CN112980857A

CN112980857A - Nucleotide composition for coding secretory wild type GAA protein, adeno-associated virus vector, and medicine and application thereof

Info

Publication number: CN112980857A
Application number: CN202110451637.2A
Authority: CN
Inventors: 罗雁红; 何健峰; 刘锐敏; 田杰; 朱骅
Original assignee: Childrens Hospital of Chongqing Medical University
Current assignee: Childrens Hospital of Chongqing Medical University
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2021-06-18

Abstract

The invention discloses a nucleotide composition for coding secretory wild type GAA protein, an adeno-associated virus vector and a medicament thereof. The nucleotide comprises nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO. 2. The application designs and prepares recombinant adeno-associated virus (rAAV), the skeletal muscle cells of a patient are infected by the rAAV at one time, the wild GAA is continuously expressed and secreted in the muscle cells, the purpose of continuously increasing the wild GAA in vivo is achieved, and the defects that the half-life period of the recombinant GAA enzyme replacement therapy in blood is short and life-long treatment is needed are fundamentally solved.

Description

Nucleotide composition for coding secretory wild type GAA protein, adeno-associated virus vector, and medicine and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a nucleotide composition for coding secretory wild type GAA protein, an adeno-associated virus vector, and a medicament and application thereof.

Background

The glycogen storage disease type II is also called Pompe disease, is reported by Pompe pathologist in Netherlands in 1932 for the first time, is a rare autosomal recessive genetic disease and is caused by a defect of acid alpha-Glucosidase (GAA) gene. GAA is an enzyme essential for glycogen degradation in lysosomes and is capable of breaking down oligosaccharides and glycogen into glucose. When the enzymatic activity is decreased, glycogen is not decomposed, and glycogen accumulation in lysosomes causes rupture of lysosomes, which are organelles that decompose biological macromolecules such as proteins, nucleic acids, and polysaccharides, and contain many hydrolases, and if the lysosomes are ruptured, the released hydrolases destroy cells, resulting in cell death, and thus, tissue damage such as skeletal muscle, cardiac muscle, and liver is caused. Clinically, it can be manifested as myasthenia, cardiac hypertrophy, cardiac insufficiency, etc., and serious patients die due to exhaustion of respiratory cycle.

The GAA gene is located at 17q25.2-q25.3, has a total length of about 28kb and comprises 20 exons; the exon 1 is not involved in coding protein, the cDNA total length is about 3.6kb, and the GAA protein is composed of 952 amino acids. To date, nearly 300 pathogenic mutations of the GAA gene have been discovered, with the mutation sites being concentrated primarily in 3 functional regions of the gene. Pathogenic mutations mainly result in a variable degree of loss of GAA activity, with lower GAA activity leading to earlier onset of disease and more severe clinical manifestations.

Currently, human recombinant acid alpha-glucosidase (rhGAA) replacement therapy is the only therapy that can effectively treat glycogen storage disease type II. rhGAA (brand name: Lumizyme) developed by Xenophilus was approved by the FDA on the market at 8 months 2014, and this therapy required intravenous infusion of rhGAA enzyme (Lumizyme 20mg/kg) by patients every 1 week. Although the rhGAA has obvious curative effect, the patient needs life-long treatment, the cost is high (50 ten thousand RMB/year), most of the patients are difficult to bear at home, the rhGAA is an active enzyme, the storage, transportation and use (1mg/kg/hr, 20 hours are needed for one intravenous injection) of the rhGAA have strict requirements, and the rhGAA has the defects of poor targeting of the rhGAA to skeletal muscle, immunological rejection reaction of the body to foreign proteins and the like, so that the curative effect is influenced. Therefore, the development of a safe, long-lasting, sustained and stable GAA that provides active to patients is an urgent clinical need for the treatment of gsdii.

The safe and efficient introduction of a target gene into a human target cell is one of the keys to successful gene therapy. Although classical gene introduction methods (microinjection, electroporation, calcium phosphate transfection, etc.) have played a certain role in scientific research, their application is limited because the problems of transfection efficiency, targeting, safety, etc. cannot be solved well. With the development of related disciplines such as virology, various viruses are widely used as vectors in gene therapy at present, and adeno-associated virus (AAV) vectors are a novel vector developed in the last two decades. As an important member of viral vectors, AAV has no obvious cytotoxic effect and does not cause strong immune response of cells like other viral vectors, so that its application potential in gene therapy has attracted more and more interest and attention, and only human gene therapy drugs using AAV as a vector for human have begun to emerge.

Jerry Mendell medical doctor, the first investigator at the gene therapy center of the Abigail Wexner institute, delivered to patient cells in a first clinical trial of gene therapy for symptoms of spinal muscular atrophy type 1 (SMA1) (life must be maintained using a ventilator for life in the first year) by administering to a child having a mutated SMA1 gene (the SMNgene) Zolgensma (modified adeno-associated virus serotype 9(AAV9) as a vector capable of introducing the function of a human wild-type SMN gene). Almost all patients receiving a high dose of single intravenous injection in the study had a rapid improvement in motor function, achieved major milestones associated with child motor development including swallowing, tumbling, sitting, running and jumping independently without other drugs, further verifying that human gene therapy drugs with AAV as a carrier are feasible.

Adeno-associated virus belongs to the genus dependovirus of the parvoviridae family, is the smallest and simplest animal virus, and the genomic structure of AAV is short (4.7kb) and simple, but sufficient to perform all its functions of the life cycle. The AAV genome encodes two large open reading frames (ORE) rep and cap, which are flanked by Inverted Terminal Repeats (ITRs), contain cis-acting elements required for packaging, replication, integration and circularization of the viral genome of interest, and have enhancer and/or weaker transcription promoter functions. The replication proteins Rep and the capsid protein Cap of AAV are usually supplied in trans from a packaging plasmid carrying their genes.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a nucleotide composition for coding a secretory wild type GAA protein, an adeno-associated virus vector, a medicament thereof and a construction method of a recombinant adeno-associated virus, which fundamentally overcome the defects that the substitute treatment of the recombinant GAA enzyme has short half-life period in blood and needs lifelong treatment.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a nucleotide composition encoding a secreted wild-type GAA protein comprising the nucleotide sequence set forth in SEQ ID No. 1.

