A recombinant adeno-associated virus, an anti-inflammatory drug and use thereof in the treatment of septic encephalopathy

Abstract

The present invention provides a recombinant adeno-associated virus, an anti-inflammatory drug and its use in the treatment of septic encephalopathy, belonging to the field of biological drug technology. A recombinant adeno-associated virus, including a modified capsid protein and an adeno-associated virus genome containing an ALK1 gene expression element wrapped inside the modified capsid protein; the modified capsid protein is a capsid protein formed after the 663rd amino acid of the coding frame is mutated to valine on the basis of the type 6 adeno-associated virus capsid protein VP1. The present invention directly injects the recombinant adeno-associated virus into the cerebellar medullary cisterna magna to introduce the expression element into the microglia of the central nervous system, thereby achieving overexpression of the ALK1 gene in the microglia and inhibiting its inflammatory response. At the same time, the adeno-associated virus carrying the ALK1 gene is verified in a mouse model of septic encephalopathy to alleviate septic encephalopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicines, and in particular relates to a recombinant adeno-associated virus, an anti-inflammatory drug and applications thereof in the treatment of septic encephalopathy. Background Art

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis and its related complications are the leading causes of death in critically ill patients. Septic encephalopathy is one of the most common sepsis-related complications, which is associated with poor prognosis and long-term cognitive dysfunction in patients with sepsis. It is also the most common type of encephalopathy in critically ill patients. In addition to the clinical manifestations related to sepsis, septic encephalopathy is mainly manifested by changes in mental state, especially changes in consciousness level and cognitive function. The treatment of septic encephalopathy is extremely challenging, and there is currently no effective method to prevent and treat septic encephalopathy. For septic encephalopathy that occurs clinically, the main treatment is symptomatic. It is of great significance to develop new and effective methods to treat septic encephalopathy.

[0003] The systemic inflammatory response under the pathological conditions of sepsis further activates immune cells in the central nervous system and triggers a neuroinflammatory response. Studies have shown that neuroinflammatory response is an important factor affecting septic encephalopathy. Microglia are the main immune cells in the central nervous system and play a key role in regulating neuroinflammatory responses and maintaining nervous system homeostasis. The activation of microglia is an important factor in the occurrence of septic encephalopathy. Therefore, alleviating the symptoms of septic encephalopathy by inhibiting the activation of microglia is a very promising therapeutic strategy.

[0004] Unfortunately, however, there are currently no reports on the preparation of drugs for alleviating or treating septic encephalopathy by inhibiting the activation of microglia. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant adeno-associated virus, which can efficiently infect microglia and regulate the inflammatory response in the cells, thereby providing a new and effective strategy for the clinical preparation of drugs for the treatment of septic encephalopathy.

[0006] The present invention provides a recombinant adeno-associated virus, comprising a modified capsid protein and an adeno-associated virus genome containing an ALK1 gene expression element encapsulated in the modified capsid protein;

[0007] The modified capsid protein is a capsid protein formed after the 663rd amino acid in the coding frame is mutated to valine based on the capsid protein VP1 of type 6 adeno-associated virus.

[0008] In the adeno-associated virus genome containing the ALK1 gene expression element, the ALK1 gene expression element is located between two ITRs of the adeno-associated virus genome.

[0009] Preferably, the adeno-associated virus genome is an adeno-associated virus type 2 genome.

[0010] Preferably, the type 2 adeno-associated virus genome is a type 2 self-complementary adeno-associated virus genome;

[0011] The type 2 self-complementary adeno-associated virus genome is a partial sequence of the type 2 adeno-associated virus genome with the 3' end ITR missing;

[0012] The partial sequence of the deleted 3' ITR is shown in SEQ ID NO:1.

[0013] Preferably, the amino acid sequence of the modified capsid protein is shown in SEQ ID NO:2.

[0014] Preferably, the ALK1 gene expression element includes polII promoter, ALK1 gene and polII terminator in sequence.

