WO2023178909A1 - Use of dhcr24 gene in preparation of medicine for treating neurodegenerative disease - Google Patents

Abstract

The present invention relates to use of an AAV9 vector complex containing a DHCR24 gene in the preparation of a medicine for treating a neurodegenerative disease. The neurodegenerative disease is Alzheimer's disease (AD), Parkinson's disease, brain aging (brain atrophy), aging-related cognitive impairments, autism, Niemann-Pick disease, Huntington's disease, motor neuron disease, frontotemporal dementia, dementia with Lewy bodies, multiple sclerosis, or Rett syndrome. The present invention also provides a medicine for treating a neurodegenerative disease. The present invention is based on a novel theory of deficiency of cholesterol in the brain for AD morbidity. By means of a DHCR24 gene therapy method (DHCR24 gene function enhancement), the present invention enhances the synthesis and supply of cholesterol in the brain and helps to correct the deficiency (or lack) of cholesterol in the brain, so that the nerve function defect (including a learning memory function) of the neurodegenerative disease such as AD is improved.

Description

Application of DHCR24 gene in the preparation of drugs for the treatment of neurodegenerative diseases Technical field

The present invention relates to the technical field of gene therapy, specifically to the application of DHCR24 gene in the preparation of drugs for the treatment of neurodegenerative diseases.

Background technique

Alzheimer's disease is a degenerative disease that is more common among dementias, accounting for more than 60% of them. Memory impairment is the most common clinical condition, characterized by a comprehensive decline in the patient's cognitive, behavioral, language and other brain functional activities, which is irreversible.

24-Dehydrocholesterolreductase (DHCR24) is a key gene that controls cellular cholesterol synthesis: research has found that the 24-Dehydrocholesterolreductase (DHCR24) gene is about 46.4kb in length and is located on chromosome 1 1p31.1 -p33, containing 9 exons and 8 introns, encodes a protein molecule containing 516 amino acid residues. In the cellular cholesterol synthesis pathway, DHCR24 is a key regulatory gene for cellular cholesterol synthesis (Figure 1). If the expression of DHCR24 gene in nervous tissue (cells) is increased, cholesterol synthesis in nervous tissue (cells) can be increased, the cholesterol level in nervous tissue (cells) can be increased, and the cholesterol deficiency in nervous tissue (cells) can be corrected. So far, there are no drugs or technologies targeting the DHCR24 gene at home or abroad.

Clinical research progress of gene therapy and clinically approved gene therapy drugs: Over the past 20 years, gene therapy technology using Adeno-Associated Virus (AAV) as a gene carrier has become increasingly mature, and European and American countries have carried out more and more More and more clinical gene therapy trials are being carried out and applied to the clinical treatment of many diseases that were previously unable to be effectively treated. Over the past 20 years, the importance of gene therapy in clinical treatment has continued to increase. In 2018 alone, the United States approved more than 200 clinical trials of gene therapy. See Figure 2 (2000-2018, the number of gene therapy drugs in the United States clinical trial statistical reporting).

The first AAV-based gene therapy approved for clinical use in Europe by the European Medicines Agency (EMA) is alipogene tiparvovec (AT) for the treatment of lipoprotein lipase deficiency (LPLD), a Rare autosomal recessive disorder. The drug was approved in 2012 under the brand name Glybera. AT gene therapy delivers a variant of the LPL gene, LPLS447X, to correct the function of LPL in patients with LPL. In a phase III clinical trial, peak chylomicron levels were reduced by 79% following a low-fat diet 14 weeks after AT administration.

So far, AAV-based gene therapies approved by the U.S. Food and Drug Administration (FDA) include Luxturna from Sparktherapeutics and Zolgensma from Avexis, which are used to treat inherited retinal diseases and spinal muscular atrophy respectively. . AAV is considered to have a better safety profile due to its lower immunogenicity and site-specific integration ability. There are currently 17 U.S. FDA-approved gene therapies, including the AAV vector voretigene neparvovec rzyl (VN), developed by Spark Therapeutics in 2017 under the trade name Luxturna. VN contains an AAV2 that encapsulates the RPE6 gene and is used to treat biallelic RPE65-associated retinal dystrophy, a rare genetic disorder that results in impaired visual function that declines with age and ultimately leads to blindness. In a phase III clinical trial conducted in 2012 (NCT00999609), 65% of participants achieved maximal improvement 1 year after subretinal injection of VN in patients with Leber congenital amaurosis type 2. Before the approval of VN, there were no drugs to treat retinal dystrophies. The second AAV-based gene therapy approved by the FDA in 2019 is Onasemogene abeparvovec xioi (OA), developed by AveXis under the trade name Zolgensma. OA utilizes AAV9 expressing a functional SMN1 transgene to treat spinal muscular atrophy type I (SMA1) in children younger than 2 years of age.

Therefore, gene therapy technology has become a new and innovative clinical treatment technology that large foreign pharmaceutical companies and biotechnology companies are competing to develop. At present, a few domestic biotechnology companies have carried out exploratory research in this area, and the clinical application prospects are broad.

