CN114515341A - Application of DHCR24 gene in preparation of medicines for treating neurodegenerative diseases - Google Patents

Abstract

The invention relates to application of an AAV9 vector compound containing DHCR24 gene in preparation of a medicament for treating neurodegenerative diseases. The neurodegenerative disease is Alzheimer Disease (AD), Parkinson disease, brain aging, aging-related cognitive disorder, autism, Niemann-pick disease, Huntington disease, motor neuron disease, frontotemporal dementia, dementia with Lewy bodies, multiple sclerosis (multiple sclerosis), Rett syndrome. The invention also provides a medicament for treating neurodegenerative diseases. The invention is based on the new cholesterol theory of AD onset, enhances the synthesis and supply of cholesterol in brain by a DHCR24 gene therapy method (DHCR24 gene function enhancement), helps to correct the shortage (or lack) of cholesterol in brain, and thereby improves the learning and memory functions of AD.

Description

Application of DHCR24 gene in preparation of medicines for treating neurodegenerative diseases

Technical Field

The invention relates to the technical field of gene therapy, in particular to application of a DHCR24 gene in preparation of a medicament for treating neurodegenerative diseases.

Background

Alzheimer disease is a variant disease, which is common in dementia, accounting for more than 60% of them. The memory disorder is the most common in clinic, and is characterized by the overall decline of the brain function activities of the patients, such as cognition, behavior, language and the like, and is irreversible.

24-dehydrocholesterol reductase (DHCR24) is a key gene controlling cellular cholesterol synthesis: the research shows that the 24-dehydrocholesterol reductase (DHCR24) gene is about 46.4kb in length, is positioned on chromosome 1p31.1-p33 and contains 9 exons and 8 introns, and 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 (fig. 1). If the expression of DHCR24 gene of nerve tissue (cell) is increased, the synthesis of cholesterol in nerve tissue (cell) can be increased, the cholesterol level in nerve tissue (cell) can be increased, and the cholesterol deficiency in nerve tissue (cell) can be corrected. So far, no medicine or technology aiming at the DHCR24 gene exists at home and abroad.

Clinical research progress in gene therapy and clinically approved gene therapy drugs: over 20 years, gene therapy technology using Adeno-Associated Virus (AAV) as a gene vector has matured more and more, and more clinical gene therapy trials have been conducted in the european and american countries and are applied to the clinical treatment of many diseases that cannot be effectively treated before. Over the past 20 years, the importance of gene therapy in clinical treatment has increased, and over 200 clinical trials of gene therapy have been approved in the us in 2018 alone, see figure 2 (2000-2018, statistical report of clinical trials of U.S. gene therapy drugs).

The first AAV-based gene therapy drug approved for clinical use in europe by the European Medicines Agency (EMA) is adipogene kinetic (AT), which is a rare autosomal recessive disease for the treatment of lipoprotein lipase deficiency (LPLD). The drug was approved in 2012 under the trade name Glybera. AT gene therapy provides a variant of the LPL gene, LPLs447X, to correct LPL function in LPL patients. In the phase III clinical trial, peak chylomicron levels were reduced by 79% after a low-fat diet 14 weeks after administration of AT.

To date, AAV-based gene therapies approved by the united states Food and Drug Administration (FDA) include lucurna by Sparktherapeutics and Zolgensma by Avexis for the treatment of hereditary retinal diseases and spinal muscular atrophy, respectively. AAV is considered to have better safety due to its lower immunogenicity and site-specific integration capability. There are currently 17 U.S. FDA approved gene therapies, including AAV vector voretigene neuropvec ryl (VN), developed in 2017 by Spark Therapeutics, inc. VN comprises an AAV2 encapsulating the RPE6 gene for use in the treatment of biallelic RPE 65-associated retinal dystrophy, a rare genetic disease, resulting in impaired visual function, decline with age and ultimately blindness. In a phase III clinical trial conducted in 2012 (NCT00999609), 65% of participants received the greatest improvement 1 year after the subretinal injection of VN in Leber congenital amaurosis 2 patients. No drug to treat retinal dystrophy had been available before VN was approved. The second AAV-based gene therapy approved by the FDA in 2019 was onasegene abeparvovec xiao (OA), developed by AveXis under the trade name Zolgensma. OA treats spinal muscular atrophy type I (SMA1) in children under 2 years of age using AAV9 expressing a functional SMN1 transgene.

Therefore, the gene therapy technology becomes a novel and innovative clinical treatment technology which is competitively developed by overseas large-scale medicine companies and biotechnology companies, and a small number of biotechnology companies develop research and research on the aspect at home at present, so that the clinical application prospect is wide.

A key consideration for the success of 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 cumbersome manufacturing processes. To address these limitations, non-viral vectors have been developed that avoid immune responses, have extensive tunability, and quality control, but they are inefficient at transfection and result in passive expression of transgenes. In summary, its clinical use is still limited by gene vector immunogenicity, carcinogenicity, off-target effects and therapeutic efficacy.

Disclosure of Invention

Our years of research have found that insufficient (or lack of) cholesterol in the brain plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's Disease (AD). It has been found that insufficient synthesis of cholesterol in the brain is one of the main causes of insufficient (or deficient) cholesterol in the brain. We found that by increasing the expression (function enhancement) of DHCR24 gene in nervous system, the synthesis of cholesterol in brain can be increased obviously, and the supply of cholesterol can be increased, thereby correcting the insufficient or lack of cholesterol level in brain. In the study of an AD mouse model, the DHCR24 gene function enhancement (knock-in) is found to be effective in improving the learning and memory functions of AD mice (see the study result later).