Comprises a nucleotide sequence which is shown as SEQ ID NO.1 and expresses the wild type GAA protein, and a nucleotide sequence which enables GAA to have the secretion function.

Further, the nucleotide sequence for making the GAA protein have a secretion function is shown in SEQ ID NO.2, and the nucleotide sequence can express tPA protein for making the GAA protein have a secretion function.

Furthermore, a nucleotide sequence with the function of cutting peptide is inserted between the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.

Further, the amino acid sequence having a function of cleaving a peptide includes a sequence encoding two amino acids SQ.

A recombinant adeno-associated virus vector for expressing secretory wild type GAA protein comprises the nucleotide composition.

The construction method of the recombinant adeno-associated virus vector for expressing the human secretory wild type GAA protein comprises the following steps:

(1) artificially synthesizing the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, inserting the nucleotide sequence into a pAAV-CMV-LacZ.bGH vector to construct a pAAV-CMV-tPA-GAA vector;

(2) and (2) co-transfecting the pAAV-CMV-tPA-GAA vector constructed in the step (1) with an RC vector pAAV2/9n (serotype 9, Addgene 112865) and a helper plasma pAdDeltaF6(Addgene 112867) packaging vector to obtain 293T cells, and culturing.

A pharmaceutical composition comprises the protein produced by the expression of the nucleotide composition or a recombinant adeno-associated virus vector, and a pharmaceutically acceptable carrier/auxiliary component thereof.

An expression cassette for expressing a secreted wild-type GAA protein comprising the above nucleotide composition operably linked to an expression control element.

The nucleotide composition, the recombinant adeno-associated virus vector and/or the pharmaceutical composition are applied to preparation of medicines for preventing and/or treating GSDII diseases.

The invention has the beneficial effects that:

1. the application designs and prepares recombinant adeno-associated virus (rAAV), and the rAAV infects skeletal muscle cells of patients to continuously express and secrete wild GAA in the muscle cells, thereby achieving the purpose of continuously increasing the wild GAA in vivo.

2. The application introduces a target gene into cells by taking adeno-associated virus (AAV) as a vector, and secretes GAA with normal function by using the skeletal muscle cells of a patient, thereby avoiding higher technical requirements and expensive economic burden of intravenous injection of human recombinant GAA.

3. Because the main target organ of the glycogen accumulation disease is skeletal muscle, the muscle function can be improved and the muscle disease can be relieved by injecting rAAV into the muscle to express normal active GAA; meanwhile, the muscle continuously expresses the secretable wild GAA, and can provide effective enzyme to each organ of the body for a long time (generally only 1 time of multi-point injection treatment of skeletal muscle is needed), thereby fundamentally solving the defects of short half-life period in blood and life-long treatment requirement of the replacement treatment of the recombinant GAA enzyme.

Drawings

FIG. 1 shows the process of introducing the secretory wild-type GAA (tPA-GAA) gene into an adeno-associated virus (AAV) vector;

FIG. 2 is a view of a vector structure; wherein A is a vector map of pAAV-CMV-LacZ.bGH; b is a structural map of the constructed pAAV-CMV-tPA-GAA vector;

FIG. 3 is a Boyden chamber experiment;

FIG. 4 is a Dunn chamber experiment;

FIG. 5 is a schematic diagram of a GSD II animal model gene therapy experiment;

FIG. 6 shows GSDII proband kidney epithelial cells in urine;

FIG. 7 shows renal epithelial cells in normal human urine;

FIG. 8 shows glycogen staining of renal epithelial cell lines in urine of normal persons and GSDII probands;

FIG. 9 is a graph showing the growth curves of the kidney epithelial cell line in urine of a normal human and a GSDII proband;

FIG. 10 is a vector map; wherein A is pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector map; b is a structural map of LV-hGAA carrier plasmid;

FIG. 11 is a graph showing the results of fluorescent plaques formed by the prepared lentiviruses;

FIG. 12 shows the result of AAV vector-introduced secretory MG53 gene for treating progressive muscular dystrophy in guinea pigs;

FIG. 13 shows the treatment of guinea pig peripheral blood MG53 protein expression by introducing a secretory MG53 gene into a lentivirus vector;

figure 14 is the result of Adenovirus (AV) as vector CRISPR/Cas9 gene editing technology gene therapy in mice with progressive muscular dystrophy.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

EXAMPLE 1 construction of recombinant adeno-associated Virus vector

The nucleotide sequence of the amino acid sequence expressing the human wild-type GAA protein (hGAA protein) is shown in SEQ ID NO: 1, the nucleotide sequence of the amino acid sequence (tPA protein) for expressing the human wild type GAA protein to have the secretion function is shown in SEQ ID NO: 2, simultaneously, SQ amino acid is added between the tPA protein and the hGAA protein.

Based on the amino acid sequence, the nucleotide sequence capable of expressing tPA and hGAA protein is artificially designed, and the nucleotide sequence is shown in SEQ ID NO: 1 and SEQ ID NO: 2. the designed nucleotide sequence was synthesized by Hanhengzheng Biotechnology (Shanghai) Co., Ltd., and the synthesized sequence was inserted between the 5 'NotI and 3' BlpI cleavage sites of the pAAV-CMV-LacZ. bGH (FIG. 2A) vector, and the constructed vector was named pAAV-CMV-tPA-GAA, and its structural diagram is shown in FIG. 2B.

EXAMPLE 2 preparation of recombinant adeno-associated Virus

293T cells are passaged to 100mm dish according to a conventional method, the cell density is observed after the passage, the transfection can be carried out when the confluence rate reaches 70-80%, pAAV-CMV-tPA-GAA carrier plasmids, RC vector pAAV2/9n (serotype 9, Addge 112865) and helper plasmid pAdDeltaF6(Addge 112867) are transfected by using transfection reagents (Lipofeter transfection reagents, purchased from Hantaheng Biotech, Ltd.), and lipofectamine is used as the transfection reagents, 75 mu L, 10 mu g of pAAV-CMV-tPA-GAA plasmids, 10 mu g of pAAV-RC plasmids and 5 mu g of pHelper plasmids are used. Mixing lipid with complex, incubating at room temperature for 15min, slowly adding into 293T cell, adding 5% CO at 37 deg.C₂Culturing in a cell culture box. The cell status was observed daily.

