia brain injury is obtained.

The invention also discloses application of the adeno-associated virus capable of passing through the blood-brain barrier and relieving acute hypoxia brain injury in preparing a medicament for relieving brain injury caused by acute hypoxia exposure.

The invention also discloses a medicine capable of relieving acute hypoxia brain injury, and the main active ingredient of the medicine is adeno-associated virus which can pass through the blood brain barrier and relieve acute hypoxia brain injury.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

Preferably, the medicament is in the form of injection or freeze-dried powder.

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

the AAVPHP.B plasmid is used as a vector, so that the adeno-associated virus can break through the blood brain barrier in an intravenous injection mode, and is transferred to the whole nervous system of an organism, and the adeno-associated virus targets the nervous system in a global mode, thereby having the advantages of noninvasive, extensive and durable whole nervous system expression; the MICU1 coding gene is used as a target gene, so that the MICU1 expression in vascular smooth muscle cells can be up-regulated, and brain injury caused by acute hypoxia exposure can be reduced. The experiment of acute hypoxia brain injury shows that the adeno-associated virus provided by the invention can infect brain tissues through the blood brain barrier of the mice, obviously improve the brain injury condition caused by the acute hypoxia exposure of the mice, and obviously reduce the apoptosis cell number of the brain tissues of the mice.

Further, the vector contains a vascular smooth muscle specific promoter SM22a, so that the targeting of adeno-associated virus can be enhanced, and the vector has tissue specificity or cell specificity.

Drawings

FIG. 1 is a diagram of pHBAAV-SM22A-3flag-T2A-ZsGreen vector of the present invention;

FIG. 2 is a diagram showing the comparison and analysis of the sequencing results of positive clones of the present invention; wherein, the green area is the matching part with the target sequence;

FIG. 3 is a flow chart of an experiment of the adeno-associated virus package of the invention;

FIG. 4 is a graph showing the results of mycoplasma detection according to the present invention; wherein, the left side is a marker, and the right side is a detection result;

FIG. 5 is a graph of the results of fluorescence imaging of organs of mice not exposed to hypoxia in accordance with the present invention; wherein, the acute hypoxia-blanc group, the acute hypoxia-control virus group, the acute hypoxia-target virus group and the Color Scale are sequentially arranged from left to right;

FIG. 6 is a graph showing the results of immunofluorescent staining of vascular smooth muscle cells, endothelial cells, pericytes and astrocytes, respectively, using α -SMA, CD31, CD13 and AQP4 according to the present invention;

FIG. 7 is a graph showing the results of pathological histological evaluation of acute hypoxia brain injury by Nile-type staining according to the present invention; wherein A is an acute hypoxia-blanc group, B is an acute hypoxia-control virus group, C is an acute hypoxia-objective virus group, the scale is 50 μm, D is the proportion of damaged cells to total cells in the area, n=6, the numerical value is expressed by mean+ -SD, ^ns P＞0.05， ^* P＜0.05；

FIG. 8 is a graph showing the evaluation results of TUNEL staining of the hippocampus of the brain of the mice according to the present invention on acute hypoxia brain injury; wherein A is TUNEL staining fluorescence diagram of each group, wherein red fluorescence represents staining positive cells, blue represents DAPI, scale is 50 μm, B is brain cell apoptosis rate comparison of each group, n=6, the values are expressed by mean+ -SD, ^ns P＞0.05， ^* P＜0.05。

Detailed Description

The invention is described in further detail below with reference to the attached drawing figures:

the invention provides an adeno-associated virus capable of passing through a blood brain barrier and relieving acute hypoxia brain injury, which mainly comprises the following construction procedures: constructing a corresponding target vector, and designing a target fragment PCR primer; the BamHI and MluI are selected as restriction enzymes to carry out enzyme digestion of the vector at a proper position, and agarose gel is recovered to obtain a purified linearization vector; performing target fragment PCR according to the designed primer, and recovering agarose gel to obtain a target fragment with correct size; ligating the linearization vector and the target fragment according to a homologous recombination method; converting competent DH5 alpha, plating bacterial liquid, and culturing for 12-16 h; selecting a monoclonal travelling colony for verification; selecting positive clones with correct colony verification for sequencing; plasmid extraction is carried out on the cloned sample with correct sequencing, and then the adeno-associated virus is obtained through packaging.

1. Preparation of overexpressed adenovirus vectors

1. Vector and target gene information

The pHBAAV-SM22A-3flag-T2A-ZsGreen vector (available from Hantao Biotechnology Co., ltd.) is shown in FIG. 1; the gene encoding MICU1 (available from Hantao Biotechnology Co., ltd.) was used as the target gene, and the sequence thereof is shown in SEQ ID NO.1 of Table 1.