The basic objects of the present technology are: the DHCR24 gene is added to specific nerve tissues (or cells) by an intracerebral transfection method of DHCR24 genes carried by adeno-associated virus (AAV) vectors and the like (including various vectors such as lentivirus, adenovirus, adeno-associated virus, modified adeno-associated virus, liposome and the like), the method further promotes the expression of the DHCR24 gene of the specific neuron tissues (or cells), improves the expression level of the DHCR24 of the specific neuron tissues (or cells), promotes the synthesis and release of cholesterol of the specific neuron tissues (or neuron cells), and helps to correct the insufficient (or lack) cholesterol in the brain. A technical method for improving the cholesterol level of a neuron tissue (or a neuron cell) by a DHCR24 gene addition (gene function enhancement) technical method. In summary, this technical approach can help correct the cholesterol deficiency or lack of neural tissue (or cells). So far, no medicine and treatment technology aiming at the DHCR24 gene exists at home and abroad at present, and the technology has potential and important clinical application prospect on AD and other neurodegenerative diseases.

The first purpose of the present invention is to provide the use of AAV9 vector complex containing DHCR24 gene, which is directed to the deficiencies of the prior art.

The second purpose of the invention is a medicine for treating neurodegenerative diseases.

In order to realize the first purpose, the invention adopts the technical scheme that:

an application of AAV9 carrier compound containing DHCR24 gene in preparing medicine for treating neurodegenerative disease is disclosed, wherein the AAV9 carrier compound containing DHCR24 gene is obtained by directly synthesizing SEQ ID NO.1 sequence to pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector.

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

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

In order to achieve the second purpose, the invention adopts the technical scheme that: the drug is an AAV9 vector compound containing DHCR24 gene, and the AAV9 vector compound containing the DHCR24 gene is a pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector directly synthesized by an SEQ ID NO.1 sequence.

As a preferred example, the neurodegenerative disease is alzheimer's disease, parkinson's disease, brain aging, aging-related cognitive disorders, autism, niemann-pick disease, huntington's disease, motor neuron disease, frontotemporal dementia, lewy body dementia, multiple sclerosis (multiple sclerosis), Rett syndrome.

The invention has the advantages that:

to date, the pathogenesis of Alzheimer's Disease (AD) has not yet been elucidated. AD is a progressive degenerative disease of nervous system with hidden onset, about 5500 more than ten thousand patients worldwide are in rapid rise along with aging of population, and serious social and health problems are caused.

Over the past 30 years, researchers of many large pharmaceutical companies around the world have relied on a variety of different theoretical hypotheses for the onset of AD, such as the hereditary mechanism, the cholinergic hypothesis, the amyloid-beta hypothesis, the Tau protein hypothesis, and others. The development of a large number of drug designs and studies for different hypotheses, particularly treatments for the beta-amyloid hypothesis, has almost completely failed in various clinical trials costing billions of dollars. To date, all traditional therapeutic approaches for AD, such as anti-beta-amyloid, anti-Tau, anti-oxidant, etc., have not achieved good therapeutic results. Thus, treatment of AD is currently in a state of stasis.

Compared with the traditional AD treatment technical method in the past, the invention belongs to a novel gene therapy technical method which is completely different from the traditional AD treatment technical method in the past. The effect of the technical method for treating AD is very obvious and is obviously superior to the traditional AD treatment technical method.

The invention is based on the new cholesterol theory of AD onset, enhances the synthesis and supply of cholesterol in brain by a DHCR24 gene therapy method (DHCR24 gene function enhancement), helps to correct the deficiency (or lack) of cholesterol in brain, and thereby improves the learning and memory functions of AD. The present invention may be applied to the treatment of neurodegenerative diseases such as alzheimer's Disease, parkinson's Disease, brain aging (brain atrophy), aging-related cognitive impairment (cognitive impairment of the old), autism, niemann-pick's Disease, Huntington's Disease (HD), motor neuron Disease, frontotemporal dementia, Lewy body Dementia (DLB), multiple sclerosis (multiple scleresis), Rett syndrome (Rett syndrome), etc., and may have very important social and economic values for the treatment of neurodegenerative diseases such as AD in the future.

Drawings

Figure 1 key mode of action of DHCR24 in cellular cholesterol synthesis and homeostatic regulation DHCR 24: 24-dehydrocholesterol reductase; cholesterol Cholesterol.

FIG. 2 summary report of clinical trial of gene therapy drugs in the United states (2000-2018).

FIG. 3 AAV 9-mediated transfection of DHCR24 gene by brain stereotactic guided injection.

FIG. 4 AAV9 mediated by adeno-associated virus (AAV) mediated transfection of DHCR24 gene into hippocampal region of AD mouse experiment AAV9 empty vector and AAV9-DHCR24 gene successfully transfect hippocampal region of AD model mouse. FIG. 2 shows that the fluorescence of wild type mouse control group, empty vector transfected AD mouse group and DHCR24 transfected AD mouse group is the auto-fluorescence of AAV9 virus.

FIG. 5AAV9 mediated DHCR24 gene transfection AD mouse hippocampal experiment AAV9-DHCR24 gene transfection AD model mouse hippocampal cell, and DHCR24 of CA1, CA2/CA3 region showed obvious positive over-expression (brown is DHCR24 positive expression).

FIG. 6AAV9 mediated effect of DHCR24 gene transfection on DHCR24 mRNA expression in hippocampal of AD model mice on the AD mouse model transfected with DHCR24 gene, PCR detection shows that the expression level of hippocampal DHCR24 mRNA of the AD model mice after transfection is obviously increased compared with that of a control group. N: a wild-type mouse; NC, transfecting an AD mouse by using an empty vector; the DHCR24 gene transfected AD mice.