16 hours after transfection, the fresh complete medium containing 10% fetal bovine serum FBS was replaced, and two virus supernatants were collected 48h and 72h after transfection (48h after collection fresh complete medium was replaced). At 48h for detoxification, the medium in 100mm dish was poured into a 50mL centrifuge tube and carefully incubatedThe vessel walls were not touched to the centrifuge tube to prevent bacterial contamination, and then 10mL of fresh complete medium containing 10% fetal bovine serum FBS was supplemented and placed at 37 ℃ smoothly with 5% CO₂The culture is continued in the constant temperature incubator. When virus is collected for 72h, directly pouring the culture medium in 100mm dish into a 50mL centrifuge tube, and also paying attention to the fact that the wall of the culture dish does not contact with the opening of the centrifuge tube to prevent bacterial pollution; centrifuging the virus supernatant in a 50mL centrifuge tube at 4 ℃ for 10min at 2000g to remove cell debris; then collecting the virus stock supernatant, placing the virus stock supernatant into an ultracentrifuge tube, centrifuging the ultracentrifuge tube for 120min at 4 ℃ at 82700g, fully culturing the suspended virus, finally subpackaging the ultracentrifuge suspension into sterilized virus tubes, and counting the virus into recombinant adeno-associated virus (AAV tPA/hrGAA, rAAV 1). Subpackaging virus, marking (virus name, year-month-day), and storing in 80 deg.C refrigerator.

Example 3 quality testing of recombinant adeno-associated viruses (AAV tPA/hrGAA, rAAV1)

1. Sterility testing

The detection method comprises the following steps: and (3) adding 10 mu L of virus into Hela cells of a 96-well plate for verification, and performing microscopic examination after culturing for 24 h: the culture medium needs to be clear and transparent, has no obvious particles in intercellular spaces, and has no any bacterial and fungal pollution.

2. Mycoplasma detection

The detection method comprises the following steps: taking 10 mu L of virus, carrying out water bath at 96 ℃ for 15min, and then configuring a PCR reaction system in a super clean bench. Electrophoresis after PCR reaction was performed to determine whether there was mycoplasma contamination. The PCR gel should have no significant mycoplasma bands.

3. Titer assay

Viral titer detection using a dilution count assay:

titer unit: TU/mL, refers to the number of biologically active viral particles contained per mL. "TU" is an abbreviation for "transducing units" and in Chinese "transducing units" and represents the number of viral genomes that can infect and enter target cells. IU/mL, refers to the number of active virus particles contained per mL. "IU" is an abbreviation for integration units, and Chinese is an integration unit.

Cell preparation

Will grow to the stateGood 293T cell digestion count and dilution to 1X 10⁵PermL, add to 96-well plate, 100. mu.L/well, prepare 6 wells for each virus. Put at 37 ℃ with 5% CO₂Culturing in an incubator.

Adding virus II

The following day, 6 1.5mL EP tubes were prepared, and 10. mu.L of virus solution was added to the first EP tube, followed by 3-fold gradient dilution for a total of 6 dilutions.

III. addition of culture Medium

On the third day, wells requiring puromycin screening were aspirated 100mL of virus-containing medium and 100. mu.L of 10% FBS complete medium containing 1.5. mu.g/mL puromycin was added.

Observe the results and calculate the titer

On the fifth day, the titer of the virus stock was determined by Southern blotting using a biotin-labeled probe produced by Prime-A-Gene labeling kit (Promega, Madison, Wis., USA). The viral vector library was processed according to the NIH published biohazard safety class 2 guidelines.

Titer (TU/mL) cell number × positive clone percentage × MOI (1) × virus dilution × 10³TU/mL。

Example 4 determination of Effect of AAV tPA/hrGAA (rAAV1) Gene on GSDII patient cells

1. According to the method of the Urineasy urine cell amplification culture kit provided by the saber organisms, a large number of kidney epithelial cells are obtained from the urine of normal people and proband through amplification culture.

2. Efficacy and safety of treatment with secretory wild-type GAA Gene introduced into human cell model

rAAV1 infects kidney epithelial cells of normal human and proband, measures the activity of wild type GAA in culture solution, content of glycogen in cells, observes and compares the change of shape and function before and after gene therapy of kidney epithelial cells of normal human and proband. To confirm that the wild-type GAA has a secretory function, cells expressing secretable GAA (tPA-GAA) and cells not expressing tPA-GAA are cultured together and subjected to a Boyden chamber test (see FIG. 3) or a Dunn chamber test (see FIG. 4) to test the growth and function of the cells not expressing tPA-GAA.

(1) Determination of the copy number of cellular viral vectors

DNA was extracted from cells expressing secretable GAA (tPA-GAA) using Wizard genomic DNA purification kit (Promega, Madison, Wis., USA). Real-time quantitative PCR detection of hGAA was performed using primer pairs 50-AGTGCCCACACAGAGTGCGACGT-30 and 50-CCTCGTAGCGCCTGTTAGCTG-30 of SYBR Green (Roche, Basel, Switzerland). AAV tPA/hrGAA plasmid DNA was used to generate a curve for calculating the viral vector copy number.

(2) Cell growth conditions: and observing the growth conditions of normal human and proband kidney epithelial cells expressing and not expressing tPA-GAA, photographing under a microscope, digesting the cells for 5min by using Accutase, stopping, centrifuging for 5min by 200g, removing supernatant, resuspending the cells by using a stock culture solution, and counting under the microscope.

