WO2023164964A1

WO2023164964A1 - Genetically engineered drug for treating brain injuries and preparation method therefor

Info

Publication number: WO2023164964A1
Application number: PCT/CN2022/080253
Authority: WO
Inventors: 林俊; 高诚
Original assignee: 林俊
Priority date: 2022-03-01
Filing date: 2022-03-11
Publication date: 2023-09-07

Abstract

Provided are a genetically engineered drug for treating brain injuries and a preparation method therefor. The preparation method specifically comprises the following operation steps: S1: biological enzyme sampling, and extraction and mixing; S2: gene transferring, and mother cell cultivating; and S3: preliminary isolation, and an in-vivo experimental study. Brain injuries often lead to various pathological changes such as brain edema, nerve cell death, and neuroinflammation, and these pathological changes often occur simultaneously. Therefore, at present, only the symptomatic treatment is performed on these complex pathological processes. By detecting main death-associated proteins, especially caspase-8, and representative inflammatory factors after the injuries, it is found that the genetically engineered drug can block the occurrence of nerve cell death after brain injuries, and meanwhile effectively inhibit the inflammatory response, and therefore can effectively inhibit neurodegenerative changes and brain edema formation caused by brain injuries.

Description

A kind of genetic engineering drug for treating brain injury and its preparation method

This application claims the priority of an invention patent application submitted to the China Patent Office on March 1, 2022, with the application number 2022101940447, and the title of the invention is "a genetically engineered drug for treating brain injury and its preparation method", the entire content of which Incorporated in this application by reference.

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering medicine for treating brain injury and a preparation method thereof.

Background technique

Brain injury refers to the destruction and death of glial cells (including neurons and glial cells) in brain tissue caused by various factors, which in turn leads to the occurrence of neurological dysfunction. Due to the non-renewable nature of neurons, brain injury is often a high-risk factor for various neuropsychiatric diseases and neurodegenerative diseases; at the same time, brain injury has a high incidence, high mortality rate and high disability rate, which seriously affects the quality of life of patients , causing a huge burden on society. In the United States, approximately 2.6 million patients with brain injuries are diagnosed each year. According to the different causes of brain injury, brain injury can be divided into traumatic brain injury and acquired brain injury. Because the severity of brain damage varies greatly, and brain damage in different regions can cause different symptoms, for example, focal brain damage often causes related symptoms including abnormalities in movement, sensation, speech, vision, hearing, etc., while diffuse brain damage Damage often affects memory, sleep, or causes confusion and coma. Therefore, there is no clear and unified method for the treatment of patients with brain damage at this stage.

At present, the treatment of brain injury is mainly symptomatic treatment: analgesics can be given for headaches, but narcotics or morphine drugs should not be used to avoid addiction, such as L-stephanidine, enteric-coated aspirin, naproxen, ibuprofen, etc. Dizziness can be given diphenhydramine, chlorobutanol, etc.; autonomic dysfunction can be given oryzanol, promethazine, γ-aminobutyric acid (γ-aminobutyric acid), methylphenidate (methylphenidate), Atropine (atropine sulfate), scopolamine, etc.; excited patients can be given perphenazine, diazepam (stable), oxazepam (norazepam), etc.; depressed patients can be given glutamic acid, γ-aminobutyric acid.

The present invention takes traumatic brain injury (but not limited to traumatic brain injury) in brain injury as an example, and explains the effect of the invention from the aspects of nerve cell death, inflammation, motor function, learning and memory ability and autonomous exploration ability after brain trauma .

Contents of the invention

The object of the present invention is to provide a lignin-in-phenolic resin molding compound to solve the problems raised in the above-mentioned background technology.

In order to achieve the above object, the present invention provides the following technical solutions: a genetically engineered drug for treating brain injury and a preparation method thereof, specifically according to the following steps:

S1: Biological enzyme sampling, extraction and mixing

(a) glycosylated monomeric metalloprotease extracted and expressed from mammalian organisms;

(b) using a reducing solution with a mixing ratio of 1:2-1:3 to activate the activity of the above-mentioned glycosylated monomeric metalloprotease to optimize its activity, and use proteolysis;

S2: Gene transfer, cultivating mother cells

The 978bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to produce pAAV9 SP146:ACP5 as the expression framework;

S3: Preliminary separation, in vivo experiment

(a) Routinely coat and operate the transfection product in an LB plate containing 80-100mg/L Amp, and the constructed plasmid is confirmed by sequencing and amplified for production;

(b) Centrifuge the transfection product at 10000-25000 rpm for 10-30 minutes at 0-8°C to obtain the supernatant and precipitate: the supernatant enters the chromatographic separation and purification procedure;

(c) take virus, in vitro experiment

By injecting recombinant adeno-associated virus (rAAV) virus into mice, and injecting the above-mentioned synthesized glycosylated monomeric metalloprotease;

(d) Recording the biological characteristics of the mice.

