RU2812468C1 - Expression vectors based on adeno-associated virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is described: an expression vector which is a viral particle of an adeno-associated virus (AAV) having AAV capsid proteins , in which a single-stranded DNA molecule is packaged as a genome, at the ends of which the AAV ITR sequences are located, within the AAV ITR the promoter of the first eukaryotic elongation factor EF1 alpha is sequentially located, histidine tag sequence, intracellular nuclear localization signal 5'-SV40 NLS, codon-optimized sequence of the Streptococcus pyogenes Cas9 gene, another intracellular nuclear localization signal 3'-SV40 NLS, a synthetic polyA signal was used as a transcription terminator. The following is also described: an expression vector which is a viral particle of an adeno-associated virus (AAV) having AAV capsid proteins , in which a single-stranded DNA molecule is packaged as a genome, at the ends of which the AAV ITR sequences are located ; within the AAV ITR, the eukaryotic polymerase III promoter — U6 is sequentially located, after which there is a guide RNA expression cassette, including a guide RNA sequence in crRNA format, complementary to the targeted locus, linked to the trackRNA sequence, a region of homology with the human genome locus AAVS1 is the left homologous arm, a transgene expression cassette: the EFS-NS promoter, under the control of which The protein-coding sequence of the hCYP21A2 gene is 78–99% identical to the sequence SEQ ID NO: 3, transcription terminator is polyA signal, region of homology with the human genome locus AAVS1 is the right homologous arm.

EFFECT: group of inventions expands the arsenal of means for producing proteins in mammalian cells.

9 cl, 3 dwg, 4 tbl, 4 ex

Description

Field of technology to which the invention relates

The group of inventions relates to biotechnology, namely to expression vectors for producing proteins in mammalian cells. The group of inventions is represented by expression vectors based on the viral particle of the adeno-associated virus (AAV), which can find their use in medicine, in particular for the treatment of congenital dysfunction of the adrenal cortex.

State of the art

Congenital adrenal dysfunction (CAD, congenital adrenal hyperplasia) is a group of diseases associated with a deficiency of cortisol and/or aldosterone (steroid hormones). The deficiency is caused by genetic mutations in an enzyme involved in the formation of cortisol and aldosterone from cholesterol. The enzyme may be completely absent or produced in insufficient quantities. The most well-known forms of congenital adrenal hyperplasia are 21-hydroxylase enzyme deficiency and 11-beta-hydroxylase deficiency. 21-hydroxylase (CYP21A2) deficiency can cause decreased cortisol/aldosterone levels and increased androgen concentrations. Excess androgens lead to male-type changes in the external genitalia in newborn girls (so-called virilization). Boys with this genetic defect do not show characteristic clinical symptoms at birth, but are susceptible to early puberty due to excess androgens. People with this genetic defect may develop early facial and body hair, acne, and menstrual irregularities. Pathology can also lead to infertility. 21-hydroxylase enzyme deficiency occurs due to mutations in the CYP21A2 gene. Patients may have classic (more severe) and non-classical variants of the disease.

A classic deficiency may be:

1. Salt wasting - a severe form of the disease, which is caused by extremely low enzyme activity, which causes a deficiency of cortisol and aldosterone. Aldosterone deficiency leads to a pathological decrease in sodium (hyponatremia) and an increase in potassium (hyperkalemia) in the body. If left untreated, it can lead to acute adrenal insufficiency and shock.

2. Simple - enzyme activity is reduced, but remains sufficient to maintain aldosterone synthesis at a normal or slightly reduced level.

The non-classical variant of the pathology is the most common variant, which causes a less severe form of the disease. Occurs in approximately one in one and a half thousand newborns in the Caucasian population. With this variant of the development of pathology, the enzyme activity is approximately 50%, which avoids critical sodium deficiency and severe consequences. Androgen concentrations are slightly increased.