FIG. 7AAV9 mediated DHCR24 gene transfection effect on AD model mouse hippocampal DHCR24 protein expression on the AD mouse model transfected by DHCR24 gene, compared with the control group, the DHCR24 protein expression level of the AD model mouse after transfection is very obviously increased by Western blot detection.

Figure 8 effect of DHCR24 overexpression on cognitive function in AD model mice (water maze test) compared to AD model mice, platform latency was significantly reduced in AD model mice overexpressing DHCR24 (figure 8B), platform crossing times (figure 8C), and target quadrant percentage were also significantly improved (figure 8A) as detected by the water maze method. WT: wild-type C57 mice; APP/PS1 APP/PS1 double transgenic AD model mice; H-AAV: AD mice transfected with hippocampal AAV9-DHCR24 gene; H-NC: AD mice transfected by an empty vector of a hippocampal AAV-9 virus; L-AAV, AD mice transfected by lateral ventricle AAV9-DHCR24 gene; L-NC: AD mice transfected with an empty vector of lateral ventricle AAV 9.

FIG. 9DHCR24 is involved in the pathological regulation mechanism of AD pathogenesis by regulating cellular cholesterol metabolism.

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

FIG. 11 plasmid map of m-dhcr 24:

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

The technical content of the invention is as follows: 1) firstly, a target gene is designed and constructed aiming at a target gene DHCR24, the construction of a mouse DHCR24(mouse-DHCR24, m-DHCR24) gene overexpression adeno-associated virus 9(AAV9) vector (provided with technical assistance by Hanhensheng constant biotechnology (Shanghai) Limited company) is completed, and finally the preparation of an AAV 9-encapsulated DHCR24 gene medicine (namely, an AAV9 vector compound containing a DHCR24 gene) is completed. 2) The prepared AAV9 medicine carrying the DHCR24 gene (the medicine of the AAV9 wrapping the DHCR24 gene) is transfected to a specific nerve tissue (or cell) by an AAV 9-mediated DHCR24 gene transfection method to improve the DHCR24 expression level of the specific nerve tissue (or cell), so that the cholesterol synthesis of the nerve tissue (or cell) is improved, the cholesterol supply is increased, and the technical method for correcting the insufficient (lack) cholesterol of the nerve tissue (or cell) is facilitated.

By the method, a plurality of nervous system diseases caused by deficiency or insufficiency of cholesterol can be corrected. In AD animal model studies, we have demonstrated that DHCR24 gene overexpression (functional enhancement, knock-in) can significantly improve cognitive function in AD mice. So far, no medicine or technology aiming at the DHCR24 gene exists at home and abroad.

Experimental materials and methods

1.2.1 the experimental materials and instruments mainly comprise experimental animals of a C57 mouse, an APP/PS1 double-transgenic AD mouse, an AAV9 wrapped DHCR24 gene medicine, a plurality of antibodies and the like, and the experimental instruments comprise a brain stereotaxic apparatus, a fluorescence confocal microscope and the like. The construction process and identification report of m-dhcr24 overexpression adeno-associated virus vector are shown below.

1.2.2 Experimental methods

1) Brain stereotaxic, directional injection and AAV 9-mediated DHCR24 gene transfection

The hippocampal region is the memory center and is also the vulnerable brain region for AD development, so we chose the hippocampal CA1 region as the brain region we studied for directional injection and gene therapy. In the research, an APP/PS1 double-transgene AD mouse is used as a research model of AD, and a brain stereotaxic positioning method is utilized to complete the drug hippocampal injection and transfection expression of AAV9 wrapped DHCR24 gene of the AD mouse (figure 3). The technology is one of the high-efficiency and convenient intracerebral administration methods which are commonly used abroad at present.

2) Mouse hippocampal AAV 9-mediated DHCR24 gene transfection experiment (brain stereotactic injection method)

Through brain stereotaxic and directional injection methods, AAV9 virus-mediated DHCR24 gene (medicament of AAV9 wrapping DHCR24 gene) is directionally injected and transfected into CA1 region of APP/PS1 double-transgenic AD mouse hippocampus, and AAV9-DHCR24 gene medicament successfully transfects AD model mouse hippocampus (figure 4).

Stereotactic injection method for mouse brain

The experimental animals are divided into 6 different groups, namely a wild type group, an AD model group (AD control group), an empty vector group, a DHCR24 over-expression AD mouse group, a DHCR24 over-expression group (lateral ventricle group), an empty vector group (lateral ventricle group) and the like, and each group contains 30 animals. According to the results of preliminary experiments in the previous subject groups, injection site 1(AP: -2.3 mm; ML. + -. 1.8 mm; DV: -1.8mm) of hippocampal CA1 region was located with reference to a stereotactic map of the mouse brain. The injection dose was made according to the results of the previous experiments (1.0 microliter of CA1 per hippocampus).

Wild type group and AD control group were not treated and used as control group. The AAV9-DHCR24 gene viral vector and the corresponding empty vector thereof are respectively given to the DHCR24 overexpression group and the empty vector group, and the AAV9-DHCR24 gene viral vector and the corresponding empty vector thereof are subjected to bilateral hippocampal CA1 area stereotactic microinjection. In addition, the group of the virus vectors of AAV9-DHCR24 gene, which were overexpressed in DHCR24 (lateral ventricle group) and the group of the empty vectors (lateral ventricle group), were administered at the same dose and the corresponding empty vectors were injected into the lateral ventricle, respectively, to study the cells as a control group for different injection routes. After injection, the needle was left for 10 minutes, and then the needle was removed and observed for another 30 minutes. After the injection experiment, the mice are sent to an animal room for feeding and observation.