Table 1 sequence listing

2. Enzyme cutting of carrier

Sequentially adding each reagent according to the sequence in the table 2, gently sucking and beating, uniformly mixing, and placing in a water bath kettle at 37 ℃ for reaction for 1-2 h; and (3) after the enzyme digestion is finished, agarose gel electrophoresis is carried out, and the linearized carrier fragment is recovered and is subjected to insertion of the target fragment.

TABLE 2 vector cleavage System

3. Acquisition of fragments of interest

1) The sequences of the primers AAV-M-MICU1-B/M-F (i.e., F primer) and AAV-M-MICU1-B/M-R (i.e., R primer) are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively, in Table 1.

2) Preparing an amplification system in Table 3, lightly mixing, and placing in a PCR instrument for reaction; the reaction procedure is shown in Table 4, and the target fragment was amplified.

TABLE 3 PCR amplification System

TABLE 4PCR amplification procedure

Note that: 1) The annealing temperature is the Tm value of the primer, and the amplification specificity is directly determined by the annealing temperature; 2) If the amplification specificity is found to be poor, the annealing temperature can be properly increased by +2 ℃ each time; 2) Proper extension of the extension time helps to increase amplification yield.

4. Ligation of the fragment of interest with the vector

The connection system of Table 5 was prepared in an ice-water bath using HB infusion ^TM Cloning the connection system in one step, reacting the connection reaction liquid at 50 ℃ for 30min, and placing on ice for 5min, immediately converting to obtain a connection product. If the liquid is stuck to the pipe wall carelessly, the liquid can be sunk into the pipe bottom by short centrifugation.

Table 5 connection system

Note that: 1) It is recommended that the total amount of the target gene fragment used is 0.02 to 0.5 pmoles when 2 to 3 fragments are ligated (typically 100 to 150ng of the fragment is added and 50 to 100ng of the linearized vector is added), and the total amount of DNA added is 0.2 to 1.0 pmoles when 4 to 6 fragments are ligated. The DNA splice efficiency gradually decreases as the number of splice fragments increases or the length of the splice fragments increases.

5. Transformation

1) Taking out DH5 alpha competent cells from a refrigerator at the temperature of-80 ℃, immediately putting the cells on ice for melting, and carrying out competent split charging process, wherein the competent split charging process needs to be operated gently, so that the mechanical damage to the cells is reduced;

2) After competent melting, split charging with a volume of 50 μl per tube (20 μl is sufficient for plasmid transformation), adding the ligation product obtained in step 4 (currently adding 5 μl ligation product) in an amount not exceeding 1/10 of the competent volume, and standing on ice for 20-30 min;

3) Heat-shock the mixture of the ligation product of step 2) and competence at 42 ℃ for 90 seconds (the time is very strict), and immediately inserting the mixture into ice for 2-3 minutes after heat shock; then adding 500 mu L of antibiotic-free LB culture medium into an ultra clean bench, and gently reversing the culture medium upside down for 3 to 5 times; then shake culturing for 45-60 min at 37 ℃ and 230rpm to obtain bacterial liquid;

4) Coating the bacterial liquid obtained in the step 3) on a solid flat plate with corresponding resistance, uniformly coating, and then inversely placing the solid flat plate in a 37 ℃ incubator for culturing for 12-16 hours;

6. identification of bacterial liquids Using PCR

Referring to Table 6, the bacterial liquids were identified by PCR, the sequences of primer 1 and primer 2 are shown in SEQ ID NO.5 and SEQ ID NO.6 in Table 1, and the identification results show successful detection of the target DNA fragment, referring to Table 7.

TABLE 6 bacterial liquid PCR identification system

TABLE 7 identification procedure for bacteria

7. Sequencing

Two clones were selected from the positive clones selected for sequencing, and the sequencing results were compared with the analysis see SEQ ID NO.4 and FIG. 2 in Table 1, and the sequencing results showed that: the sequencing result is consistent with the target sequence, and the target plasmid is successfully constructed.

8 plasmid extraction

After sequencing was successful, bacterial liquid amplification was performed, plasmid extraction purification was performed, and the plasmid extraction protocol was used to transfect cells after QC validation was passed according to the instructions of the general plasmid extraction kit. The principle of plasmid QC is that the concentration is greater than 200 ng/. Mu.L and 260-280 is between 1.8 and 2.0 (the detailed data varies according to the kit).