A key consideration for successful gene therapy is the vector used to deliver the therapeutic nucleic acid to express the gene. AAV viruses have been used as gene therapy vectors and are more effective in cellular uptake, gene integration, and long-term gene expression, especially in vivo, but they are immunogenic, potentially carcinogenic, and have cumbersome manufacturing processes. To address these limitations, nonviral vectors that avoid immune responses, have broad adjustability and quality control have been developed, but they suffer from low transfection efficiency and result in passive expression of transgenes. In short, its clinical application is still limited by the immunogenicity, carcinogenicity, off-target effects and efficacy of gene vectors.

Contents of the invention

The present invention is based on the new brain cholesterol deficiency theory of AD onset. Our years of research have found that insufficient (or lack of) cholesterol in the brain plays a key role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Research has found that insufficient cholesterol synthesis in the brain is one of the main causes of insufficient (or lack of) cholesterol in the brain. Our research has found that by increasing the expression of DHCR24 gene in the nervous system (functional enhancement), the synthesis of cholesterol in the brain can be significantly increased and the supply of cholesterol can be increased, thereby correcting the insufficient or deficient cholesterol levels in the brain and improving the neurological deficits in neurodegenerative diseases ( Including learning and memory functions). In AD mouse model research, we found that DHCR24 gene function enhancement (knock-in) can effectively improve the learning and memory functions of AD mice (see subsequent research results).

The basic purpose of this technology is: intracerebral transfection method of carrying DHCR24 gene through adeno-associated virus (AAV) vectors (including lentivirus, adenovirus, adeno-associated virus, modified adeno-associated virus, liposome and other vectors) Methods for adding the DHCR24 gene to specific neural tissue (or cells) to further promote DHCR24 gene expression in specific neuronal tissues (or cells), increasing the DHCR24 expression level of specific neuronal tissues (or cells), and promoting specific neuronal tissues (or cells) Synthesis and release of cholesterol from neuronal cells) to help correct the cholesterol deficiency (or lack thereof) in the brain. A technical method to increase cholesterol levels in neuronal tissues (or cells) through DHCR24 gene addition (gene function enhancement). In summary, this technical approach can help correct cholesterol deficiencies or deficiencies in neural tissue (or cells). So far, there are currently no drugs or treatment technologies targeting the DHCR24 gene at home or abroad. This technology has potential and important clinical application prospects for neurodegenerative diseases such as AD.

The first object of the present invention is to provide the use of an AAV9 vector complex containing the DHCR24 gene to address the deficiencies in the prior art.

The second object of the present invention is a drug for treating neurodegenerative diseases.

In order to achieve the above-mentioned first purpose, the technical solution adopted by the present invention is:

The application of an AAV9 vector complex containing the DHCR24 gene in the preparation of drugs for the treatment of neurodegenerative diseases. The AAV9 vector complex containing the DHCR24 gene is directly synthesized from the SEQ ID NO.1 sequence into pHBAAV-CMV-MCS-3flag -T2A-ZsGreen vector.

As a preferred example, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging (brain atrophy), aging-related cognitive impairment, autism, Niemann-Pick's disease, Huntington's disease , motor neurone disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis, Rett syndrome.

As a preferred example, the neurodegenerative disease is Alzheimer's disease.

In order to achieve the above second purpose, the technical solution adopted by the present invention is: a drug for treating neurodegenerative diseases, the drug is an AAV9 vector complex containing the DHCR24 gene, and the AAV9 vector complex containing the DHCR24 gene The SEQ ID NO.1 sequence is directly synthesized into the pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector.

As a preferred example, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging (brain atrophy), aging-related cognitive impairment, autism, Niemann-Pick's disease, Huntington's disease , motor neurone disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis, Rett syndrome.

The advantages of the present invention are:

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. AD is a neurodegenerative disease with insidious onset and progressive development. Currently, there are more than 55 million patients worldwide. With the aging of the population, the number of cases in the global population is still rising rapidly, causing serious social and health problems.

Over the past 30 years, researchers from many major pharmaceutical companies in the world have relied on a variety of different theoretical hypotheses for the onset of AD, such as genetic mechanisms, cholinergic hypothesis, β-amyloid hypothesis, tau protein hypothesis, etc. A large number of drug designs and studies have been carried out for different hypotheses, especially for the treatment of β-amyloid protein hypothesis. Various clinical trials costing tens of billions of dollars have almost completely failed. So far, all traditional treatments for AD, such as anti-β-amyloid, anti-Tau, antioxidant, etc., have not achieved good therapeutic effects. Therefore, the treatment of AD is currently at a standstill.

Comparing the past traditional AD treatment techniques and methods, the present invention belongs to a new gene therapy technique that is completely different from the past AD traditional treatment techniques. The effect of this technical method in treating AD is very significant, which is significantly better than the traditional treatment methods for AD in the past.