(3) GAA activity assay: the GAA activity of normal human and proband kidney epithelial cells expressing and not expressing tPA-GAA and their culture solutions were measured. The collected cells were homogenized in distilled water (1mg/20mL) using a homogenizer, then sonicated for 15s, and centrifuged at 1800g for 15 minutes at 4 ℃. For GAA activity measurement, 20mL of 2.2mM 4-methylumbelliferyl anti-a-D-glucoside (Sigma-Aldrich, St. Louis, Mo., USA) in 0.2M sodium acetate buffer (pH 4.3) was added to 10mL of the above supernatant and the stock culture in a petri dish, collected, centrifuged, and placed in another tube to give a supernatant while measuring GAA activity. The mixture was incubated at 37 ℃ for 1 hour and the reaction was stopped by adding 0.5M carbonate buffer (pH 10.5). The amount of fluorescence (360nm excitation, 465nm emission) was measured on a victor X multi-label plate reader. And was standardized to 4-methylumbelliferone (Sigma-Aldrich, St. Louis, Mo.).

(4) Measurement of glycogen content: mixing the following components in parts by weight: 5 dilutions of normal human and proband kidney epithelial cell lysates expressing and not expressing tPA-GAA were boiled for 3 minutes (to inactivate endogenous enzymes) and mixed with 0.175U/mL (final concentration in the reaction) of amyloglucosidase (Sigma-Aldrich, St.Louis, Mo., USA) for 90 minutes at 37 ℃. The reaction mixture was then boiled again for 3 minutes to terminate the reaction. 30mL of the mixture was incubated with 1mL of Point Scientific glucose (hexokinase) liquid reagent (Fisher, Hanpton, N.H., USA) at Room Temperature (RT) for at least 10 minutes. The absorbance at 340nm was read on a Shi madzu UV-1700 PharmaSpec UV-VIS spectrophotometer. Protein concentrations were determined by BCA assay and used to normalize the data.

(5) Micro Western Blot experiment (Western Blot): normal human and proband kidney epithelial cells expressing and not expressing tPA-GAA, the amount of hGAA is determined. The collected cells were homogenized on ice in RIPA buffer (PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and protease and phosphatase inhibitor mix [ Cell Signaling Technology, Danvers, MA, USA) using a glass homogenizer. Lysates were cleared by centrifugation at 18,000g for 15min at 4 ℃. The protein concentration in the supernatant was measured using a bicinchoninic acid (BCA) assay. Equal amounts of protein were run on SDS-PAGE gels and then transferred to nitrocellulose membranes. The membrane was blocked with Tween 20(PBST) in phosphate buffer containing 3% BSA, incubated with primary antibody overnight at 4 ℃, washed, incubated with secondary antibody, washed again, and then developed using ECL kit (Bio-Rad, Herc. mu. Les, Calif., USA). Images were obtained by image analyzer (Bio Rad, Herc. mu. Les, CA, USA). The following antibodies were used: anti-hGAA from Sigma-Al drich (st louis, missouri, usa).

(6) PAS dyeing: normal human and proband kidney epithelial cells expressing and not expressing tPA-GAA were fixed by immersion in 10% Neutral Buffer Formalin (NBF) for 48 hours. After the initial immersion fixation, the samples were mixed with 1% Periodic Acid (PA) in 10% NBF at 4 ℃ for 48 hours. The samples were then washed with PBS, dehydrated with high purity alcohol, and washed with xylene. Oxidation with freshly prepared 0.5% PA for 5min followed by rinsing with distilled water for 1 min. The staining was then performed again for 15 minutes with Schiff reagent and washed with tap water for 10 minutes. Counterstaining with hematoxylin and rinsing with tap water, soaking with bluing agent for 1 min, dehydrating and fixing. The image was taken under a BZ X710 microscope to observe the area of glycogen accumulation.

Example 5 validation of the efficacy and safety of AAV tPA/hrGAA (rAAV1) Gene therapy at the GAA knockout transgenic mouse animal model level

Ready-made GAA knockout transgenic mice (GAAKO76 mice) hermaphrodite and normal wild type mice (C57BL/6J WT mice) hermaphrodite mice were purchased and bred to the number required for the experiment (40 GAAKO76 mice, 20C 57BL/6J WT mice). rAAV1 is infected with GAA knockout transgenic mouse skeletal muscle (GAAKO76 mouse skeletal muscle is directly injected at multiple points by rAAV1), Genotyping detects whether a target gene is successfully introduced into GAAKO76 mouse skeletal muscle cells and the efficiency of the target gene, Western Blot is used for detecting the expression quantity of wild type GAA in blood and tissues and measuring the GAA activity, whether the wild type GAA in the blood of the transgenic mouse is stably expressed is observed, the clinical performances (spirit, appetite, weight, activity state, mating condition and the like) of GAAKO76 mice and C57BL/6J WT mice infected by GAAKO76 mice and rAAV1 which are treated by rAAV1 gene, infected by unloaded AAV, treated by human recombinant GAA (Lumizyme intravenous injection) and the improvement of laboratory and auxiliary examination (various metabolic indexes, toxicity experiments, mouse accelerated rotation tests and the like) are carried out, and color ultrasonography, liver and heart organ conditions and the like are also examined (see figure 5).

(1) Animal care and experiments were performed according to guidelines approved by the committee for animal care and use of the secondary children hospital, university of Chongqing medicine.

Female and male GAAKO76 mice 2 weeks old received a dose of 5X 10¹²Vector genome (vg)/kg of rAAV1 and empty AAV were injected intramuscularly (via hind leg skeletal muscle) and human recombinant GAA enzyme (Lumizyme 20mg/kg)2 weeks 1 times intravenously (via retro-orbital sinus) and sex and age matched C57BL/6J WT mice received a dose of 5X 10¹²Vector genome (vg)/kg of rAAV1 were injected intramuscularly (via hind leg skeletal muscle) in a single multiple injection. Sex and age matched untreated GAAKO and C57BL/6J WT (from Jackson laboratories) mice were used as controls. Mice were tested for all indicators to observe and then sacrificed at 4 months of age (14 weeks after injection of rAAV1) to collect tissue and blood. The fresh tissue specimen is immediately frozen and preserved with dry ice and maintained at minus 80 deg.C until used for biochemical analysis or histological examination, and the blood can be directly used for examining liver and kidney function and myocardial enzymeSpectra, etc.