Treatment of VDKN according to the Federal clinical guidelines [Federal clinical guidelines - protocols for the management of patients with congenital dysfunction of the adrenal cortex in childhood, Problems of Endocrinology, 2014;60(2): 42 50] is carried out using conservative methods.

Thus, the drug of choice for the treatment of children with CAH is tableted hydrocortisone (1+++). The use of hydrocortisone syrup and long-acting forms of glucocorticoids in children during the period of active growth is not indicated (1++). Fludrocortisone is indicated for all children with the salt-wasting form of CADC; for infants, additional table salt is added to the diet (1++).

The main principle of treatment for all forms of CAH is the administration of glucocorticoids, which replace cortisol deficiency and thereby suppress excessive secretion of adrenocorticotropic hormone (ACTH). As a result, the production by the adrenal glands of those steroids that are synthesized in excess during a particular enzymatic block decreases.

There are various medications that have glucocorticoid activity: prednisolone, cortisone, dexamethasone. Long-acting synthetic glucorticoid drugs (prednisolone, dexamethasone) negatively affect growth. For children with open growth plates, especially young children, tablet analogues of hydrocortisone should be considered the most optimal drugs. The initial daily dose of hydrocortisone required to suppress ACTH secretion in children 1 year of age can reach 20 mg/ ^m2 . On average, the daily dose of hydrocortisone in children over 1 year of age should be 10-15 mg/ ^m2 . The drug is given 3 times a day in equal doses (at 7.00, 15.00 and 22.00).

In the salt-wasting form of VDCN, therapy with a mineralocorticoid drug, fludrocortisone, is required, which is prescribed at a dose of 0.05-0.15 mg per day (1-2 times a day). In infants, the need for mineralocorticoids is higher and can reach 0.3 mg per day; the dose can be divided into 3 doses. Children without clinical manifestations of salt-wasting syndrome may have a subclinical deficiency of mineralocorticoids, the criterion of which is an increased level of renin. In such cases, fludrocortisone therapy is also indicated.

An alternative to conservative treatment may be genetic therapy.

Adeno-associated virus (AAV) is promising for use in human gene therapy. AAV has the ability to effectively infect both dividing and non-dividing human cells, with the AAV genome integrating at a single chromosomal site into the host cell genome. However, the presence of AAV in the human body is not associated with any disease.

AVV vectors are known for genetic therapy, solving the problem of creating means and methods that make it possible to obtain a stable and high large-scale yield of AAV vectors in insect cells [RU2457252 C2, 07.27.2012]. Thus, the known vectors are universal, that is, suitable for use regardless of the specific disease. However, specific tasks often require specific solutions, so for each task you have to create your own optimal design.

An AAV vector containing gRNA and Cas9 is known from the prior art, where the Cas9 molecule represents active or inactive S. pyogenes Cas9. The vector is used for antitumor therapy, including for the adrenal glands [RU2019133280 A, 04/22/2021]. However, this solution is not intended for the treatment of congenital adrenal dysfunction. An AAV vector containing Cas9, NLS, HA is also known [KR1020150056539 A, 05/26/2015].

In addition, an AAV vector containing Cas9, SV40 NLS, HA is known [US20210054405 A, 02/25/2021] and an AAV vector, including serotype 2, containing guide RNA, ITR, intended for adrenal therapy [WO2020082047 A1, 04/23/2020] . However, none of these vectors solves the problem of treating congenital dysfunction of the adrenal cortex.

It is known to use vectors for targeting the CYP21A2 gene in the human genome, containing guide RNA and Streptococcus pyogenes Cas9. NLS, eukaryotic elongation factor EF1 alpha [Neville E. Sanjana, Ophir Shalem and Feng Zhang, Improved vectors and genome-wide libraries for CRISPR screening, Nat Methods. 2014 Aug; 11(8): 783-784]. This solution is the closest to the claimed solution, however, this solution concerns a lentiviral vector, which is not suitable for the treatment of congenital dysfunction of the adrenal cortex.