This invention is based on the new theory of cholesterol deficiency in the brain that causes AD. It enhances the synthesis and supply of cholesterol in the brain through DHCR24 gene therapy (DHCR24 gene function enhancement), helps correct the deficiency (or deficiency) of cholesterol in the brain, and thereby improves AD, etc. Neurological deficits in neurodegenerative diseases (including learning and memory functions). The present invention may be suitable for the treatment of Alzheimer's disease, Parkinson's disease, brain aging (brain atrophy), aging-related cognitive impairment (some cognitive deficits typical of the old age), autism, and Niemann-Pick's disease , Huntington Disease (HD), motor neuron disease, frontotemporal lobe dementia, dementia with Lewy bodies (DLB), multiple sclerosis, Rett syndrome (Rett syndrome) and other neurodegenerative diseases The treatment of diseases may have very important social and economic value for the future treatment of neurodegenerative diseases such as AD.

Description of the drawings

Figure 1 Schematic diagram of the key role of DHCR24 in cellular cholesterol synthesis and homeostasis regulation DHCR24: 24-dehydrocholesterol reductase; Cholesterol: cholesterol.

Figure 2 Summary report of clinical trials of gene therapy drugs in the United States (2000-2018).

Figure 3 AAV9-mediated DHCR24 gene transfection through brain stereotaxic guidance and directional injection.

Figure 4 Adeno-associated virus AAV9 mediates DHCR24 gene transfection into the hippocampus of AD mice Experimental AAV9 empty vector and AAV9-DHCR24 gene were successfully transfected into the hippocampus of AD model mice. Figure 2 shows the wild-type mouse control group, the empty vector-transfected AD mouse group, and the DHCR24-transfected AD mouse group. The fluorescence is the autofluorescence of the AAV9 virus.

Figure 5 AAV9-mediated DHCR24 gene transfection into the hippocampus of AD mice Experimental AAV9-DHCR24 gene transfection into cells in the hippocampus of AD model mice. DHCR24 in the CA1 and CA2/CA3 regions showed significant positive overexpression (brown indicates positive expression of DHCR24).

Figure 6 The effect of AAV9-mediated DHCR24 gene transfection on the expression of DHCR24 mRNA in the hippocampus of AD model mice. In the AD mouse model transfected with the DHCR24 gene, compared with the control group, PCR detection found that the AD model mice after transfection had The expression level of hippocampal DHCR24 mRNA was significantly increased. N: Wild-type mice; NC: AD mice transfected with empty vector; Over exp: AD mice transfected with DHCR24 gene.

Figure 7 The effect of AAV9-mediated DHCR24 gene transfection on the expression of DHCR24 protein in the hippocampus of AD model mice. In the AD mouse model transfected with the DHCR24 gene, compared with the control group, Western blot testing found that the AD model mice after transfection The expression level of DHCR24 protein was very significantly increased.

Figure 8 The effect of DHCR24 overexpression on the cognitive function of AD model mice (water maze test). Compared with AD model mice, it was found through the water maze method that the platform latency time of AD model mice with DHCR24 overexpression was significantly shortened (Figure 8B), the number of platform crossings (Fig. 8C) and the percentage of target quadrants were also significantly improved (Fig. 8A). WT: wild-type C57 mice; APP/PS1:APP/PS1 double transgenic AD model mice; H-AAV: AD mice transfected with AAV9-DHCR24 gene in the hippocampus; H-NC: AAV-9 virus null in the hippocampus AD mice transfected with vector; L-AAV: AD mice transfected with lateral ventricle AAV9-DHCR24 gene; L-NC: AD mice transfected with lateral ventricle AAV9 empty vector.

Figure 9 DHCR24 participates in the pathological regulatory mechanism of AD by regulating cellular cholesterol metabolism.

Figure 10 pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector map.

Figure 11 m-dhcr24 plasmid map:

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Example 1

The technical content of the present invention is: 1) First, the target gene is designed and constructed for the target gene DHCR24, and the mouse DHCR24 (mouse-DHCR24, mouse-DHCR24) gene overexpression adeno-associated virus 9 (AAV9) vector is completed. Construction (with technical assistance provided by Hanheng Biotechnology (Shanghai) Co., Ltd.), and finally completed the preparation of AAV9-encapsulated DHCR24 gene drug (i.e., AAV9 vector complex containing DHCR24 gene). 2) We will use the AAV9-mediated DHCR24 gene transfection method to transfect the prepared AAV9-carrying DHCR24 gene drug (AAV9-encapsulated DHCR24 gene drug) into specific neural tissues (or cells) to improve specific neural tissues (or cells). ) DHCR24 expression level, thereby increasing cholesterol synthesis in nervous tissue (or cells) and increasing cholesterol supply, and helping to correct the technical method of cholesterol deficiency (deficiency) in nervous tissue (or cells).

Through this technical method, many neurodegenerative diseases caused by cholesterol deficiency or deficiency in the brain can be corrected. In AD animal model studies, we have demonstrated that DHCR24 gene overexpression (function enhancement, knock-in) can significantly improve the cognitive function of AD mice. So far, there are no drugs or technologies targeting the DHCR24 gene at home or abroad.