1.3 results of the experiment

1.3.1 mouse Hippocampus AAV 9-mediated DHCR24 Gene transfection assay (immunohistochemistry)

Through AAV9 virus mediated DHCR24 gene directed injection and transfection, DHCR24 gene successfully transfects AD model mouse hippocampus. Immunohistochemistry showed that the CA1, CA2/CA3 regions showed significantly strong positive DHCR24 overexpression (FIG. 5, brown staining positive for DHCR 24). The result indicates that AAV9 mediates the successful transfection and expression of DHCR24 gene in mouse hippocampus.

1.3.2 Effect of AAV9 Virus-mediated HCR24 Gene transfection on AD mouse Hippocampus DHCR24 mRNA expression

After 5 weeks of mouse hippocampal targeted injection (surgery), PCR detection showed that DHCR24 over-expressed AD model mice had a very significant increase in DHCR24 mRNA expression level, approximately 19-fold, in comparison with wild-type mice and empty vector AD mice, in the AD mouse model with DHCR24 over-expression (FIG. 6). The results indicate that AAV9 mediates the transfection and overexpression of DHCR24 gene in hippocampus of AD mice.

1.3.3 Effect of AAV9 Virus-mediated HCR24 Gene transfection on overexpression of Hippocampus DHCR24 protein in AD model mice

At 5 weeks after surgery, in the AD mouse model with the over-expressed DHCR24, the expression level of DHCR24 protein of the AD mouse with the over-expressed DHCR24 was significantly increased by about 313% compared with the wild-type mouse and the AD mouse with the empty vector (fig. 7). The results indicate that AAV9 mediates the transfection and overexpression of DHCR24 gene in hippocampus of AD mice.

1.3.4 Effect of DHCR24 overexpression on cognitive function in AD model mice (Water maze assay)

After 5 weeks of operation, compared with AD control group mice and AD mice in an empty vector group, the incubation time of the AD model mice after DHCR24 gene treatment is obviously shortened, and the crossing times and the target quadrant percentage are also obviously improved by water maze detection on an APP/PS1 double-rotation AD mouse model with DHCR24 overexpression. Compared with wild mice (normal mice), the learning and memory functions of AD mice after DHCR24 gene therapy (gene function enhancement) are obviously close to the cognitive functions of normal mice (figure 8). The above results suggest that DHCR24 gene therapy (overexpression) significantly improved cognitive function in AD mice. Therefore, the DHCR24 gene therapy method is inferred to have important and potential clinical application value for AD patients.

1.4 discussion

Through more than 10 years of research, the research of the subject group shows that the 24-dehydrocholesterol reductase (DHCR24) gene knockout obviously causes the inhibition of the synthesis of the neuron cholesterol and the obvious reduction of the levels of the neuron membrane cholesterol and intracellular cholesterol, and the DHCR24 is a key regulatory gene for the steady-state regulation of the cholesterol. In addition, DHCR24 deficiency induces decrease in neuronal cholesterol levels, leading to abnormalities and dysfunction of cellular membrane lipid raft structure, and further induces key pathological lesions of Alzheimer's Disease (AD) such as increased release of neuronal β amyloid and abnormal phosphorylation of Tau protein. The above-mentioned findings strongly support the possible involvement of AD-related key pathological impairment mechanisms via a deficiency in cholesterol (fig. 9).

We further developed animal model studies of AD mice. In the study of AD animal models, the AD mice with APP/PS1 double-transgenic genes are used as the study AD models, AAV9 mediated DHCR24 genes (namely DHCR24 gene drugs wrapped by AAV 9) are transfected into APP/PS1 mouse hippocampal CA1 regions through brain stereotaxic and orientation, the DHCR24 genes are successfully transfected and over-expressed in the hippocampal CA1 regions, and the cognitive function of the AD mice is obviously improved. The research result shows that the DHCR24 gene overexpression (function enhancement) obviously improves the cognitive function of AD. Therefore, the present study further proves that DHCR24 influences AD pathological activities by increasing cholesterol synthesis and supply, regulating neuronal cholesterol metabolism, and further improves cognitive function of AD mice (fig. 9).

Based on the 10 years of research and accumulation of our topic groups on the pathogenesis of cholesterol in AD, we first proposed internationally the "cholesterol theory that brain cholesterol deficiency may be involved in the pathogenesis of AD" which is about to be published in the european authority journal, "neuropathology Communications. We find that the cholesterol deficiency mechanism in the brain of AD patients and AD animal models can relate to three links of cholesterol synthesis reduction, cholesterol transport obstacle and cholesterol cleavage obstacle.

In conclusion, through the research results of cell models and AD mouse models of more than 10 years in our subject group, we further determined that DHCR24 has important theoretical breakthrough and instructive significance in the treatment of AD by regulating neuronal cholesterol metabolism and intervening AD (fig. 9). In addition, the DHCR24 gene can be used as a new target for future treatment of AD, has important and potential clinical application value as improving AD cognitive function by regulating the DHCR24 gene target, and provides a brand-new theoretical basis and technical method for DHCR24 gene treatment.