(2) Determination of skeletal muscle tissue viral vector copy number

DNA was extracted from skeletal muscle tissue expressing tPA-GAA using Wizard genomic DNA purification kit (Promega, Madison, Wis., USA). Real-time quantitative PCR detection of hGAA was performed using primer pairs 50-AGTGCCCACACAGAGTGCGACGT-30 and 50-CCTCGTAGCGCCTGTTAGCTG-30 of SYBR Green (Roche, Basel, Switzerland). AAV tPA/hrGAA plasmid DNA was used to generate a curve for calculating the viral vector copy number.

(3) PAS dyeing: cells were replaced with tissues from different sites in experimental mice as before. The images were taken under a BZX710 microscope.

(4) GAA activity assay: cells were replaced with tissues from different sites in experimental mice as before.

(5) Measurement of glycogen content: cells were replaced with tissues from different sites in experimental mice as before.

(6) Micro Western Blot experiment (Western Blot): cells were replaced with tissues from different sites in experimental mice as above.

(7) Accelerated rotation test: mice were trained by means of a rotator (ENV-577M, Med Associates, fel fakes, buddle usa) by first resting them on a rotating drum rotating at a constant speed of 4 revolutions per minute (waiting mode) for 3 minutes. The mice were then trained twice on rotating horses (4.0-40rpm) with gradual acceleration. Trained mice were then tested in three sessions using an accelerated spin protocol and the latency to fall was recorded. A rest period of at least 5 minutes is provided between each exercise.

(8) And (3) cylinder testing: mice were placed in 800mL glass beakers and 3 minutes of vertical exploration movements were recorded for each animal. A mirror is placed behind the cylinder to record all dorsal activity (vertical movement of one or both forelimbs contacting the cylinder wall). The amount of posterior activity during the vertical survey is recorded. The asymmetry in spontaneous forelimb usage was calculated by a one-to-one forelimb usage ratio.

(9) Light beam walking test: some modifications were made to the beam walking test described previously. Briefly, a mouse was placed at one end of the beam (12 mm wide) and the time required for the mouse to traverse the black box (800 mm) to reach the other end was measured. Prior to testing, mice were trained twice. On average, two mice did not stall on the beam.

(10) Safety check of gene therapy: observing the performance (spirit, appetite, weight, activity state, mating condition, etc.) of the mice subjected to gene therapy, checking the peripheral blood liver and kidney functions and myocardial zymogram of each experimental mouse, and checking the heart, liver, etc. of the animals subjected to inspection by color ultrasonography.

Example 6 proband clinical data Collection

Firstly, patients are diagnosed with oliguria, profuse sweating and dysphoria when the patients are 5 months old, find that the liver and spleen are swollen, the cardiac muscle is hypertrophic and the cardiac function is incomplete, misdiagnosis is hypertrophic cardiomyopathy and treatment is carried out, gene detection is carried out when the patients are 2 years old, and a homozygous mutation point is unexpectedly found in the GAA gene exon region: c.2015g > a (guanine > adenine), resulting in an amino acid change p.r672q (arginine > glutamine), is a missense mutation in exon 14. Therefore, a dry blood strip method (DBS) is adopted to collect a peripheral blood sample of the infant patient and detect that the activity of the leucocyte acid GAA of the infant patient is obviously lower than a normal value; and detecting GAA genes of parents in the family, grandparents and grandparents, and finding that the parents in the family, the mothers, the grandparents and the fathers are carriers of the disease-causing gene heterozygotes and accord with the recessive inheritance rule of the autosomal genes. The diagnosis was unequivocally gsdii. When the predecessor is 4 years old, come to our hospital again to see a doctor, and ask the medical history: the infant can walk alone in 1 year or half, can also hold up the railing and go up and down stairs about 2 years or half, and the back of squatting supports the knee and can stand by oneself, but can only walk the level road now, can not go up and down stairs, can not stand by oneself after squatting, easily wrestle, and two upper limbs can not hold heavier object, and the unclear mouth and teeth can not say the sentence, and respiratory infection repeats, and cough is powerless, and face and two low limbs are edema repeats. Physical examination: 15Kg of body weight, special face appearance (wide eye distance, collapse of nose bridge, thick mouth and lip), smooth breathing, slightly coarse breathing sound of both lungs, audible II/II SM in precordial region, 3.0cm below hepatic rib, soft texture, and sharp edge. The muscle strength of both lower limbs is grade 4, the muscle tension is slightly low, and the gastrocnemius muscles on both sides are pseudohypertrophic. And (3) reviewing a myocardial zymogram, a myocardial marker, abnormal liver function, abnormal electrocardiogram, abnormal heart color ultrasound and abnormal heart nuclear Magnetic Resonance (MRI). The abnormal condition of electromyogram of double lower limbs is found by reexamination, and the fasting blood sugar is normal. The disease condition of the infant patient slowly progresses, and the diagnosis accords with the non-classical infant GSDII diagnosis.

Example 7 establishment of Normal human and proband urinary Kidney epithelial cell lines

Separating and culturing urine cells of normal people and probands:

according to the method of the Urineasy urine cell separation culture kit provided by the siperie organisms: after the sampler discharges aged urine, a large amount of water is drunk, and more than 100mL of urine is discharged as much as possible. Urine cells, mainly renal epithelial cells, were isolated according to the kit method.

Urine cell amplification culture of proband and normal people:

according to the method of a Urineasy urine cell amplification culture kit provided by Seebine organisms, a large number of kidney epithelial cells are obtained by amplification culture (see figure 6 and figure 7).

Example 8 glycogen staining of Normal human and GSDII proband urorenal epithelial cell lines

Normal human and GSDII proband urine kidney epithelial cell line are stained with glycogen, and it can be seen that GSDII proband kidney epithelial cells are obviously hypertrophied compared with normal human kidney epithelial cells, and the intracellular glycogen content is high (see figure 8).