Experimental materials and methods

1.2.1 Experimental materials and instruments mainly include experimental animals C57 mice, APP/PS1 double transgenic AD mice, AAV9-wrapped DHCR24 gene drugs and several antibodies. Experimental instruments include brain stereotaxic instruments, fluorescence confocal microscopes, etc. The construction process and identification report of m-dhcr24 overexpression adeno-associated virus vector are shown below.

1.2.2 Experimental methods

1) Brain stereotaxy, targeted injection and AAV9-mediated DHCR24 gene transfection

The hippocampus is the memory center and is also a brain area susceptible to the onset of AD. Therefore, we chose the CA1 area of the hippocampus as the brain area for our targeted injection and gene therapy research. In this study, we used APP/PS1 double-transgenic AD mice as AD research models, and used the brain stereotaxic positioning method to complete hippocampal injection and transfection expression of AAV9-encapsulated DHCR24 gene in AD mice (Figure 3). This technology is one of the most efficient and convenient intracerebral drug delivery methods commonly used abroad.

2) AAV9-mediated DHCR24 gene transfection experiment in mouse hippocampus (brain stereotaxic injection method)

Through brain stereotaxic and directional injection methods, AAV9 virus-mediated DHCR24 gene (AAV9-encapsulated DHCR24 gene drug) was directionally injected and transfected into the CA1 area of the hippocampus of APP/PS1 double-transgenic AD mice. The AAV9-DHCR24 gene drug was successfully transfected. The hippocampi of AD model mice were stained (Figure 4).

Stereotaxic injection method into mouse brain

The experimental animals were divided into six groups: wild-type group, AD model group (AD control group), empty vector group, DHCR24 overexpression AD mouse group, DHCR24 overexpression (lateral ventricle group), and empty vector group (lateral ventricle group). Different groups, 30 animals in each group. According to the preliminary experimental results of the previous research group, the injection site 1 in the CA1 area of the hippocampus was located with reference to the stereotaxic atlas of the mouse brain (AP: -2.3mm; ML±1.8mm; DV: -1.8mm). The injection dose was based on the results of previous experiments (1.0 μl was injected into CA1 of each side of the hippocampus).

The wild-type group and the AD control group were not treated and served as the control group. The DHCR24 overexpression group and the empty vector group were respectively given stereotaxic microinjection of the AAV9-DHCR24 gene viral vector and its corresponding empty vector in the CA1 area of the hippocampus. In addition, the DHCR24 overexpression (lateral ventricle group) and empty vector group (lateral ventricle group) were given the same dose of AAV9-DHCR24 gene viral vector and its corresponding empty vector for lateral ventricular injection as control groups for different injection routes. Leave the needle in for 10 minutes after injection, then remove the needle and observe for 30 minutes. After the injection experiment, the mice were sent to the animal room for feeding and observation.

1.3 Experimental results

1.3.1 AAV9-mediated DHCR24 gene transfection experiment in mouse hippocampus (immunohistochemistry)

Through AAV9 virus-mediated targeted injection and transfection of DHCR24 gene, DHCR24 gene was successfully transfected into the hippocampus of AD model mice. Immunohistochemistry showed that the CA1 and CA2/CA3 regions showed significantly strong positive DHCR24 overexpression (Figure 5, brown indicates DHCR24 positive staining). The results suggest that AAV9 mediates the successful transfection and expression of DHCR24 gene in mouse hippocampus.

1.3.2 Effects of AAV9 virus-mediated HCR24 gene transfection on DHCR24 mRNA expression in hippocampus of AD mice

Five weeks after hippocampus-directed injection (surgery) of mice, in the DHCR24-overexpressing AD mouse model, compared with wild-type mice and empty vector AD mice, PCR detection found that DHCR24-overexpressing AD model mice had DHCR24 The mRNA expression level increased very significantly, approximately 19 times (Figure 6). The above results suggest that AAV9 mediates the successful transfection and overexpression of DHCR24 gene in the hippocampus of AD mice.

1.3.3 Effects of AAV9 virus-mediated HCR24 gene transfection on overexpression of DHCR24 protein in hippocampus of AD model mice

Five weeks after surgery, in the AD mouse model with overexpression of DHCR24, compared with wild-type mice and empty vector AD mice, Western blot testing found that the expression of DHCR24 protein in the AD model mice with overexpression of DHCR24 increased significantly. , an increase of approximately 313% (Figure 7). The above results suggest that AAV9 mediates the successful transfection and overexpression of DHCR24 gene in the hippocampus of AD mice.

1.3.4 Effects of DHCR24 overexpression on cognitive function in AD model mice (water maze test)

Five weeks after surgery, in the APP/PS1 double-transformed AD mouse model overexpressing DHCR24, compared with AD control mice and empty vector group AD mice, the water maze method was used to detect that the AD model after DHCR24 gene therapy The latency of mice was significantly shortened, and the number of crossings and target quadrant percentage were also significantly improved. Compared with wild-type mice (normal mice), the learning and memory functions of AD mice after DHCR24 gene treatment (enhanced gene function) are significantly close to the cognitive functions of normal mice (Figure 8). The above results suggest that DHCR24 gene therapy (overexpression) significantly improves the cognitive function of AD mice. Therefore, we infer that DHCR24 gene therapy may have important and potential clinical application value for AD patients.