As preferably, the coding sequence of the virus is:

Gene ID: 54

acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase.

Technical effect and advantage of the present invention:

In the present invention, brain injury often leads to various pathological changes such as cerebral edema, nerve cell death, and neuroinflammation, and these pathological changes often occur simultaneously. Therefore, the current treatment for these complex pathological processes is only symptomatic. After detecting the main death-related proteins (especially caspase-8) and representative inflammatory factors after injury, it is found that the present invention can block the occurrence of nerve cell death after brain injury, and can effectively inhibit the inflammatory response at the same time. Based on this, the present invention can effectively inhibit the neuron degenerative changes and the formation of cerebral edema caused by brain injury.

reference sign

Fig. 1 is based on ring segment among the present invention;

Fig. 2 is the gene fragment image among the present invention;

Fig. 3 is the Wire-grip experimental result figure among the present invention;

Fig. 4 is the result figure of Escapegoat latency experiment among the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

A genetically engineered drug for treating brain damage and a preparation method thereof, specifically according to the following steps:

S1: Biological enzyme sampling, extraction and mixing

(b) Using a reducing solution with a mixing ratio of 1:3, activate the activity of the above-mentioned glycosylated monomeric metalloprotease to optimize its activity, and use proteolysis;

S2: Gene transfer, cultivating mother cells

S3: Preliminary separation, in vivo experiment

(a) Routinely coat the transfection product in an LB plate containing 100mg/L Amp, and the constructed plasmid is confirmed by sequencing and amplified for production;

(b) Centrifuge the transfection product at 25,000 rpm for 30 minutes at 8°C to obtain the supernatant and precipitate: the supernatant enters the chromatographic separation and purification procedure;

(c) take virus, in vitro experiment

By injecting recombinant adeno-associated virus (rAAV) virus into mice, and injecting the above-mentioned synthetic glycosylated monomeric metalloprotease, the coding sequence of the virus is:

Gene ID: 54

acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase;

(d) Recording the biological characteristics of the mice.

Embodiment two

S1: Biological enzyme sampling, extraction and mixing

(b) using a reducing solution with a mixing ratio of 1:2 to activate the activity of the above-mentioned glycosylated monomeric metalloprotease to optimize its activity, and use proteolysis;

S2: Gene transfer, cultivating mother cells

S3: Preliminary separation, in vivo experiment

(a) Routinely coat the transfection product in an LB plate containing 80mg/L Amp, and the constructed plasmid is confirmed by sequencing and amplified for production;

(b) The transfection product was centrifuged at 10,000 rpm for 10 minutes at 0°C to obtain the supernatant and precipitate: the supernatant entered the chromatographic separation and purification procedure;

(c) take virus, in vitro experiment

Gene ID: 54

(d) Recording the biological characteristics of the mice.

Embodiment three

S1: Biological enzyme sampling, extraction and mixing

(b) using a reducing solution with a mixing ratio of 1:2.5 to activate the activity of the above-mentioned glycosylated monomer metalloprotease to optimize its activity, and use proteolysis;

S2: Gene transfer, cultivating mother cells

S3: Preliminary separation, in vivo experiment

(a) Routinely coat the transfection product in an LB plate containing 85mg/L Amp, and the constructed plasmid is confirmed by sequencing and amplified for production;

(b) The transfection product was centrifuged at 23000rpm at 5°C for 27 minutes to obtain the supernatant and precipitate: the supernatant entered the chromatographic separation and purification procedure;

(c) take virus, in vitro experiment

Gene ID: 54

(d) Recording the biological characteristics of the mice.

Tartrate-resistant acid phosphatase (TRAP or TRAPase), also known as ACP5, is a glycosylated monomeric metalloprotease expressed in mammals. It has a molecular weight of approximately 35kDa, a basic isoelectric point (7.6–9.5), and optimal activity under acidic conditions. TRAP is synthesized as a potential zymogen and activated by proteolytic cleavage and reduction. It differs from other mammalian acid phosphatases by its inhibition of tartrate and by its molecular weight.

Mammalian TRAP is encoded by a gene located on chromosome 19 (19p13.2–13.3) in humans and chromosome 9 in mice. As expected from protein sequencing, TRAPDNA is highly conserved across mammalian classes. Human, mouse and pig TRAP genes all contain 5 exons, and have ATG codon at the beginning of exon 2, and exon 1 is non-coding. Among the exon 1 promoters, there are three different "tissue-specific" promoters: 1A, 1B and 1C. This will allow tight control of TRAP expression. Transcribed from this gene is a 1.5 kb mRNA with an open reading frame (ORF) of 969-975 bp, encoding a protein of 323-325 amino acids. In rat, the ORF has a length of 981 bp and encodes a protein of 327 amino acids. TRAP is translated as a single polypeptide. TRAP gene transcription is regulated by microphthalmia-associated transcription factors.