2. Adeno-associated virus packaging and quality detection

Referring to the experimental flow chart of FIG. 3, after high-purity endotoxin-free extraction was performed on each of the three plasmid vectors, hantaan Lipofilter was used ^TM Transfection reagents (purchased from hantao biosciences limited) co-transfected 293T cells with three plasmids. Cell pellet was collected 72h after transfection. And (3) obtaining high-titer adeno-associated virus preservation solution by adopting a column purification mode, and finally determining various indexes of adeno-associated virus according to strict quality standards. The method comprises the following specific steps:

1. experimental material and instrument

1) Cell strain and strain

Packaging cell lines: 293T, packaging cells for adeno-associated virus, are anchorage dependent epithelioid cells, and the growth medium is DMEM (10% FBS). The adherent cells are grown by culture to form monolayer cells.

Strains: coli strain Stbl3 for amplifying adeno-associated viral vectors and helper packaging vector plasmids.

2) Three-plasmid adeno-associated virus packaging system

Three plasmid system: pAAV-RC vector plasmid, pHelper vector plasmid (all available from Hantao Biotech Co., ltd.) and shuttle plasmid carrying the gene of interest.

2. Adeno-associated virus packaging

The first day: 293T cells for transfection were passaged into 100mm dishes, then placed at 37℃in 5% CO ₂ And 95% relative humidity in an incubator.

Third day: extracting three plasmid vectors with high purity endotoxin-free solution, and using Lipofiter ^TM Transfection reagents (purchased from hantao) three plasmids were co-transfected into 293T cells.

Liquid replacement: 6h after transfection, fresh complete medium containing 10% fetal bovine serum FBS was changed.

Cell collection: 72h after transfection, cells containing AAV particles were gently scraped off with a cell scraper, collected in a 15mL centrifuge tube, centrifuged at 150 Xg for 3min to collect cells, the culture supernatant was removed, the collected cell pellet was washed once with PBS, and finally the cells were resuspended in 300. Mu.L of PBS.

Cell disruption: preparing a 37 ℃ constant-temperature water bath kettle and liquid nitrogen, and repeatedly freezing and thawing the centrifuge tube filled with cells in the liquid nitrogen and the 37 ℃ water bath for three times. Centrifugation was performed at 2000 Xg for 5min at 4℃to remove cell debris and the lysates containing AAV particles were collected.

3. Adeno-associated virus purification

Treatment with omnipotent nuclease: to 1mL of the crude virus extract, 0.1. Mu.L of Benonase enzyme was added, and the virus solution was subjected to water bath at 37℃for 1 hour to remove the cell genome and the residual plasmid DNA. Centrifuge at 4℃and 600 Xg for 10min, and collect the supernatant.

Column purification: purification was performed according to the Biomiga adeno-associated virus purification kit V1469-01; adding 4mL of AAV virus sample liquid obtained by column purification into an ultrafiltration tube, and centrifuging at 1400 Xg for 30min to obtain about 1mL of AAV; the finally obtained purified virus was collected and stored at-80 ℃.

4. Adenovirus quality detection

The quality control key points of adenovirus include sterility test, mycoplasma test and virus titer test.

1) Sterility testing

The detection method comprises the following steps: 10. Mu.L of virus was added to a 96-well plate containing HeLa cells for verification, and after culturing for 24 hours, microscopic examination was performed.

QC standard: the culture medium needs to be clear and transparent, has no obvious particles in cell gaps and no bacterial and fungal pollution.

Detection result: meets QC standard.

2) Mycoplasma detection

The detection method comprises the following steps: 10 mu L of virus is taken, water bath is carried out for 15min at 96 ℃, and a PCR reaction system is prepared in an ultra clean bench. Electrophoresis after PCR reaction determines whether mycoplasma contamination is contained.

QC standard: the PCR gel pattern has no obvious bands.

Detection result: FIG. 4 shows that around 500bp positions have bands, e.g.1, 2, 3, 6 indicate that the sample has mycoplasma contamination, and 4, 5 have no bands, indicating that there is no mycoplasma contamination.

3) Titer detection

In this experiment, the SYBRGreen method was used to measure AAV genome content and thus AAV titer.

Virus of interest: 1.5X10 ¹² vg/mL

Control virus: 1.3X10 ¹² vg/mL

Conclusion: the virus titer is high, and the experimental requirement can be met.

3. Verification of adeno-associated virus to alleviate acute hypoxia brain injury

Male C57BL/6J mice of 4 weeks of age were randomly divided into 3 groups: mice of the acute hypoxia-blanc group, the acute hypoxia-control virus group and the acute hypoxia-objective virus group were injected with 100. Mu.L (viral titer not less than 1.2X10) from the canthus angle ¹² vg/ml) adeno-associated virus, and acute hypoxia-control virus group mice are injected with control diseasesThe virus (the control virus differs from the virus of interest in that the gene of interest is not inserted), and the mice of the acute hypoxia-virus group are injected with the virus of interest. After virus injection, all mice were kept in an animal house at 20-25 ℃ and the laboratory was given a 12h light-dark alternation time during which the mice were given free food and water. The normal feeding was continued for 28 days to allow stable expression of the virus. 3 animals are taken from each group to verify virus infection efficiency, and the other mice (6 animals in each group) are put into a hypoxia chamber (aviation medical system of air force medical university) for animal experiment&Macro oxygen company limited) was exposed for 5 days at a simulated altitude of 6000 m.