1.4 Discussion

After more than 10 years of research, our research group found that 24-dehydrocholesterol reductase (DHCR24) gene knockout significantly inhibited neuronal cholesterol synthesis, and significantly decreased neuronal membrane cholesterol and intracellular cholesterol levels, suggesting that DHCR24 is responsible for cholesterol homeostasis. Key regulatory genes for regulation. In addition, DHCR24 function deficiency induces a decrease in neuronal cholesterol levels, leading to abnormalities and dysfunction of cell membrane lipid raft structures, which in turn induces Alzheimer's disease (AD) such as increased release of amyloid-beta protein in neurons and abnormal phosphorylation of Tau protein. of key pathological lesions. Many of the above-mentioned research evidences strongly support that cholesterol deficiency in Sertoli cells may be involved in the key pathological damage mechanisms related to AD (Figure 9).

We further carried out animal model research on AD mice. In AD animal model research, we use APP/PS1 double-transgenic AD mice as AD models, and transfect APP/PS1 mice with AAV9-mediated DHCR24 gene (that is, AAV9-wrapped DHCR24 gene drug) through brain stereotaxic positioning and orientation. In the CA1 region of the mouse hippocampus, the DHCR24 gene was successfully transfected and overexpressed in the CA1 region of the hippocampus, and significantly improved the cognitive function of AD mice. The results of the study show that overexpression (functional enhancement) of DHCR24 gene significantly improves the cognitive function of AD. Therefore, this study further proves that DHCR24 affects AD pathological activities by increasing the synthesis and supply of cholesterol in the brain and regulating neuronal cholesterol metabolism, thereby improving the cognitive function of AD mice (Figure 9).

Based on our research group's more than 10 years of research and accumulation on the cholesterol pathogenesis of AD, we have proposed for the first time in the world the "cholesterol theory that lack of cholesterol in the brain may be involved in the pathogenesis of AD." The paper will be published in the authoritative European journal Neuropathology Communications for the first time. Published in Acta Neuropathologica Communications. Our research has found that in AD patients and AD animal models, the mechanism of cholesterol deficiency in the brain may involve three links: reduced cholesterol synthesis, cholesterol transport disorders, and cholesterol cleavage disorders.

In summary, through more than 10 years of research on cell models and AD mouse models by our research group, we have further clarified that DHCR24 regulates neuronal cholesterol metabolism to intervene in AD, which may have important theoretical breakthroughs and guiding significance for the treatment of AD ( Figure 9). In addition, the DHCR24 gene may serve as a new target for the future treatment of AD. Modulating the DHCR24 gene target has important and potential clinical application value in improving AD cognitive function, providing a new theoretical basis and technical method for DHCR24 gene therapy.

m-dhcr24 overexpression adeno-associated virus vector construction process and identification report

An experimental material

Experimental reagents

Reagent name	Manufacturer
carrier	Hanbio Biotechnology
E. coli strain DH5α	Invitrogen
restriction endonuclease	Thermo Fisher Scientific
HB-infusionTM seamless cloning kit	Hanbio Biotechnology CO.LTD.
Plasmid DNA small and large amount extraction kit	Beijing ComWin Biotech
Gel recovery kit	Shanghai Generay Biotech
agarose, agar powder	Sangon Biotech
DNA ladder	Thermo Fisher Scientific
KOD-Plus Kit	TOYOBO CO.LTD.

Vector and target gene information

1. The pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector map is as follows:

See Figure 10

2. For m-dhcr24 sequence information, see SEQ ID NO.1

Note: This sequence is directly synthesized into the pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector.

2. Experimental process

1. The vector is digested at 37°C and the gel is recovered.

2. Recover the fragments after PCR

3. Connect the processed target fragment to the vector, and the reaction system (20μl)

4. Transformation (competent cells: DH5α). For specific steps, see the transformation section in the appendix.

5. Resistance: Amp, 37℃, 230rpm/min, culture overnight

6. Pick bacteria from the transformed plate, shake the bacteria at 37°C and 230 rpm/min for 14 hours, use the bacterial liquid for PCR identification, and send the positive clone liquid to a sequencing company for sequencing.

3. Experimental results

1. Sequencing results of m-dhcr24 overexpression vector: see SEQ ID NO.2

2. m-dhcr24 plasmid map:

See Figure 11.

DNA purification kit (column centrifugal type)

(DNA Pure-Spin Kit)

How to operate:

1) Add 3 to 4 times the volume of Buffer PSB (binding solution) to the DNA solution, invert or suspend and shake to mix.

2) Transfer less than 700 μl of the solution to the spin column inserted into the cannula, centrifuge at high speed for 1 minute on a desktop centrifuge, discard the waste liquid in the cannula, and then insert the spin column into the cannula.