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

An experimental material

Experimental reagent

Name of reagent	Manufacturer of the product
		Carrier	Hanbio Biotechnology
Escherichia coli strain DH5 alpha	Invitrogen
		Restriction enzyme	Thermo Fisher Scientific
HB-infusation seamless cloning kit	Hanbio Biotechnology CO.LTD.
		Small and large quantity extraction kit for plasmid DNA	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. pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector map as follows:

see fig. 10

2. The sequence information of m-dhcr24 is shown in SEQ ID NO.1

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

Second, Experimental procedure

1. Digesting the carrier at 37 ℃ and recovering the glue

2. Recovery of fragments after PCR

3. The treated target fragment was ligated with the vector, reaction system (20. mu.l)

4. Transformation (competent cells: DH 5. alpha.) details were given in the appendix section on transformation

5. Resistance is Amp, 37 ℃, 230rpm/min, cultured overnight

6. Selecting transformed flat plate, shaking at 37 deg.C and 230rpm/min for 14 hr, and inoculating with bacterial liquid

Performing PCR identification, and sending the positive clone bacterial liquid to sequencing company for sequencing

Third, experimental results

1. The sequencing result of the m-dhcr24 overexpression vector is shown in SEQ ID NO.2

2. m-dhcr24 plasmid map:

see fig. 11.

DNA purification kit (column centrifugal type)

(DNA Pure-Spin Kit)

The operation method comprises the following steps:

1) adding 3-4 times volume of Buffer PSB (binding solution) into the DNA solution, and reversing or suspending, shaking and uniformly mixing.

2) Transferring less than 700 μ l of the solution to a cannulated spin column, centrifuging at high speed for 1min in a desktop centrifuge, discarding the waste fluid in the cannulation, and cannulating the spin column.

3) More than 700. mu.l of the remaining solution was added to the same spin column and step 2 was repeated.

4) Adding 700 μ l Buffer PW (washing solution) into the centrifugal column, centrifuging at high speed for 1min, discarding waste solution in the casing, and inserting the centrifugal column into the casing.

5) This step can be omitted and step 6 can be performed directly. Adding 200 mul of Buffer PW (washing solution) into the centrifugal column, and centrifuging at high speed for 1-2 min.

6) After high-speed centrifugation for 1-2 min, carefully taking out the centrifugal column without adhering to waste liquid in the sleeve. The cannula was discarded.

7) Inserting the centrifugal column into a new 1.5ml centrifugal tube, adding 30-50 μ l of an Elution Buffer (eluent) into the central position of the silica gel membrane in the centrifugal column, and paying attention to not touch the silica gel membrane; standing at room temperature for 2-5 min, centrifuging at high speed for 1min, and centrifuging in a centrifuge tube to obtain the purified DNA solution.

8) The obtained DNA solution can be directly applied to subsequent experiments or stored at-20 ℃ for later use.

Note that:

1) the DNA recovery rate depends on the size of the DNA fragment. The 50 bp-10 kb fragment can be recovered with high efficiency. The recovery rate is also reduced when the DNA content is too low. The binding capacity of a centrifugal column can be 10-15 mu g.

2) Step 5 is omitted, and usually has no effect on the purification result, but sometimes a trace amount of salt remains in the DNA solution.

DNA gel recovery and purification kit (column centrifugal type)

(Gel-Spin DNA Extraction Kit)

The operation method comprises the following steps:

1) after the completion of DNA electrophoresis (agarose gel electrophoresis using TAE buffer is recommended), the corresponding fragment was excised with a clean blade under an ultraviolet lamp. Carefully remove the DNA-free portion of the gel.

2) The agarose gel blocks containing the DNA were loaded into 1.5ml centrifuge tubes and the volume was estimated. Add 500. mu.l (< 150. mu.l gel) or 3-4 times (> 150. mu.l gel) volume of Buffer PS (sol gel).

3) Placing the centrifugal tube in a water bath at 50-60 ℃ for 5-10 min, taking out the centrifugal tube at intervals of 2-3 min, suspending and shaking for 10sec until the agarose gel is completely dissolved, and placing the centrifugal tube at room temperature for 5min and cooling.

4) Transferring less than 700 μ l of the thawed gelatin solution to a centrifugal column inserted into the cannula, centrifuging at high speed for 1min on a desktop centrifuge, discarding the waste solution in the cannula, and inserting the centrifugal column into the cannula.

5) More than 700. mu.l of the remaining thawed gel was added to the same column and step 4 was repeated.

6) Adding 700 μ l Buffer PW (washing solution) into the centrifugal column, centrifuging at high speed for 1min, discarding waste liquid, and inserting the centrifugal column into a sleeve.

7) This step can be omitted and step 8 can be performed directly. Adding 200 mul of Buffer PW (washing solution) into the centrifugal column, and centrifuging at high speed for 1-2 min.

8) After high-speed centrifugation for 1-2 min, carefully taking out the centrifugal column without adhering waste liquid in the sleeve. The cannula was discarded.

9) Inserting the centrifugal column into a new 1.5ml centrifugal tube, and adding 30-50 μ l of an Elution Buffer (eluent) into the central position of the silica gel membrane in the centrifugal column without touching the silica gel membrane; standing at room temperature for 2-5 min, and centrifuging at high speed for 1min to obtain a purified DNA solution.

10) The obtained DNA solution can be directly applied to subsequent experiments or stored at-20 ℃ for later use.

Note that:

1) the DNA gel recovery efficiency depends on the size of the DNA fragment. The 50 bp-10 kb fragment can be recovered with high efficiency. The recovery efficiency of 20-50 bp fragments is obviously reduced. The DNA content in the gel also affects recovery. The recovery efficiency is also lowered by the too small amount of DNA. The binding capacity of a centrifugal column can be 10-15 mu g.

2) Step 7 is omitted, and usually has no effect on the purification result, but sometimes a trace amount of salt remains in the DNA solution.