The technical problem solved by the claimed group of inventions is the creation of genetic constructs containing optimized sequences, including Cas9 S. pyogenes, ensuring a high level of transgene expression.

Disclosure of the invention

When implementing the claimed group of inventions, the following technical results are achieved:

1. fairly easy production;

2. degree of nonspecific cutting 0.01%;

3. wide tropism for different tissues;

4. high level of transgene expression: the number of viral genomes per cell is from 1 to 100, expression is from 10 ^-4 to 10 ^-1 relative to the control gene GAPDH.

Technical results are achieved due to the following.

One of the group's inventions is the pAAV-nEFCas9 expression vector.

This vector represents viral particles of adeno-associated viruses, capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hu14, rh10 and others) or serotypes obtained by synthetic biology methods (DJ/8, DJ, anc32 and other).

As a genome, a single-stranded DNA molecule is packaged into particles, at the ends of which ITRs (internal terminal repeats) of the AAV virus are located, in the preferred variant of serotype 2. These AAV ITRs are well studied, their use gives a stable expected effect. However, it is possible to use other AAV ITRs as there is no prior art indicating that ITRs from other AAV serotypes will produce incorrect results when used. Within the AAV ITRs, the promoter of the first eukaryotic elongation factor EF1 alpha is located sequentially. The selection of this particular promoter is the result of a series of experiments in which it was shown that it gives a high level of transgene expression in the vector construct of the present invention. Moreover, it is eukaryotic and not viral, therefore, it will not be methylated and inhibited, which is also an additional advantage of the present invention. In addition, the promoter is constitutive, i.e. it will work in all cell types. After the mentioned promoter there is a histidine tag sequence (HA (human influenza hemagglutinin) epitopetag), which serves for easy identification of the transgene by Western blot, cytometry and immunohistochemistry. The label is followed by an intracellular localization signal in the nucleus of the 5'-SV40 NLS. The advantage of this nuclear signal is its small size and, as has been shown, a high degree of protein transfer into the nucleus. Next comes the codon-optimized sequence of the Cas9 gene of Streptococcus pyogenes. The Cas9 effector is relatively small in size and is capable of introducing a double-strand break into the target locus in the genome. The CRISPR/Cas9-based genome editing system is easy to use and design, the degree of nonspecific cutting is 0.01% with the need to select a specific RNA guide that is easy to synthesize. This is the main task on the solution of which the achievement of the specified specificity depends. An optimal RNA guide has been developed that allows maximum use of all the advantages of the CRISPR/Cas9-based genome editing system. An incorrectly selected RNA guide will lead to a decrease in the effects of using such a system. For more efficient synthesis of bacterial protein in human cells, codon optimization was carried out (sequence SEQ ID NO: 5), which also made it possible to obtain the above effects. However, other options for codon optimization are possible, so the option used in this development is demonstrative and shows the fundamental possibility of achieving the effects of the invention by optimizing this sequence. Following the codon-optimized sequence is another intracellular localization signal in the nucleus of the 3'-SV40 NLS. A synthetic polyA signal was used as a transcription terminator.

Another object of the invention is the AAV-hCYP21A2 expression vector.