3) Add more than 700 μl of remaining solution to the same spin column and repeat step 2.

4) Add 700μl of Buffer PW (washing solution) into the spin column, centrifuge at high speed for 1 minute, discard the waste liquid in the casing, and insert the spin column into the casing.

5) This step can be omitted and proceed directly to step 6. Add 200 μl of Buffer PW (washing solution) into the spin column and centrifuge at high speed for 1 to 2 minutes.

6) After high-speed centrifugation for 1 to 2 minutes, carefully remove the spin column to avoid getting the waste liquid in the casing. Discard the casing.

7) Insert the spin column into a new 1.5ml centrifuge tube, add 30 to 50 μl Elution Buffer (eluent) to the center of the silica gel membrane in the spin column, be careful not to touch the silica gel membrane; leave it at room temperature for 2 to 5 minutes, and centrifuge at high speed for 1 minute. The purified DNA solution is obtained in the centrifuge tube.

8) The obtained DNA solution can be directly used in subsequent experiments, or stored at -20°C for later use.

Precautions:

1) DNA recovery rate depends on the size of DNA fragments. Generally, fragments of 50bp to 10kb can be recovered efficiently. If the DNA content is too small, the recovery rate will also decrease. The binding amount of a spin column can generally reach 10 to 15 μg.

2) Omitting step 5 usually has no effect on the purification results, but sometimes trace amounts of salt will remain in the DNA solution.

DNA gel recovery and purification kit (column centrifugal type)

(Gel-Spin DNA Extraction Kit)

How to operate:

1) After DNA electrophoresis (it is recommended to use TAE buffer for agarose gel electrophoresis), use a clean blade to cut out the corresponding fragments under UV light. Carefully remove the DNA-free portion of the gel block.

2) Put the agarose gel block containing DNA into a 1.5ml centrifuge tube and estimate its volume. Add 500 μl (<150 μl gel) or 3 to 4 times (>150 μl gel) gel volume of Buffer PS (sol binding solution).

3) Place the centrifuge tube in a 50-60°C water bath for 5-10 minutes. Take out the suspension and shake it for 10 seconds every 2-3 minutes until the agarose gel is completely dissolved. Leave it at room temperature for 5 minutes to cool down.

4) Transfer less than 700 μl of the melted glue solution into the spin column inserted into the sleeve, centrifuge at high speed for 1 minute on a desktop centrifuge, discard the waste liquid in the sleeve, and then insert the spin column into the sleeve.

5) Add more than 700 μl of remaining melted glue into the same spin column and repeat step 4.

6) Add 700 μl of Buffer PW (washing solution) into the spin column, centrifuge at high speed for 1 minute, discard the waste liquid, and insert the spin column into the cannula.

7) This step can be omitted and proceed directly to step 8. Add 200 μl of Buffer PW (washing solution) into the spin column and centrifuge at high speed for 1 to 2 minutes.

8) After high-speed centrifugation for 1 to 2 minutes, carefully take out the spin column and avoid contaminating the waste liquid in the casing. Discard the casing.

9) Insert the spin column into a new 1.5ml centrifuge tube, add 30 to 50 μl Elution Buffer (eluent) to the center of the silica gel membrane in the spin column, do not touch the silica gel membrane; leave it at room temperature for 2 to 5 minutes, and centrifuge at high speed for 1 minute, that is Obtain purified DNA solution.

10) The obtained DNA solution can be directly used in subsequent experiments, or stored at -20°C for later use.

Precautions:

1) DNA gel recovery efficiency depends on the size of DNA fragments. Generally, fragments of 50bp to 10kb can be recovered efficiently. The recovery efficiency of 20-50bp fragments dropped significantly. The amount of DNA in the gel also affects recovery. If the DNA content is too small, the recovery efficiency will also decrease. The binding amount of a spin column can generally reach 10 to 15 μg.

2) Omitting step 7 usually has no effect on the purification results, but sometimes trace amounts of salt will remain in the DNA solution.

Plasmid small/medium extraction and purification kit

(Plasmid Mini/Midiprep Kit)

How to operate:

1) Take 0.5 to 3 ml of overnight bacterial culture, put it into a 1.5 ml or 2 ml centrifuge tube, centrifuge at 12,000g for 2 minutes at room temperature to precipitate the bacteria, and completely discard the supernatant.

2) Add 150μl Buffer P1, fully suspend and shake the bacterial sediment for 10 _to 15 seconds to completely disperse it until no floccules exist.

3) Add 150μl Buffer P2, gently invert the centrifuge tube 3 to 5 times, and leave it at room temperature for 1 minute to completely lyse the bacteria and make the solution transparent. When there are many bacteria, it can be left for 3 to 5 minutes to fully lyse the bacteria.

4) Add 150μl Buffer P3, immediately invert the centrifuge tube 4 to 6 times quickly, mix thoroughly, and leave it at room temperature for 1 minute. White floc will be visible. When there are many bacteria, the viscous mass is difficult to mix. You can hold the mouth of the centrifuge tube upside down and flick the tip of the tube twice with your fingers to mix it into a flocculent shape.