Example 9 growth curves of Normal human and proband urinary Kidney epithelial cell lines

The growth curves of the urinary epithelial cell lines of the proband normal persons show that the GSDII proband renal epithelial cells grow obviously slower than the normal human renal epithelial cells (see figure 9)

Example 10 h-GAA Lentiviral vector construction and viral packaging

1. Destination carrier information

pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector map (see FIG. 10A):

2. vector cleavage

Sequentially adding each reagent according to the sequence in the following table, slightly sucking, beating and uniformly mixing, and placing in a water bath kettle at 37 ℃ for reaction for 1-2 h; carrying out agarose gel electrophoresis after enzyme digestion is finished, and recovering a target fragment; the vector cleavage system is shown in Table 1 below.

TABLE 1 vector cleavage System

3. The acquisition of the target fragment, the primer design and the PCR amplification of the target fragment are carried out by conventional means.

4. Ligation of the fragment of interest to the vector

Ligation system was cloned in one step using HB infusion (TM): the following reaction system was prepared in an ice-water bath. If the liquid is inadvertently stuck to the tube wall, it can be sunk to the bottom of the tube by brief centrifugation. The ligation reaction solution was reacted at 50 ℃ for 30min, then placed on ice for 5min, and immediately transformed, and the ligation system is shown in Table 2.

TABLE 2 connection System

5. Transformation of

(1) Immediately putting DH5 alpha competent cells on ice to melt after the cells are taken out from a refrigerator at the temperature of-80 ℃, and gently operating the competent split charging process to reduce the mechanical damage to the cells;

(2) after competent thawing, aliquots were dispensed in volumes of 50 μ L per tube (20 μ L is sufficient for plasmid transformation), and after aliquots were added the ligation product in amounts not exceeding the competent volume 1/10 (currently 5 μ L ligation product), placed on ice for 20-30 min;

(3) heat shock is carried out for 90s at 42 ℃ (the time is very strict), and ice is inserted into the water to carry out ice incubation for 2-3min immediately after the heat shock is finished;

(4) in a clean bench, 500. mu.L of LB medium (note that there must be no antibody LB medium) was added, gently inverted 3-5 times;

(5) shaking and culturing at 37 deg.C and 230rpm for 45-60 min;

(6) coating the bacterial liquid on a solid plate with corresponding resistance, uniformly coating, and then inversely placing the plate in a constant temperature box at 37 ℃ for culturing for 12-16 h;

6. and carrying out PCR identification on the bacterial liquid.

7. Sequencing

And selecting two screened positive clones for sequencing, wherein the sequencing result is consistent with the target sequence, and the target plasmid is successfully constructed.

8. Plasmid extraction

After the sequencing is successful, bacterial liquid amplification, plasmid extraction and purification are carried out, and the plasmid extraction scheme is subject to the instruction of a conventional extraction kit. The extracted plasmids were used to transfect cells after QC validation was required. Construction of a successful LV-hGAA vector map (see FIG. 10B)

9. Lentivirus packaging and concentration purification

(1) 293T cells were passaged in advance for transfection (provided that the cells were cultured to meet the requirements of subsequent transfection experiments). After the operation is finished, the mixture is placed at 37 ℃ and 5 percent CO₂The incubator of (1);

(2) observing the cell density before transfection, and performing transfection when the confluence rate reaches 70-80%; three plasmid System, pSPAX2, pMD2G and LV-hGAA shuttle plasmid carrying the Gene of interest (lentiviral vectors and helper packaging vector plasmids amplified beforehand with E.coli strain DH 5-alpha)

(3) The composition of the complex transfected into 100mm dishes after lipofection into the complex is shown in Table 3.

TABLE 3 complete composition

Note: lipofiter transfection reagent is Hexantrone, and the instructions refer to Lipofiter instructions. The shuttle plasmid is LV-hGAA carrier plasmid containing target gene fragment.

Mixing lipid with complex, incubating at room temperature for 15min, slowly adding into 293T cell, adding 5% CO at 37 deg.C₂Culturing in a cell culture box;

(4) fresh complete medium containing 10% Fetal Bovine Serum (FBS) was replaced 16h after transfection;

(5) toxic materials recovering: two virus supernatants were collected 48h and 72h post transfection (48h collection followed by replacement of fresh complete medium). At 48h harvest, the medium in 100mm dish was poured into a 50mL centrifuge tube, taking care that the dish wall did not touch the centrifuge tube orifice to prevent bacterial contamination, then 10mL fresh complete medium containing 10% fetal bovine serum FBS was supplemented and placed smoothly at 37 ℃ with 5% CO₂The culture is continued in the constant temperature incubator. When virus is collected for 72h, directly pouring the culture medium in 100mm dish into a 50mL centrifuge tube, and also paying attention to the fact that the wall of the culture dish does not contact with the opening of the centrifuge tube to prevent bacterial pollution;

(6) ultracentrifugation: centrifuging the virus supernatant in a 50mL centrifuge tube at 4 ℃ for 10min at 2000g to remove cell debris; then collecting the virus stock solution supernatant, placing the virus stock solution supernatant into an ultracentrifuge tube, centrifuging the ultracentrifuge tube for 120min at 4 ℃ at 82700g, fully culturing the suspended virus precipitate, and finally subpackaging the ultracentrifuge heavy suspension solution into sterilized virus tubes.

(7) And (3) virus preservation: subpackaging virus, marking (virus name, year-month-day), and storing in 80 deg.C refrigerator.

10. Lentiviral quality detection

(1) Sterility testing

The detection method comprises the following steps: and (3) adding 10 mu L of virus into Hela cells of a 96-well plate for verification, and performing microscopic examination after culturing for 24 h:

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

(2) Mycoplasma detection

The detection method comprises the following steps: taking 10 mu L of virus, carrying out water bath at 96 ℃ for 15min, and then configuring a PCR reaction system in a super clean bench. Electrophoresis after PCR reaction was performed to determine whether there was mycoplasma contamination.