Recombinant adeno-associated virus (rAAV) has extremely low immunogenicity, high safety, wide range of host cells (capable of infecting both dividing cells and non-dividing cells), strong diffusion ability, and long time for expressing genes in vivo. One of the most promising vectors for gene research and gene therapy. Recombinant adeno-associated virus pseudotype 9, the key structure of the packaging genome is as follows: artificially synthesized macrophage-specific promoter SP146 drives human ACP5 gene.

Virus-associated plasmid: pAAV9-SP146-ACP5.

Construction method: the CDS sequence of ACP5 was synthesized by Genescript (Suzhou, China). The Infusion system was used to construct vectors according to the instruction manual (Clontech). The vector framework and insert were mixed at a ratio of 1/1 in a total volume of 5 μL, and the same volume was added to GeneArt ^™ Seamless Ligase Mix, mixed well with a pipette. The test tube was incubated at 50°C to complete the connection, and the Stable3 competent cells (E.coli) were transfected by heat shock. The 978 bp coding sequence was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9SP146:ACP5 as the expression framework. Transfection products were routinely coated in LB plates containing 100 mg/LAmp. The constructed plasmid was confirmed by sequencing and amplified for production. Virus production was done by Genescript (Suzhou, China), and downstream AAV was purified using the AAVpro purification kit (TakaraBioUSA).

Experimental data:

To further prove the effect (advantage) of this product or method on the data.

Mouse model: Quantitative cortical impactor (Controlled cortical impact, CCI) is a common instrument used to establish rodent traumatic brain injury (Traumatic brain injury, TBI) model, the animal model is recognized as a model for simulating brain injury one. Male, 6-8 week-old C57BL6/J mice were used as the research object, and the CCI instrument was used to adjust the blow depth to 1 mm to establish a mouse TBI model. After the injury, AAV-ACP5 and AAV-NC (negative control) in a volume of 2 μl were administered to the contralateral ventricle, respectively, and the cell death-related proteins in the brain tissue of the injured side were detected 1 day after the injury (1dpi), And changes in inflammation-related proteins; 1d–20d after injury to detect related behavioral changes.

result:

As shown in Figure 2-4

Detecting the protein changes of caspase family in the brain tissue of the injured side by immunoblotting: After AAV-ACP5 treatment, the protein expression level of the caspase family in the injured side brain tissue of mice was significantly reduced, That is, AAV-ACP5 treatment can alleviate neuronal cell death caused by traumatic brain injury;

Detection of protein changes of inflammatory factors in the brain tissue of the injured side by immunoblotting: After AAV-ACP5 treatment, the expression level of inflammatory factors in the brain tissue of the injured side of mice was significantly reduced, that is, AAV-ACP5 treatment can reduce the expression level of inflammatory factors in the brain tissue caused by traumatic brain injury. neuroinflammation.

The changes of motor function and learning and memory ability of mice after TBI were detected by related behavioral studies: mice treated with AAV-ACP5 showed significantly stronger motor ability, better learning and memory ability, that is, AAV-ACP5 treatment It can promote the recovery of motor function, learning and memory after TBI.

Claims

A genetically engineered drug for treating brain damage and a preparation method thereof, characterized in that: the specific steps are as follows:

S1: Biological enzyme sampling, extraction and mixing

(a) glycosylated monomeric metalloprotease extracted and expressed from mammalian organisms;

(b) using a reducing solution with a mixing ratio of 1:2-1:3 to activate the activity of the above-mentioned glycosylated monomeric metalloprotease to optimize its activity, and use proteolysis;

S2: Gene transfer, cultivating mother cells

The coding sequence of 978 bp was cloned into the vector pAAV9 containing the SP146 promoter to generate pAAV9 SP146:ACP5 as the expression framework;

S3: Preliminary separation, in vivo experiment

(a) Routinely coat and operate the transfection product in an LB plate containing 80-100mg/L Amp, and the constructed plasmid is confirmed by sequencing and amplified for production;

(b) Centrifuge the transfection product at 10000-25000 rpm for 10-30 minutes at 0-8°C to obtain the supernatant and precipitate: the supernatant enters the chromatographic separation and purification procedure;

(c) take virus, in vitro experiment

By injecting recombinant adeno-associated virus (rAAV) virus into mice, and injecting the above-mentioned synthesized glycosylated monomeric metalloprotease;

(d) Recording the biological characteristics of the mice.
A kind of genetic engineering drug for treating brain injury and preparation method thereof according to claim 1, characterized in that: the coding sequence of the virus is:

Gene ID: 54

acid phosphatase 5; tartrate resistant (ACP5); also known as HPAP; TRAP; TRACP5a; TRACP5b; TrATPase.