5) Add 150 μl Buffer P4, gently invert the centrifuge tube 3 _to 5 times, and leave it at room temperature for 10 minutes.

6) Centrifuge the above lysate at 12,000g for 8 minutes at room temperature, carefully aspirate the supernatant, and transfer it to a new 1.5ml centrifuge tube. The sedimentation clumps are usually attached to the bottom or side wall of the tube. Be careful not to inhale the sedimentation residue.

7) Add 600μl Buffer P5, invert the centrifuge tube 3 _to 5 times, and mix thoroughly. Leave at room temperature for 5 minutes.

8) Centrifuge at 12,000g for 8 minutes at room temperature, discard the supernatant, invert and gently drain off the remaining liquid. Small, nearly transparent precipitate clumps are generally visible on the bottom and side walls of the tube. Gently add 1ml of 70% ethanol and wash twice, drain the remaining liquid, and let the precipitate air dry for 3 to 5 minutes.

9) Add appropriate amount of TE (20~50μl) to dissolve the precipitate. The obtained DNA solution contains some residual RNA, but can be directly used in electrophoresis, enzyme digestion, ligation, transformation and sequence determination.

10) Add 20μl Buffer P6 (Precipitate B) to 50μl plasmid solution, mix well and place on ice for 10min. Centrifuge at 12,000g room temperature for 10min. Discard the supernatant. Generally, no precipitate clumps can be seen. Gently add 1ml of 70% ethanol and wash twice, drain the remaining liquid, and air-dry the precipitate for 3 to 5 minutes.

11) Add appropriate amount of TE (20~50μl) to dissolve the precipitate. The obtained DNA solution has been basically cleared of RNA and other remaining impurities, and can be directly used in cell transfection, in vitro transcription, etc.

Precautions:

1) Be gentle during operation to prevent possible loss of DNA due to mechanical shearing.

2) TE with pH 8.0 should be used as the dilute solution to measure the OD and ratio. The OD ratio measured in pure water may be lower.

3) The OD260/280 ratio of the finally obtained plasmid solution is generally in the range of 1.7 to 1.8. If the plasmid DNA content is relatively

Low, the OD260/280 ratio will also be low, but it does not affect the use in transfection and other work

CaCl2 method conversion experiment

(Transformation of Plasmid DNA Using CaCl2)

How to operate:

1) Take 100 μl of competent cell suspension from the -70°C refrigerator, thaw it at room temperature, and place it on ice immediately after thawing.

2) Add plasmid DNA solution (content does not exceed 1 μg, volume does not exceed 10 μl), shake gently, place on ice for 30 minutes, and heat shock in a 42°C water bath for 90 seconds. Do not move the centrifuge tube during the heat shock process. Then quickly place it on ice to cool for 3-5 minutes.

3) Add 1 ml of LB liquid culture medium (without antibiotics) to the tube, mix well by pipetting, and culture with shaking at 37°C and 220 rpm for 1 hour to restore the bacteria to normal growth and express the antibiotic resistance gene encoded by the plasmid. .

4) Shake the above bacterial solution evenly, centrifuge, remove 900 μl of supernatant, mix the remaining culture medium by pipetting, and spread 100 μl on a screening plate containing antibiotics.

5) Place the plate face up for half an hour. After the bacterial liquid is completely absorbed by the culture medium, invert the Petri dish and incubate at 37°C for 16-24 hours.

Precautions:

1) Quality and concentration of plasmid DNA: The plasmid DNA used for transformation should mainly be in a superhelical state. Generally, the volume of the DNA solution should not exceed 5% of the volume of the competent cells.

2) Prevent contamination from bacteria and DNA

3) The entire operation must be performed on ice and cannot leave the ice bath, otherwise the cell conversion rate will be reduced.

4) The time of heat shock should not be too long or too short, and the movements should be gentle

Plasmid large-scale extraction and purification kit

(Plasmid Maxprep Kit)

How to operate:

Plasmid extraction:

1) Take less than 150ml of overnight cultured bacterial liquid, put it into a suitable centrifuge bottle, centrifuge at 4,500-6,000g for 10 minutes at 4°C to precipitate the bacterial cells, and completely discard the supernatant.

2) Add 5ml Buffer I, fully suspend and shake the bacterial sediment to completely disperse it until no floccules exist. Transfer the bacterial suspension into a 50ml centrifuge tube.

3) Add 5ml Buffer II, gently invert the centrifuge tube 6 to 8 times, and leave it at room temperature for 5 minutes to completely lyse the bacteria and make the solution transparent.

4) Add 5ml Buffer III, immediately invert the centrifuge tube 6 to 8 times, and mix thoroughly until white floc is produced. Place on ice for 10 _to 15 minutes.

5) Centrifuge the above lysate at 12,000~16,000g for 15 minutes at 4°C, carefully aspirate the supernatant, and transfer it to a new 50ml centrifuge tube.