[0015] Preferably, the polII promoter is selected from one of CMV, PGK, EF1a, SV40, and Ubc promoters;

[0016] The polII terminator is selected from one of SV40pA, bGHpA, TKpA and rGBpA.

[0017] Preferably, the nucleotide sequence of the ALK1 gene is shown in SEQ ID NO:3.

[0018] The invention provides an anti-inflammatory drug, comprising the recombinant adeno-associated virus and medically acceptable adjuvants.

[0019] The present invention provides the use of the recombinant adeno-associated virus in preparing a medicine for preventing and / or treating septic encephalopathy.

[0020] The present invention provides a recombinant adeno-associated virus, including a modified capsid protein and an adeno-associated virus genome containing an ALK1 gene expression element wrapped inside the modified capsid protein; the modified capsid protein is a capsid protein formed after the 663rd amino acid of the coding frame is mutated to valine on the basis of the 6-type adeno-associated virus capsid protein VP1. Since adeno-associated virus (Adeno-associated virus) is a virus widely used in gene therapy, its safety is generally recognized. Adeno-associated viruses are divided into many subtypes, and each subtype of virus has a unique preference for infected cells. Among the various subtypes of adeno-associated viruses, the 6-type adeno-associated virus has a high infection efficiency for macrophages, dendritic cells, microglia, etc., and the infection efficiency of microglia cells is further improved after the 663rd amino acid of the 6-type adeno-associated virus capsid protein VP1 is mutated from serine to valine. Therefore, the present invention uses the 6-type adeno-associated virus capsid protein after the 663rd amino acid mutation to package the virus to achieve efficient introduction into microglia. ALK1 is a receptor for the anti-inflammatory factor TGF-β. The present invention has found that under the pathological state of sepsis, the expression of ALK1 in microglia is significantly reduced, indicating that the regulatory effect of TGF-β on microglia is inhibited under the state of sepsis. Therefore, the present invention carries ALK1 expression elements in adeno-associated viruses, and encapsulates the viral genome through the type 6 adeno-associated virus capsid protein (AAV6 CapS663V) with the 663rd amino acid mutation, and directly injects these elements into the cerebellar medullary pool to efficiently introduce these elements into the microglia of the central nervous system, thereby achieving overexpression of the ALK1 gene in microglia and inhibiting its inflammatory response. The present invention further verifies the alleviating effect of adeno-associated viruses carrying the ALK1 gene on septic encephalopathy in a mouse model of septic encephalopathy. It can be seen that the present invention uses adeno-associated viruses that efficiently infect microglia to introduce the ALK1 gene into microglia, thereby enhancing TGF-β signals, inhibiting the activation of microglia in sepsis, and alleviating the occurrence of septic encephalopathy. The recombinant adeno-associated virus provided by the present invention provides a new idea for the treatment of septic encephalopathy.

[0021] Furthermore, the present invention specifically defines the type of adeno-associated virus genome as a type 2 self-complementary adeno-associated virus genome. After infecting target cells, adeno-associated viruses need to replicate to form double strands before they can begin to express the target gene they carry, and this step limits the speed at which the target gene is expressed after the virus infects cells. The present invention can form a self-complementary adeno-associated virus by deleting a segment of bases from the 3'-end ITR of the adeno-associated virus, further accelerating the speed and efficiency of expression after the virus infects cells, thereby improving the inhibition of microglial activation and inhibiting inflammatory responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The results are for the expression of ALK1 gene in microglia under the pathological conditions of sepsis;

[0023] Figure 2 is the structure of the scAAV2-ALK1 viral genome;

[0024] Figure 3 The results show the effect of overexpression of ALK1 on the inflammatory state of microglia.

[0025] Figure 4 The results show the effect of scAAV2-ALK1 virus injection on microglial activation in septic mice.