QC standard: PCR gel pattern has no obvious strip

(3) Titer assay

Lentivirus titer detection dilution count assay was used:

I. Cell preparation

The 293T cells with good growth state are digested and counted, and then diluted to 1 × 10⁵PermL, add to 96-well plate, 100. mu.L/well, prepare 6 wells for each virus. Put at 37 ℃ with 5% CO₂Culturing in an incubator.

Adding virus II

III. addition of culture Medium

Observe the results and calculate the titer

On the fifth day, the results were observed under a fluorescence microscope, and the fresh 10% FBS complete medium was replaced 6 hours before the observation, 80. mu.L of the medium was aspirated from the wells, and 80. mu.L of the fresh 10% FBS complete medium was added thereto, and the wells were placed at 37 ℃ and 5% CO₂Culturing in an incubator. After 6h, the results were observed under a fluorescence microscope (see FIG. 11), and the virus titer was calculated in wells with a fluorescence percentage of 10 to 50%.

Titer (TU/mL) cell number × positive clone percentage × MOI (1) × virus dilution × 10³TU/mL

Example 11 treatment of progressive muscular dystrophy in guinea pig by introducing secretable MG53 Gene into Virus vector

Guinea pig for treating progressive muscular dystrophy by introducing secretory MG53 gene into AAV as vector

1. Hematoxylin-eosin (H)&E) Staining assessed histological improvement. In newborn mice, AAV-MG53 (1X 10) was injected¹³vg/kg) 2 months later, TO-2 hamsters were analyzed. The scale bar is 100 μm.

2. The percentage of centrally located nuclei (CN) of the treated muscle fibers described in step (1) was significantly reduced. The percentage of CN in the muscle fibers of each of the above groups is shown. Values represent mean ± sd. P <0.01, compared TO untreated TO-2 (n-5/group, 300 fibers analyzed per muscle) (see two leftmost panels in fig. 12).

3. Masson trichrome staining demonstrated decreased skeletal muscle and myocardial fibrosis. Treatment in newborn hamsters (1X 10)¹³vg/kg) were collected 4.5 months and 7.5 months after the start of the test. The scale bar is 100 μm.

4. Newborn hamster (1X 10)¹³vg/kg) or adult hamster (2X 10)¹³vg/kg) increased the percent left ventricular shortening (% LVFS) shown by cardiac function improvement. P<0.05 compared TO untreated TO-2 (n-5). (see the middle part of FIG. 12 for two drawings)

5. Newborn hamster (1X 10)¹³vg/kg) (Ms, month) treated TO-2 diseased hamsters left ventricular end systolic and end diastolic diameters (LVID; s and LVID; d) decreased, indicating improved cardiac function. P<0.05 compared TO untreated TO-2 (n-5). Values represent mean ± sd; all hamsters were male. (see the two rightmost figures in FIG. 12)

As shown in fig. 12, in the present application, when the AAV was used as a vector TO introduce the secretable MG53 gene TO treat progressive muscular dystrophy in guinea pigs, it was found that impaired myocyte and intermuscular fibrosis and muscle function were significantly improved, indicating that introduction of the MG53 gene system using AAV as a vector could improve skeletal muscle and myocardial degeneration and fibrosis of the muscular dystrophy TO-2 hamster and improve cardiac function. (see FIG. 12, the bars in each set of data in the figure are WT-F1B, TO-2, and TO-2+ AAV in order from left TO right, where WT F1B is wild-type normal control hamster, TO-2 is muscular dystrophy hamster, and TO-2+ AAV is adeno-associated virus hamster).

In the present application, the slow virus is used as a vector to introduce the secretory MG53 gene to treat guinea pig progressive muscular dystrophy peripheral blood MG53 protein assay, and it can be seen that wild type guinea pig peripheral blood MG53 protein is very low, while the slow virus is used as a vector to introduce the secretory MG53 gene to treat guinea pig peripheral blood MG53 protein expression level is very high, and the slow virus is used as a vector to introduce the secretory MG53 gene successfully (see FIG. 13).

Example 12 Gene therapy of mice with progressive muscular dystrophy Using the CRISPR/Cas9 Gene editing technique with Adenovirus (AV) as vector

The CRISPR/Cas9 gene editing technology using Adenovirus (AV) as a vector treats mice suffering from progressive muscular dystrophy, successfully enables the mice without the ability to walk to recover walking, and the result is shown in figure 14.

In FIG. 14, a is a genomic position map of mouse X chromosome and gRNA target, and mutated exon 23 is highlighted in yellow (not shown); b Polymerase Chain Reaction (PCR) analysis of genomic DNA extracted from C2C12 cells treated with or without gRNA and Cas9 constructs; c is RT-PCR analysis of dystrophin transcript expression in C2C12 cells; d is PCR analysis of mdx myoblast genomic DNA after introduction of gRNA with and without adenovirus introduction (Ad-i20/i23) and Cas9 constructs; e is RT-PCR analysis of dystrophin transcript expression in mdx myoblasts treated in d; f is DNA sequencing analysis of the smaller RT-PCR product (475bp) in c and e; arrows indicate expected bands after gene editing. All data were from at least three experiments.

Sequence listing

<110> Chongqing medical university affiliated children hospital

<120> a nucleotide composition for coding secretory wild type GAA protein, adeno-associated virus vector, and medicament and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2859