Plasmid small/medium extraction and purification kit

(Plasmid Mini/Midiprep Kit)

The operation method comprises the following steps:

1) taking 0.5-3 ml of overnight cultured bacteria, putting the overnight cultured bacteria into a 1.5ml or 2ml centrifuge tube, centrifuging 12,000g at room temperature for 2min to precipitate bacteria, and completely removing supernatant.

2) Adding 150 mu l of Buffer P1, fully suspending and shaking the thalli to precipitate for 10-15 sec, and completely dispersing until no floc exists.

3) Adding 150 mu l of Buffer P2, slightly inverting the centrifuge tube for 3-5 times, and standing at room temperature for 1min to completely crack bacteria, wherein the solution is transparent. When the bacteria are more, the bacteria can be placed for 3-5 min to fully crack the bacteria.

4) Adding 150 mu l of Buffer P3, immediately and quickly reversing the centrifuge tube for 4-6 times, fully and uniformly mixing, and standing at room temperature for 1min to show that white floccules are generated. When the thallus is abundant, the viscous mass is not easy to mix uniformly, the mouth of the centrifugal tube holding tube can be inverted, and the tip of the centrifugal tube is flicked by fingers for 2 times, so that the viscous mass is mixed uniformly into floccules.

5) Add 150. mu.l Buffer P4, gently invert the tube 3-5 times, and stand at room temperature for 10 min.

6) The above lysate was centrifuged at 12,000g for 8min at room temperature, the supernatant carefully aspirated and transferred to a new 1.5ml centrifuge tube. The pellet is typically attached to the bottom or side wall of the tube, taking care not to suck in the pellet residue.

7) Adding 600 mu l of Buffer P5, inverting the centrifuge tube for 3-5 times, and fully and uniformly mixing. Standing at room temperature for 5 min.

8) Centrifugation at 12,000g for 8min at room temperature discarded supernatant, inversion gently drained residual liquid, and small near-transparent pellet was typically visible on the bottom wall of the tube. And (3) lightly adding 1ml of 70% ethanol, washing for 2 times, draining residual liquid, and air-drying the precipitate for 3-5 min.

9) Adding a proper amount of TE (20-50 mu l) to dissolve the precipitate. The obtained DNA solution has partial RNA residue, but can be directly applied to the work of electrophoresis, enzyme digestion, connection, transformation, sequence determination and the like.

10) Mu.l of plasmid solution was added to 20. mu.l of Buffer P6 (precipitation B), mixed well and left on ice for 10min, centrifuged at 12,000g at room temperature for 10min, and the supernatant was discarded, and no pellet was observed. And (3) slightly adding 1ml of 70% ethanol, washing twice, draining residual liquid, and air-drying the precipitate for 3-5 min.

11) Adding a proper amount of TE (20-50 mu l) to dissolve the precipitate. The obtained DNA solution has RNA and other residual impurities removed basically, and can be directly applied to cell transfection, in vitro transcription and other works.

Note that:

1) the operation is gentle to prevent the loss of DNA due to mechanical shearing.

2) The OD and ratio should be determined using TE at pH 8.0 as the dilution. The OD ratio measured in pure water may be low.

3) The OD260/280 ratio of the finally obtained plasmid solution is generally in the range of 1.7-1.8. Such as plasmid DNA content

Low OD260/280 ratio will be low, but will not affect the use in transfection etc

CaCl2 method transformation experiment

(Transformation of Plasmid DNA Using CaCl2)

The operation method comprises the following steps:

1) from the-70 ℃ refrigerator, 100 u l competent cell suspension, at room temperature to thaw, thawing immediately after ice.

2) Adding plasmid DNA solution (the content is not more than 1 μ g, and the volume is not more than 10 μ l), shaking gently, placing on ice for 30 minutes, then thermally shocking in water bath at 42 ℃ for 90 seconds without moving a centrifugal tube in the thermal shock process, and rapidly placing on ice for cooling for 3-5 minutes after thermally shocking.

3) Adding 1ml LB liquid culture medium (without antibiotic) into the tube, sucking, mixing evenly, shaking and culturing for 1 hour at 37 ℃ and 220rpm in a shaking table, so that the bacteria recover to normal growth state, and express antibiotic resistance gene coded by plasmid.

4) Shaking the bacterial liquid evenly, centrifuging, removing 900 mul of supernatant, sucking and evenly mixing the rest culture medium, and then coating 100 mul of the mixture on a screening plate containing antibiotics.

5) The plate is placed for half an hour with the front face upward, the culture dish is inverted after the bacteria liquid is completely absorbed by the culture medium, and the culture is carried out for 16 to 24 hours at 37 ℃.

Note that:

1) plasmid DNA mass and concentration: the plasmid DNA used for transformation should be predominantly supercoiled, and generally the volume of the DNA solution should not exceed 5% of the volume of competent cells

2) Preventing contamination by bacteria and DNA

3) The whole operation needs to be carried out on ice, and the ice bath cannot be left, otherwise, the cell conversion rate is reduced

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

Plasmid mass extraction and purification kit

(Plasmid Maxprep Kit)

The operation method comprises the following steps:

plasmid extraction:

1) taking the overnight cultured bacteria with the bacterial liquid of less than 150ml, putting the overnight cultured bacteria into a proper centrifugal bottle, centrifuging 4,500-6,000 g at 4 ℃ for 10min to precipitate bacteria, and completely removing supernatant.

2) 5ml of Buffer I is added, and the thalli precipitation is fully suspended and shaken to be completely dispersed until no floc exists. The bacterial suspension was transferred into a 50ml centrifuge tube.

3) Adding 5ml of Buffer II, slightly inverting the centrifuge tube for 6-8 times, and standing at room temperature for 5min to completely crack bacteria, wherein the solution is transparent.