[0026] Figure 5 The results of the effects of injection of scAAV2-ALK1 virus on the behavior of septic mice;

[0027] Figure 6 is the structure of the AAV2-ALK1 viral genome;

[0028] Figure 7 The results show the behavioral effects of septic mice after injection of AAV2-ALK1 virus. DETAILED DESCRIPTION

[0029] The present invention provides a recombinant adeno-associated virus, comprising a modified capsid protein and an adeno-associated virus genome containing an ALK1 gene expression element encapsulated in the modified capsid protein;

[0030] The modified capsid protein is a capsid protein formed after the 663rd amino acid in the coding frame is mutated to valine based on the capsid protein VP1 of type 6 adeno-associated virus.

[0031] In the present invention, the ALK1 gene expression element in the adeno-associated virus genome containing the ALK1 gene expression element is located between two ITRs of the adeno-associated virus genome. The ALK1 gene expression element is inserted between two ITRs of the adeno-associated virus genome in a manner of replacing the original sequence.

[0032] In the present invention, the adeno-associated virus genome is preferably a type 2 adeno-associated virus genome. The type 2 adeno-associated virus genome is preferably a type 2 self-complementary adeno-associated virus genome. The type 2 self-complementary adeno-associated virus genome is a type 2 adeno-associated virus genome in which a partial sequence of the 3'-terminal ITR is deleted; the partial sequence of the deleted 3'-terminal ITR is shown in SEQ ID NO: 1 (GTGGCCAACTCCATCACTAGGGGTTCCT).

[0033] In the present invention, the amino acid sequence of the modified capsid protein preferably is as shown in SEQ ID NO: 2 (MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLV LPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDKDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNLQSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAEFVATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL).

[0034]

[0035] In the present invention, the method for constructing the recombinant adeno-associated virus preferably comprises the following steps:

[0036] The sequence between two ITRs in pscAAV-GFP or pAAV.CMV.Luc.IRES.EGFP.SV40 is removed to obtain pscAAV or pAAV vector, and the ALK1 gene expression element is cloned between two ITRs of pscAAV or pAAV to obtain a recombinant plasmid;

[0037] The amino acid 663 at VP1 coding frame of AAV6 cap gene was site-directedly mutated to valine to obtain packaging plasmid pAAV6 Rep-CapS663V;

[0038] The recombinant plasmid, pAAV6 Rep-CapS663V and pHelper are co-transfected into HEK293 cells, cultured, and the virus is isolated to obtain the recombinant adeno-associated virus.

[0039] In the present invention, the method for removing the sequence between the two ITRs in pscAAV-GFP or pAAV.CMV.Luc.IRES.EGFP.SV40 is preferably: removing the sequence between the two ITRs of pscAAV-GFP by restriction endonucleases AvrII and SpeI; the method for removing the sequence between the two ITRs in pAAV.CMV.Luc.IRES.EGFP.SV40 is preferably removing the sequence between the two ITRs in pAAV.CMV.Luc.IRES.EGFP.SV40 by restriction endonucleases BglII and ClaI. The ALK1 gene expression element is cloned between the two ITRs of pscAAV or pAAV through the AvrII / SpeI or BglII / ClaI restriction sites, respectively. The pscAAV-GFP was purchased from Addgene, with the catalog number Addgene#32396. The pAAV.CMV.Luc.IRES.EGFP.SV40 plasmid was purchased from Addgene, with the catalog number Addgene#105533.

[0040] In the present invention, the primers used for site-directed mutation of the amino acid at position 663 of the VP1 coding frame of the AAV6 cap gene to valine preferably include a forward primer having a nucleotide sequence as shown in SEQ ID NO: 4 (AATCCTCCGGCAGAGTTTgtGGCTACAAAGTTTGCTTC) and a reverse primer having a nucleotide sequence as shown in SEQ ID NO: 5 (GAAGCAAACTTTGTAGCCacAAACTCTGCCGGAGGATT). The template used is pAAV6 Rep-Cap, which was purchased from GENEMEDI. When performing site-directed mutation, PCR amplification reaction is preferably used. The reaction system of the PCR amplification is preferably PrimeSTAR GXL Premix Fast 25μl, 10μM forward primer 1μl, 10μM reverse primer 1μl, 2ng / μl template DNA 0.5μl, and ultrapure water is added to 50μl. The reaction program of the PCR amplification is preferably 98℃10s, 60℃5s, 68℃1min, for a total of 12 cycles. After the PCR reaction, 1 μl of DpnI enzyme was added to the PCR product and incubated at 37°C for 1 h. Then 1 μl of the treated PCR product was transformed into Escherichia coli DH5α, and the plasmid was extracted to obtain pAAV6 Rep-CapS663V.