6) Add 10ml of isopropyl alcohol, invert the centrifuge tube and mix thoroughly. Place on ice for 10 to 15 minutes.

7) Centrifuge at 412,000°C ~ 16,000g for 10 minutes, carefully discard the supernatant, invert and gently drain the remaining liquid, add 0.5ml Buffer I to completely dissolve the precipitated clumps (use a wide-mouth pipette to gently pipet to assist dissolution). Transfer to a new 1.5ml centrifuge tube and place at room temperature for 10 to 20 minutes.

8) Centrifuge the crude plasmid extract in a desktop centrifuge at room temperature for 2 minutes at high speed, and transfer the supernatant to a new 1.5 ml centrifuge tube. Plasmid purification:

1) Add 100μl Buffer IV (Impurity Scavenging Solution A) to 0.5ml plasmid crude extract, mix gently, centrifuge at 12,000g for 2 minutes, and transfer the supernatant to a new centrifuge tube.

2) Add 100μl Buffer IV (Impurity Removal Liquid A), mix gently, centrifuge at 12,000g for 5 minutes, and transfer the supernatant to a new centrifuge tube.

3) Add 70μl Buffer V (Impurity Cleaning Solution B), mix gently, centrifuge at 12,000g for 5 minutes, and transfer the supernatant to a new centrifuge tube.

4) Add 0.5ml isopropyl alcohol, mix well, and leave it at room temperature for 10 minutes. Centrifuge at 12,000 g for 10 min at room temperature, discard the supernatant, wash gently with 1 ml of 70% ethanol, discard the liquid, and invert to dry at room temperature for 5 min.

5) 0.5ml TE dissolves the precipitate (can be shaken in a 37°C water bath or gently blown with a wide-mouth pipette to assist dissolution)

6) Add 200μl BufferVI (Impurity Removal Solution C), mix and place on ice for 10-30min, centrifuge at 12,000g for 10min at room temperature, discard the supernatant, gently add 1ml of 70% ethanol and wash twice, invert at room temperature to dry for 5 Allow 10 minutes for the ethanol to evaporate completely.

7) Add appropriate amount of TE (200~500μl) to dissolve the precipitate (it can be shaken in a 37°C water bath to assist dissolution).

Precautions:

1) When extracting plasmids from a large amount of bacterial liquid or cells, the dosage of BufferI, BufferII and BufferIII can be increased proportionally to fully lyse the cells and improve the recovery amount and purity of the plasmids.

2) In extraction step 7 and purification step 5, fully dissolving the precipitate is very important to improve plasmid yield and purity.

3) Be gentle during operation to prevent possible loss of DNA due to mechanical shearing.

4) Several microliters of DNA solution can be taken before and after each step of purification, and finally the extraction and purification results can be compared by electrophoresis.

5) After centrifugation in purification steps 3 and 6, no obvious precipitate may be seen. If no precipitation is seen in step 6 and you are worried about DNA loss, you can keep the supernatant and conduct electrophoresis analysis after all operations are completed to determine whether the final product is obtained (hundreds of micrograms of high-purity plasmid DNA precipitates on the side wall of the tube after centrifugation) , obvious clumps may not be visible).

6) TE or Tris with pH 8.0 should be used as the dilute solution to measure the OD and ratio. The OD ratio measured in pure water may be lower.

The above are only preferred embodiments of the present invention. It should be noted that those of ordinary skill in the art can also make several improvements and additions (including DHCR24 gene sequence optimization and vectors) without departing from the method of the present invention. Tool improvements), these improvements and additions should also be regarded as the protection scope of the present invention.

Claims (7)

1. The application of an AAV9 vector complex containing the DHCR24 gene in the preparation of drugs for the treatment of neurodegenerative diseases. The AAV9 vector complex containing the DHCR24 gene is directly synthesized from the SEQ ID NO.1 sequence into pHBAAV-CMV-MCS-3flag -T2A-ZsGreen vector.
2. The application according to claim 1, characterized in that the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging, brain atrophy, aging-related cognitive impairment, autism, Niemann's disease -Pick's disease, Huntington's disease, motor neurone disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis, Rett syndrome.
3. The application according to claim 2, wherein the neurodegenerative disease is Alzheimer's disease.
4. A drug for treating neurodegenerative diseases, characterized in that the drug is an AAV9 vector complex containing the DHCR24 gene, and the AAV9 vector complex containing the DHCR24 gene is directly synthesized from the SEQ ID NO.1 sequence into pHBAAV- CMV-MCS-3flag-T2A-ZsGreen vector.
5. The medicine according to claim 4, characterized in that the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging, brain atrophy, aging-related cognitive impairment, autism, Niemann's disease -Pick's disease, Huntington's disease, motor neurone disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis, Rett syndrome.
6. Application of a DHCR24 gene in the preparation of drugs for the treatment of neurodegenerative diseases.
7. The application according to claim 6, characterized in that the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging, brain atrophy, aging-related cognitive impairment, autism, Niemann's disease -Pick's disease, Huntington's disease, motor neurone disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis, Rett syndrome.

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