Virus infection efficiency validation: mice were sacrificed using intraperitoneal injection of 1% sodium pentobarbital and their hearts, livers, brains, and lungs were placed in a small animal living imaging system for fluorescence imaging, and the results of fig. 5 demonstrate that viruses can infect brain tissue through the blood brain barrier. Meanwhile, immunofluorescent staining was performed on vascular smooth muscle cells, endothelial cells, pericytes and astrocytes using α -SMA, CD31, CD13 and AQP4, respectively, and in combination with the virus autofluorescence ZsGreen, the results of fig. 6 found that both control virus and virus of interest acted mainly on vascular smooth muscle cells, and that a small amount of virus was also seen on endothelial cells and pericytes to initiate, but astrocytes were not seen with viral fluorescent expression. The results show that the vascular smooth muscle specificity of the virus is better. By immunofluorescence detection of MICU1, a significant increase in MICU1 expression of cerebrovascular smooth muscle in brain frozen sections of mice of the virus group of interest was found (n=3) compared to the control virus.

Hypoxia exposure experiment: the brain tissue neuron damage condition of each group of animals after hypoxia exposure was evaluated by using a Nile type staining. FIG. 7 shows that there is no significant improvement in brain injury in mice from the acute hypoxia-control virus group compared to the acute hypoxia-blanc group ^ns P > 0.05), but the virus of interest significantly reduces brain damage caused by mice under acute hypoxia exposure (P < 0.05, n=6). Brain apoptosis was assessed in groups of animals following hypoxia exposure using TUNEL staining. FIG. 8 shows the results compared to the acute hypoxia-blanc groupNo obvious change in positive cell number in brain tissue of mice in acute hypoxia-control virus group ^ns P > 0.05), but the number of apoptotic cells in brain tissue of acute hypoxia-objective virosome mice was significantly reduced (< P < 0.05).

The results show that the target virus can effectively improve brain injury caused by acute hypoxia exposure.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims

1. An adeno-associated virus plasmid is characterized in that AAVPHP.B plasmid is used as a vector, and a coding gene of MICU1 is used as a target gene for transformation;

wherein the coding gene sequence of the MICU1 is shown as SEQ ID NO. 1.

2. The adeno-associated virus plasmid of claim 1, wherein the aavphp.b plasmid has an SM22A promoter, a flag tag, a T2A junction sequence, a fluorescent protein, a WPRE post-transcriptional regulatory element, and a polyA sequence inserted in sequence in the ITR.

3. An adeno-associated virus plasmid according to claim 2, wherein the aavphp.b plasmid is a pHBAAV-SM22A-3flag-T2A-ZsGreen vector.

4. The adeno-associated virus plasmid of claim 3, wherein the construction method of the transgenic vector plasmid comprises the following steps: the pHBAAV-SM22A-3flag-T2A-ZsGreen vector is digested with BamHI and MluI, and the digested products are recovered to obtain a linearization expression vector; and recombining and transforming the target gene and the linearized expression vector to obtain the adeno-associated virus plasmid.

5. An adeno-associated virus which passes through the blood-brain barrier and alleviates acute hypoxia brain injury, characterized by being obtained by using the adeno-associated virus plasmid according to any one of claims 1 to 4 as a core plasmid.

6. An adeno-associated virus capable of passing the blood brain barrier and alleviating acute hypoxia brain injury according to claim 5, wherein the adeno-associated virus is constructed by the following method: after cotransfection of 293T cells with pAAV-RC vector plasmid, pHelper vector plasmid and adeno-associated virus plasmid according to any one of claims 1-4, adeno-associated virus that passes the blood brain barrier and alleviates acute hypoxia brain injury is obtained.

7. Use of an adeno-associated virus according to claim 5 or 6 which passes the blood brain barrier and reduces acute hypoxia brain damage in the manufacture of a medicament for reducing brain damage caused by acute hypoxia exposure.

8. A medicament for alleviating acute hypoxia brain injury, wherein the main active ingredient of the medicament is an adeno-associated virus capable of passing through the blood brain barrier and alleviating acute hypoxia brain injury as claimed in claim 5 or 6.

9. The medicament for reducing acute hypoxia brain injury of claim 8, further comprising a pharmaceutically acceptable carrier.

10. The medicine for alleviating acute hypoxia brain injury according to claim 8, wherein the medicine is in the form of injection or freeze-dried powder.