[0041] In the present invention, the mass ratio of the recombinant plasmid pscAAV2-ALK1 (or pAAV2-ALK1), pAAV6Rep-CapS663V and pHelper is preferably 9:11:23, the culture time after transforming HEK293 cells is preferably 72 hours, and the method for isolating the virus is preferably iodixanol density gradient centrifugation to purify the virus. The virus preferably includes AAV2-ALK1 virus and scAAV2-ALK1 virus. The AAV2-ALK1 virus comprises the following elements from 5' to 3' end: AAV25'ITR, ALK1 gene expression element, AAV23'ITR. The scAAV2-ALK1 virus comprises the following elements from 5' to 3' end: AAV25'ITR, ALK1 gene expression element, AAV23'ITR-△trs. AAV23'ITR-△trs is formed by the deletion of the base sequence of AAV23'ITR, and the deleted base sequence is as shown in SEQ ID NO: 1.

[0042] In the present invention, after obtaining the virus, the titer of the virus is preferably measured. The measurement method is preferably performed using a qPCR method.

[0043] The invention provides an anti-inflammatory drug, comprising the recombinant adeno-associated virus and medically acceptable adjuvants.

[0044] The present invention has no particular limitation on the types of auxiliary materials, and auxiliary materials known in the art can be used.

[0045] In the embodiment of the present invention, microglia were infected with recombinant adeno-associated virus, and 24 hours after virus infection, microglia were stimulated with LPS, and the activation state of microglia was detected by enzyme-linked immunosorbent assay. The results showed that infection with recombinant adeno-associated virus was beneficial to reduce the secretion of IFN-γ and TNF-α, and significantly increased the secretion of IL-10. This shows that recombinant adeno-associated virus is beneficial to inhibit the activation of microglia.

[0046] The present invention provides the use of the recombinant adeno-associated virus in preparing a medicine for preventing and / or treating septic encephalopathy.

[0047] In the embodiments of the present invention, the present invention takes AAV6 CapS663V capsid-coated type 2 self-complementary adeno-associated virus scAAV2-ALK1 and AAV6 CapS663V capsid-coated type 2 adeno-associated virus AAV2-ALK1 as examples to illustrate the use of recombinant adeno-associated virus in the treatment of septic encephalopathy. The results show that the recombinant adeno-associated virus is an ideal preventive / therapeutic anti-inflammatory drug and can be used as a preparation for a drug for the treatment of septic encephalopathy.

[0048] The following is a detailed description of a recombinant adeno-associated virus, an anti-inflammatory drug and their use in the treatment of septic encephalopathy provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Type 2 adeno-associated virus plasmid pAAV.CMV.Luc.IRES.EGFP.SV40 and type 2 self-complementary adeno-associated virus plasmid pscAAV-GFP were purchased from Addgene; the adeno-associated virus packaging helper plasmid pHelper and AAV6 packaging plasmid pAAV6 Rep-Cap were purchased from GENEMEDI;

[0050] The ALK1 gene was commissioned to be synthesized by GenScript Biotech.

[0051] The ALK1 gene, promoter and terminator are cloned into adeno-associated virus or self-complementary adeno-associated virus plasmid in sequence by molecular cloning.