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggagtga ggcacccgcc ctgctcccac cggctcctgg ccgtctgcgc cctcgtgtcc 60

ttggcaaccg ctgcactcct ggggcacatc ctactccatg atttcctgct ggttccccga 120

gagctgagtg gctcctcccc agtcctggag gagactcacc cagctcacca gcagggagcc 180

agcagaccag ggccccggga tgcccaggca caccccggcc gtcccagagc agtgcccaca 240

cagtgcgacg tcccccccaa cagccgcttc gattgcgccc ctgacaaggc catcacccag 300

gaacagtgcg aggcccgcgg ctgttgctac atccctgcaa agcaggggct gcagggagcc 360

cagatggggc agccctggtg cttcttccca cccagctacc ccagctacaa gctggagaac 420

ctgagctcct ctgaaatggg ctacacggcc accctgaccc gtaccacccc caccttcttc 480

cccaaggaca tcctgaccct gcggctggac gtgatgatgg agactgagaa ccgcctccac 540

ttcacgatca aagatccagc taacaggcgc tacgaggtgc ccttggagac cccgcatgtc 600

cacagccggg caccgtcccc actctacagc gtggagttct ccgaggagcc cttcggggtg 660

atcgtgcgcc ggcagctgga cggccgcgtg ctgctgaaca cgacggtggc gcccctgttc 720

tttgcggacc agttccttca gctgtccacc tcgctgccct cgcagtatat cacaggcctc 780

gccgagcacc tcagtcccct gatgctcagc accagctgga ccaggatcac cctgtggaac 840

cgggaccttg cgcccacgcc cggtgcgaac ctctacgggt ctcacccttt ctacctggcg 900

ctggaggacg gcgggtcggc acacggggtg ttcctgctaa acagcaatgc catggatgtg 960

gtcctgcagc cgagccctgc ccttagctgg aggtcgacag gtgggatcct ggatgtctac 1020

atcttcctgg gcccagagcc caagagcgtg gtgcagcagt acctggacgt tgtgggatac 1080

ccgttcatgc cgccatactg gggcctgggc ttccacctgt gccgctgggg ctactcctcc 1140

accgctatca cccgccaggt ggtggagaac atgaccaggg cccacttccc cctggacgtc 1200

cagtggaacg acctggacta catggactcc cggagggact tcacgttcaa caaggatggc 1260

ttccgggact tcccggccat ggtgcaggag ctgcaccagg gcggccggcg ctacatgatg 1320

atcgtggatc ctgccatcag cagctcgggc cctgccggga gctacaggcc ctacgacgag 1380

ggtctgcgga ggggggtttt catcaccaac gagaccggcc agccgctgat tgggaaggta 1440

tggcccgggt ccactgcctt ccccgacttc accaacccca cagccctggc ctggtgggag 1500

gacatggtgg ctgagttcca tgaccaggtg cccttcgacg gcatgtggat tgacatgaac 1560

gagccttcca acttcatcag gggctctgag gacggctgcc ccaacaatga gctggagaac 1620

ccaccctacg tgcctggggt ggttgggggg accctccagg cggccaccat ctgtgcctcc 1680

agccaccagt ttctctccac acactacaac ctgcacaacc tctacggcct gaccgaagcc 1740

atcgcctccc acagggcgct ggtgaaggct cgggggacac gcccatttgt gatctcccgc 1800

tcgacctttg ctggccacgg ccgatacgcc ggccactgga cgggggacgt gtggagctcc 1860

tgggagcagc tcgcctcctc cgtgccagaa atcctgcagt ttaacctgct gggggtgcct 1920

ctggtcgggg ccgacgtctg cggcttcctg ggcaacacct cagaggagct gtgtgtgcgc 1980

tggacccagc tgggggcctt ctaccccttc atgcggaacc acaacagcct gctcagtctg 2040

ccccaggagc cgtacagctt cagcgagccg gcccagcagg ccatgaggaa ggccctcacc 2100

ctgcgctacg cactcctccc ccacctctac acactgttcc accaggccca cgtcgcgggg 2160

gagaccgtgg cccggcccct cttcctggag ttccccaagg actctagcac ctggactgtg 2220

gaccaccagc tcctgtgggg ggaggccctg ctcatcaccc cagtgctcca ggccgggaag 2280

gccgaagtga ctggctactt ccccttgggc acatggtacg acctgcagac ggtgccagta 2340

gaggcccttg gcagcctccc acccccacct gcagctcccc gtgagccagc catccacagc 2400

gaggggcagt gggtgacgct gccggccccc ctggacacca tcaacgtcca cctccgggct 2460

gggtacatca tccccctgca gggccctggc ctcacaacca cagagtcccg ccagcagccc 2520

atggccctgg ctgtggccct gaccaagggt ggggaggccc gaggggagct gttctgggac 2580

gatggagaga gcctggaagt gctggagcga ggggcctaca cacaggtcat cttcctggcc 2640

aggaataaca cgatcgtgaa tgagctggta cgtgtgacca gtgagggagc tggcctgcag 2700

ctgcagaagg tgactgtcct gggcgtggcc acggcgcccc agcaggtcct ctccaacggt 2760

gtccctgtct ccaacttcac ctacagcccc gacaccaagg tcctggacat ctgtgtctcg 2820

ctgttgatgg gagagcagtt tctcgtcagc tggtgttag 2859

<210> 2

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgccc 66

Claims

1. A nucleotide composition encoding a secreted wild-type GAA protein, comprising a nucleotide sequence as set forth in SEQ ID No.1 which expresses the wild-type GAA protein and a nucleotide sequence which confers the GAA a secretory function.

2. The nucleotide composition of claim 1, wherein the GAA secretory nucleotide sequence is set forth in SEQ ID No.2, and the GAA secretory protein is expressed.

3. The nucleotide composition for encoding a secreted wild-type GAA protein according to claim 2, wherein a nucleotide sequence having a function of cleaving a peptide is inserted between the nucleotide sequences represented by SEQ ID No.1 and SEQ ID No. 2.

4. The nucleotide composition of claim 3, wherein said nucleotide sequence having the function of cleaving a peptide comprises a sequence encoding two amino acids SQ.

5. A recombinant adeno-associated viral vector expressing a secreted wild-type GAA protein, comprising the nucleotide composition according to any one of claims 1 to 4.

6. A pharmaceutical composition, comprising the protein expressed by the nucleotide composition of any one of claims 1 to 4, or the recombinant adeno-associated virus vector of claim 5, and a pharmaceutically acceptable carrier/auxiliary component thereof.

7. An expression cassette for expressing a secreted wild-type GAA protein comprising the nucleotide composition of any one of claims 1 to 4 operably linked to an expression control element.

8. Use of the nucleotide composition of any one of claims 1 to 3, the recombinant adeno-associated virus vector of claim 5 and/or the pharmaceutical composition of claim 6 in the preparation of a medicament for the prevention and/or treatment of gsdii disorders.