4) Adding 5ml of Buffer III, immediately reversing the centrifuge tube for 6-8 times, and fully and uniformly mixing until white floccules are generated. And standing on ice for 10-15 min.

5) The lysate is centrifuged at 12,000-16,000 g for 15min at 4 ℃, the supernatant carefully aspirated and transferred to a new 50ml centrifuge tube.

6) Add 10ml of isopropanol, reverse the tube, mix well. And placing on ice for 10-15 min.

7) Centrifugation was carried out at 412,000-16,000 g for 10min, the supernatant carefully discarded, the residual liquid was gently drained by inversion, 0.5ml Buffer I was added, and the pellet was completely dissolved (assisted by gentle pipetting with a wide-mouthed pipette). Transferring the mixture into a new 1.5ml centrifuge tube, and standing the centrifuge tube for 10-20 min at room temperature.

8) The crude plasmid extract was centrifuged at room temperature for 2min at high speed in a desk centrifuge and the supernatant was transferred to a new 1.5ml centrifuge tube. And (3) plasmid purification:

1)0.5ml plasmid crude extract with 100 u l Buffer IV (impurity removal liquid A), gently mixing, 12,000g centrifugation for 2min, supernatant transferred to a new centrifuge tube.

2) Then 100. mu.l of Buffer IV (impurity-removing solution A) was added, the mixture was gently mixed, centrifuged at 12,000g for 5min, and the supernatant was transferred to a new centrifuge tube.

3) Add 70. mu.l Buffer V (impurity scavenger B), mix gently, centrifuge at 12,000g for 5min, transfer the supernatant to a new centrifuge tube.

4) Adding 0.5ml isopropanol, mixing well, standing at room temperature for 10 min. Centrifuging at 12,000g at room temperature for 10min, discarding supernatant, gently washing with 1ml of 70% ethanol, discarding liquid, and air drying at room temperature for 5 min.

5)0.5ml TE dissolution precipitation (auxiliary dissolution by shaking in a 37 ℃ water bath or gently blowing with a wide-mouthed pipette)

6) Adding 200 mul of buffer VI (impurity removing solution C), uniformly mixing, standing on ice for 10-30 min, centrifuging 12,000g at room temperature for 10min, discarding the supernatant, slightly adding 1ml of 70% ethanol, washing twice, and reversely drying at room temperature for 5-10 min to completely evaporate the ethanol.

7) The precipitate was dissolved with an appropriate amount of TE (200-500. mu.l) (shaking in a 37 ℃ water bath to aid dissolution).

Note that:

1) when plasmids are extracted from a large amount of bacterial liquid or thalli, the using amounts of BufferI, BufferII and BufferIII can be increased in proportion so as to fully crack the thalli and improve the recovery amount and purity of the plasmids.

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

3) The operation is gentle to prevent the loss of DNA due to mechanical shearing.

4) Several microliters of DNA solution can be left before and after each step of purification, and finally the results of extraction and purification are compared by electrophoretic identification.

5) After centrifugation in purification steps 3 and 6, no significant precipitation may be seen. If no precipitation is found in step 6, there is a fear that DNA is lost, the supernatant may be retained, and after completion of the entire operation, it may be electrophoretically identified to determine whether the final product is obtained (several hundred micrograms of high purity plasmid DNA precipitated on the side wall of the tube after centrifugation, and a distinct pellet may not be visible).

6) The OD and the ratio should be determined using TE or Tris at pH8.0 as a dilution solution. The OD ratio measured in pure water may be low.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> gold mountain hospital affiliated to Fudan university (prevention and treatment institute for eye disease in gold mountain area of Shanghai city, nuclear injury emergency treatment center in gold mountain area of Shanghai city)

Application of <120> DHCR24 gene in preparation of medicines for treating neurodegenerative diseases