[0052] Example 1

[0053] Analysis of ALK1 gene expression in microglia under the pathological conditions of sepsis

[0054] 1. Establishment of sepsis mouse model

[0055] Adult C57BL / 6J mice were randomly divided into a sham operation control group and a cecal ligation and puncture model group. After anesthesia, the mice were shaved, an incision was made along the midline of the abdomen, and the cecum was separated and about 50% of the cecum was ligated using blunt forceps. A 21G needle was used to puncture the cecum, squeeze out a small amount of feces, and return the cecum to the abdominal cavity, and the abdomen was sutured layer by layer. After the operation, lidocaine cream was applied to the incision for analgesia, and normal saline preheated at 37°C was injected subcutaneously. In the sham operation control group, the mice only had their cecum exposed and their abdomens sutured and closed, without ligation or puncture. For details, please refer to the prior art (Immunodesign of experimentalsepsis by cecal ligation andpuncture, Daniel Rittirsch, doi:10.1038 / nprot.2008.214).

[0056] 2. Isolation of Microglia

[0057] Sepsis mice (CLP) and normal mice (Sham) were killed, and the brains were removed after cardiac perfusion with PBS. After digestion into single-cell suspension, microglia were separated using Miltenyi CD11b magnetic beads. Total RNA of microglia was extracted using Trizol reagent (Thermo Fisher Scientific Catalog No. 15596026), and RNA was reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher Scientific Catalog No. 4368814). Please refer to the product manual for the specific operation process.

[0058] 3. Real-time fluorescence quantitative PCR detection of ALK1 gene expression

[0059] 10ul SYBR Green premix (Thermo Fisher Scientific Catalog No. 4309155), 1ul each of forward and reverse PCR primers (concentration 1μM), and 8ul diluted cDNA template (2ng / μl) were added to each reaction tube, and the PCR reaction program was as follows: 95℃5min, 95℃30s, 60℃40s, and the reaction was carried out for 40 cycles. The forward primer: CAGGTTCCAGCATGGCCTAA (SEQ ID NO: 6), the reverse primer: CCTTGCCACGTTTCCAGTTG (SEQ ID NO: 7).

[0060] After the reaction, the expression of ALK1 gene in microglia of septic mice and normal mice was analyzed. Figure 1 It can be seen that the expression of ALK1 gene in microglia of septic mice is significantly lower than that in microglia of normal mice. This suggests that the regulatory effect of TGF-β on microglia is inhibited in the state of sepsis.

[0061] Example 2

[0062] Method for constructing type 2 self-complementary adeno-associated virus scAAV2-ALK1 carrying ALK1 gene expression elements and Cap6S663V capsid

[0063] 1. The scAAV2-ALK1 virus structure is as follows

[0064] The virus consists of a viral genome and a Cap6S663V capsid protein wrapped outside the genome, wherein the viral genome includes the following elements from the 5' to the 3' end: AAV25'ITR, EFS-ALK1-bGHpA expression element, AAV23'ITR-△trs. AAV23'ITR-△trs is formed by the AAV23'ITR missing base sequence, and the missing base sequence is as shown in SEQ ID NO: 1. Figure 2 The structure of the scAAV2-ALK1 viral genome is shown.

[0065] The EFS promoter sequence is as follows:

[0066] TAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGG GTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA(SEQ ID NO: 8);

[0067] The bGHpA sequence is as follows:

[0068] gggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatcattttgtctgactaggtgtcc ttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgggaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttgg ctcactgcaatctccgcctcctgggttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacgg ggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaaccactgctcccttccctgtcctt(SEQ ID NO: 9).

[0069] 2. Method for mutating the 663rd amino acid in the VP1 coding frame of AAV6 cap gene to valine

[0070] Using pAAV6 Rep-Cap (purchased from GENEMEDI) as a template, the 663rd amino acid of the VP1 coding frame of the AAV6 cap gene was mutated to valine by PCR-mediated site-directed mutagenesis to obtain the packaging plasmid pAAV6 Rep-CapS663V.

[0071] Among them, the primers for AAV6 CapS663V site-directed mutagenesis are:

[0072] S663V forward primer:

[0073] AATCCTCCGGCAGAGTTTgtGGCTACAAAGTTTGCTTC (SEQ ID NO: 4);

[0074] S663V reverse primer:

[0075] GAAGCAAACTTTGTAGCCacAAACTCTGCCGGAGGATT (SEQ ID NO: 5).

[0076] The reaction system for PCR-mediated site-directed mutagenesis was: PrimeSTAR GXL Premix Fast 25 μl, 10 μM forward primer 1 μl, 10 μM reverse primer 1 μl, 2 ng / μl template DNA 0.5 μl, and ultrapure water to 50 μl.

[0077] The reaction program of PCR-mediated site-directed mutagenesis was: 98°C for 10 s, 60°C for 5 s, and 68°C for 1 min, for a total of 12 cycles.

[0078] After the PCR reaction, 1 μl of DpnI enzyme was added to the PCR product and incubated at 37°C for 1 h. Then 1 μl of the treated PCR product was transformed into Escherichia coli DH5α, and the plasmid was extracted to obtain pAAV6Rep-CapS663V.

[0079] 3. Preparation of scAAV2-ALK1 virus

[0080] The pscAAV-GFP plasmid (Plasmid#32396) was obtained from Addgene, and the sequence between the two ITRs of pscAAV-GFP was removed by restriction endonucleases AvrII and SpeI. Then, the EFS-ALK1-bGHpA expression element was cloned into it through the AvrII and SpeI sites to obtain pscAAV2-ALK1.

[0081] 9 μg pscAAV2-ALK1, 11 μg pAAV6 Rep-CapS663V and 23 μg pHelper were co-transfected into a plate containing 1×10 7 scAAV2-ALK1 virus was packaged in a 150 mm cell culture dish of HEK293 cells, and the virus was collected 72 hours after transfection and purified by iodixanol density gradient centrifugation. The titer of the purified virus was determined by qPCR. The detection primer sequences used for qPCR were: AAV2 ITR for: GGAACCCCTAGTGATGGAGTT (SEQ ID NO: 10), AAV2 ITR reverse: CGGCCTCAGTGAGCGA (SEQ ID NO: 11). The detailed titer determination process can refer to the AAV titer determination method provided on Addgene.

[0082] After the above virus was packaged and purified, the virus titer was determined by qPCR to be 1.5×10 9 vg / μl.

[0083] Example 3

[0084] Application of scAAV2-ALK1 virus in the treatment of septic encephalopathy

[0085] A sepsis mouse model was established using the same method as in Example 1;

[0086] On the third day of modeling, the septic mice were divided into four groups of five. Two groups of mice were treated as follows: the drug or control reagent was injected into the cerebellar medullary cisterna magna with an injection volume of 10 μl. The first group of mice was injected with scAAV-ALK1 virus at a virus dose of 5×10 10 vg; the second group was injected with the control virus scAAV-scram, with a virus dosage of 5×10 10 vg. After one week of drug treatment, the mice were killed, and microglia were isolated using Miltenyi CD11b magnetic beads. Reverse transcription-PCR was used to detect the activation of microglia in mice under different treatments.

[0087] Test results see Figure 4 The results show that scAAV-ALK1 virus is beneficial to inhibit the activation of mouse microglia.

[0088] The behavioral abnormalities of septic encephalopathy are mainly manifested as cognitive impairment (memory, learning ability, etc.) or emotional disorders (anxiety, depression, etc.). In order to study the effect of scAAV-ALK1 on mouse behavior, the following experiments were conducted. The other two groups of septic mice were treated with scAAV-ALK1 and scAAV-scram, respectively, and the treatment method was the same as the previous step. After 3 weeks of drug treatment, the effects of different treatments on mouse behavior were analyzed through mouse behavioral experiments.

[0089] In this embodiment, the open field experiment is used to evaluate the emotional disorder level of mice after different treatments. After 7 days of modeling, an opaque box with a length / width / height of 50cm / 50cm / 40cm is used, and its bottom is divided into 25 small squares of 10cm×10cm, and the central area is circled. Before detection, let the mice adapt to the environment in the experimental room for 1-2h, adjust the light and start the experiment. Each mouse is released from the central square of the open field in the same direction, allowing it to explore freely in the box for 5min, and record the complete activities of the mouse by video. After each mouse is tested, 75% alcohol is used to thoroughly clean the open field, and the next mouse is tested after the smell disappears. The number of times and the length of stay of the mouse in the central area of ​​the open field can reflect its level of emotional disorder (anxiety). A reduction in the number of entries into the central area or a reduction in the length of stay in the central area is related to its anxiety state.