<130> /

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1551

<212> DNA

<213> Artificial sequence

<400> 1

atggagcccg ccgtgtcgct ggccgtgtgc gcgctgctct ttctgctctg ggtgcgagtg 60

aaggggttgg agttcgttct catccaccag cgctgggtgt tcgtgtgcct cttcttgctg 120

ccgctctcgc tcatcttcga tatctactac tacgtgcgcg cctgggtggt gttcaagctg 180

agcagtgcgc cgcgcctgca cgagcagcgc gtgcgggaca tccagaaaca ggtccgggaa 240

tggaaggaac agggcagtaa gaccttcatg tgcacggggc gcccaggctg gctcactgtc 300

tcgctgcgag tcggaaagta caagaagacc cataagaaca tcatgatcaa cctgatggac 360

atcctggagg tggacaccaa gaaacagatt gttcgagtgg agcccttggt gtctatgggt 420

caggtgacag ctttgctgaa ctccattggc tggaccctgc ctgtgttgcc tgagcttgat 480

gacctcacag tggggggcct gatcatgggc acaggcatcg agtcatcgtc ccacaagtat 540

ggcctgttcc aacacatttg cactgcctac gagctgatcc tggcagacgg cagctttgtg 600

cgctgcacac cgtctgaaaa ctcagacctg ttctatgccg tgccctggtc ctgtgggacc 660

ctgggcttcc tggtggctgc cgagatccgg atcatcccgg ccaagaagta tgtcaagctg 720

cggtttgagc ctgttcgggg cctggaggcc atctgtgaaa aattcacccg cgagtcccag 780

cggctggaga accacttcgt ggaagggttg ctgtactccc tggatgaggc tgtcatcatg 840

acaggggtca tgacggacga cgtagagccc agcaagctga atagcattgg cagttactac 900

aagccctggt tcttcaagca tgtggagaac tacctgaaga caaaccggga gggcctcgaa 960

tacattcccc tgagacacta ctaccaccga cacacgcgca gcatcttctg ggagctccag 1020

gacatcatcc ctttcggcaa caaccccatc ttccgctacc tcttcggctg gatggtgcct 1080

cccaagatct ccctcctgaa gctgacccag ggcgagacgc tacgcaagct gtacgagcag 1140

caccacgtgg tgcaggacat gctggtgccc atgaagtgca tgtcacaggc cctgcatacc 1200

ttccaaaatg acatccacgt ctaccccatc tggctgtgcc cattcatcct gcccagccag 1260

ccaggactag tgcatcccaa gggagatgaa gcagagctct acgtggacat cggggcatac 1320

ggggagccac gtgtgaagca cttcgaggcc aggtcctgca tgaggcagct ggagaagttt 1380

gtgcggagtg tgcacgggtt ccaaatgtta tacgccgatt gctatatgaa ccgcgaggaa 1440

ttctgggaga tgttcgatgg ctccttgtac cacaagctgc gcaagcagct gggctgccag 1500

gacgccttcc ctgaggtgta cgacaagatc tgcaaggcgg caaggcactg a 1551

<210> 2

<211> 1566

<212> DNA

<213> Artificial sequence

<400> 2

ggatccgcca ccatggagcc cgccgtgtcg ctggccgtgt gcgcgctgct ctttctgctc 60

tgggtgcgag tgaaggggtt ggagttcgtt ctcatccacc agcgctgggt gttcgtgtgc 120

ctcttcttgc tgccgctctc gctcatcttc gatatctact actacgtgcg cgcctgggtg 180

gtgttcaagc tgagcagtgc gccgcgcctg cacgagcagc gcgtgcggga catccagaaa 240

caggtccggg aatggaagga acagggcagt aagaccttca tgtgcacggg gcgcccaggc 300

tggctcactg tctcgctgcg agtcggaaag tacaagaaga cccataagaa catcatgatc 360

aacctgatgg acatcctgga ggtggacacc aagaaacaga ttgttcgagt ggagcccttg 420

gtgtctatgg gtcaggtgac agctttgctg aactccattg gctggaccct gcctgtgttg 480

cctgagcttg atgacctcac agtggggggc ctgatcatgg gcacaggcat cgagtcatcg 540

tcccacaagt atggcctgtt ccaacacatt tgcactgcct acgagctgat cctggcagac 600

ggcagctttg tgcgctgcac accgtctgaa aactcagacc tgttctatgc cgtgccctgg 660

tcctgtggga ccctgggctt cctggtggct gccgagatcc ggatcatccc ggccaagaag 720

tatgtcaagc tgcggtttga gcctgttcgg ggcctggagg ccatctgtga aaaattcacc 780

cgcgagtccc agcggctgga gaaccacttc gtggaagggt tgctgtactc cctggatgag 840

gctgtcatca tgacaggggt catgacggac gacgtagagc ccagcaagct gaatagcatt 900

ggcagttact acaagccctg gttcttcaag catgtggaga actacctgaa gacaaaccgg 960

gagggcctcg aatacattcc cctgagacac tactaccacc gacacacgcg cagcatcttc 1020

tgggagctcc aggacatcat ccctttcggc aacaacccca tcttccgcta cctcttcggc 1080

tggatggtgc ctcccaagat ctccctcctg aagctgaccc agggcgagac gctacgcaag 1140

ctgtacgagc agcaccacgt ggtgcaggac atgctggtgc ccatgaagtg catgtcacag 1200

gccctgcata ccttccaaaa tgacatccac gtctacccca tctggctgtg cccattcatc 1260

ctgcccagcc agccaggact agtgcatccc aagggagatg aagcagagct ctacgtggac 1320

atcggggcat acggggagcc acgtgtgaag cacttcgagg ccaggtcctg catgaggcag 1380

ctggagaagt ttgtgcggag tgtgcacggg ttccaaatgt tatacgccga ttgctatatg 1440

aaccgcgagg aattctggga gatgttcgat ggctccttgt accacaagct gcgcaagcag 1500

ctgggctgcc aggacgcctt ccctgaggtg tacgacaaga tctgcaaggc ggcaaggcac 1560

ggtacc 1566

Claims (5)

1. An application of AAV9 carrier compound containing DHCR24 gene in preparing medicine for treating neurodegenerative diseases is disclosed, wherein the AAV9 carrier compound containing DHCR24 gene is prepared by directly synthesizing SEQ ID NO.1 sequence to pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector.

2. The use according to claim 1, wherein the neurodegenerative disease is alzheimer's disease, parkinson's disease, brain aging, aging-related cognitive disorders, autism, niemann-pick disease, huntington's disease, motor neuron disease, frontotemporal dementia, lewy body dementia, multiple sclerosis (multiple sclerosis), Rett syndrome.

3. The use according to claim 2, wherein the neurodegenerative disease is alzheimer's disease.

4. The medicine for treating neurodegenerative diseases is an AAV9 vector compound containing DHCR24 gene, and the AAV9 vector compound containing the DHCR24 gene is obtained by directly synthesizing a SEQ ID NO.1 sequence into a pHBAAV-CMV-MCS-3flag-T2A-ZsGreen vector.

5. The medicament according to claim 4, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, brain aging, aging-related cognitive disorders, autism, Niemann-pick's disease, Huntington's disease, motor neuron disease, frontotemporal dementia, Lewy body dementia, multiple sclerosis (multiple sclerosis), Rett syndrome.

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