[0090] The analysis results are shown in Figure 5 The results showed that compared with the control group, the scAAV-ALK1 group significantly increased the number of times that septic mice entered the central area, indicating that scAAV-ALK1 has a significant effect in alleviating septic encephalopathy.

[0091] Example 4

[0092] Construction and functional verification of adeno-associated virus type 2 (AAV2-ALK1) carrying ALK1 expression elements and coated with AAV6 CapS663V capsid

[0093] 1. The AAV2-ALK1 virus structure is as follows:

[0094] The viral genome contains the following elements from 5' to 3' end: AAV 5'ITR, ALK1 gene expression element, AAV3'ITR. Figure 6 The structure of the AAV2-ALK1 viral genome is shown.

[0095] The other structures are the same as those in Embodiment 1.

[0096] 2. Preparation of AAV2-ALK1 virus

[0097] pAAV.CMV.Luc.IRES.EGFP.SV40 (Addgene #105533) was obtained from Addgene. The sequence between the two ITRs in pAAV.CMV.Luc.IRES.EGFP.SV40 was removed by restriction endonucleases BglII and ClaI. Then, the CMV-ALK1-SV40pA expression element was cloned into it through the BglII and ClaI sites to obtain pAAV2-ALK1. The other steps were the same as in Example 1, and the AAV2-ALK1 virus could be obtained.

[0098] 3. Application of AAV2-ALK1 virus in the treatment of septic encephalopathy

[0099] The sepsis mouse model was established according to the method of Example 1. AAV2-ALK1 and AAV2-scram were used respectively, and the method was the same as that of Example 2. After 3 weeks of drug treatment, the effects of different treatments on mouse behavior were analyzed by mouse behavioral experiments.

[0100] The analysis results are shown in Figure 7 The results showed that compared with the control group, the AAV-ALK1 group significantly increased the time that septic mice entered the central area, indicating that AAV-ALK1 has a significant effect in alleviating septic encephalopathy.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a recombinant adeno-associated virus in the preparation of a drug for preventing and / or treating septic encephalopathy, wherein the recombinant adeno-associated virus comprises a modified capsid protein and an adeno-associated virus genome containing an ALK1 gene expression element encapsulated in the modified capsid protein; The modified capsid protein is a capsid protein formed after the 663rd amino acid of the coding frame is mutated to valine based on the capsid protein VP1 of type 6 adeno-associated virus; The ALK1 gene expression elements are polII promoter, ALK1 gene and polII terminator in sequence.

2. The application according to claim 1, characterized in that: In the adeno-associated virus genome containing the ALK1 gene expression element, the ALK1 gene expression element is located between two ITRs of the adeno-associated virus genome.

3. The application according to claim 1, characterized in that: The adeno-associated virus genome is an adeno-associated virus type 2 genome.

4. The use according to claim 3, characterized in that: The type 2 adeno-associated virus genome is a type 2 self-complementary adeno-associated virus genome; The type 2 self-complementary adeno-associated virus genome is a partial sequence of the type 2 adeno-associated virus genome with the 3' end ITR missing; The partial sequence of the deleted 3' ITR is shown in SEQ ID NO:

1.

5. The use according to claim 1, characterized in that: The amino acid sequence of the modified capsid protein is shown in SEQ ID NO:

2.

6. The use according to any one of claims 1 to 5, characterized in that: The polII promoter is selected from one of CMV, PGK, EF1a, SV40, and Ubc promoters; The polII terminator is selected from one of SV40pA, bGHpA, TKpA and rGBpA.

7. The use according to claim 1, characterized in that: The nucleotide sequence of the ALK1 gene is shown in SEQ ID NO:3.

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