As a genome, a single-stranded DNA molecule is packaged into particles, at the ends of which there are ITRs (internal terminal repeats) of AAV, in the preferred variant of serotype 2. These ITRs of AAV are well studied, their use gives a stable expected effect. However, it is possible to use other AAV ITRs as there is no prior art indicating that ITRs from other AAV serotypes will produce incorrect results when used. Inside the vector, the eukaryotic promoter of polymerase III - U6 is located sequentially. This U6 promoter demonstrated a high level of transgene transcription in the vector construct of the invention. It is eukaryotic and not viral, which means it will not be inhibited in a human cell, and it is relatively small in size, which allows the guide RNA transcription cassette to be packaged into the AAV genome. After the U6 promoter there is a guide RNA sequence in crRNA format. This is a unique sequence of 20 nucleotides, complementary to the targeted locus, linked to the trackRNA sequence (scaffold). Following the guide RNA expression cassette is a region of homology with the human genome locus AAVS1 (left homologous arm). This locus was chosen because many experiments have shown that integration into it does not affect the functioning of the rest of the genome, which ensures safe integration. Moreover, the transgene integrated into this locus is efficiently transcribed in any cell type. Transgene expression cassette: EFS-NS promoter, it is this promoter that shows a high level of transgene expression, is eukaryotic, not viral, therefore, will not be methylated and inhibited, it is also a constitutive promoter, i.e. works in all types of cells, under the control of which the protein-coding codon-optimized sequence of the hCYP21A2 gene is located. At the same time, codon optimization made it possible to increase the efficiency of gene translation; optimization was carried out relative to the corresponding reference sequence NG_007941.3. The polyA signal of the human β-globin gene (human β-globin polyadenylation signal) was used as a transcription terminator. Following the polyA signal is a region of homology with the AAVS1 locus of the human genome (right homologous arm).

Brief description of drawings

The invention is illustrated by the following illustrative material.

Fig. 1 - diagram of the arrangement of layers of iodixanol solutions in a centrifuge beaker. 40% of the fraction is selected after centrifugation of the QuickSeal tubes, while first piercing the very top of the tube with a separate needle to allow air to enter, and the required fraction is selected with another needle and syringe, with the needle hole directed upward and the needle tip located horizontally approximately 2 mm below the 40- 60% interphase. In this way, 40% of the gradient was aspirated without touching the interphase of 25% of the layer.

Fig. 2 - AAV-hCYP21A2 map.

Fig. 3 - pAAV-nEFCas9 map.

Carrying out the invention

One of the group’s inventions is an expression vector based on adeno-associated viruses, capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hu14, rh10 and others) or serotypes obtained by synthetic biology methods (DJ/8, DJ, anc32 and others). As a genome, a single-stranded DNA molecule is packaged into particles, at the ends of which the ITRs (internal terminal repeats) of the serotype 2 virus are located, inside the promoter of the first eukaryotic elongation factor EF1 alpha, then the histidine tag sequence (HA (humanin fluenza hemagglutinin) epitopetag), after followed by the intracellular nuclear localization signal 5'-SV40 NLS, followed by the codon-optimized sequence of the Streptococcus pyogenes Cas9 gene. Then there is another intracellular localization signal in the nucleus of the 3'-SV40 NLS. A synthetic polyA signal was used as a transcription terminator. An example of the invention is SEQ ID NO:1.

Another invention of the group is an expression vector based on adeno-associated viruses, capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hu14, rh10 and others) or serotypes obtained by synthetic biology methods (DJ/8, DJ, anc32 and others).

As a genome, a single-stranded DNA molecule is packaged into particles, at the ends of which there are ITRs (internal terminal repeats) of the serotype 2 virus, inside the following are sequentially located: the eukaryotic polymerase III promoter - U6, the guide RNA sequence in crRNA format (a unique sequence of 20 nucleotides, complementary target locus, linked to the trackRNA sequence (scaffold)), behind the guide RNA expression cassette there is a region of homology with the human genome locus AAVS1 (left homologous arm) transgene expression cassette, including the EFS-NS promoter, under the control of which the protein-coding sequence of the hCYP21A2 gene is located , corresponding to the reference sequence NG_007941.3. The polyA signal of the human β-globin gene (human β-globin polyadenylation signal) was used as a transcription terminator. Following the polyA signal is a region of homology with the AAVS1 locus of the human genome (right homologous arm). An example implementation is SEQ ID NO: 2.

SEQ ID NO: 3 is the sequence of the wild type CYP21A2 gene NG_007941.3.

SEQ ID NO: 4 is a variant of the optimized sequence of SEQ ID NO: 3.

SEQ ID NO: 5 is the codon optimized sequence of the Cas9 gene of Streptococcus pyogenes.

Since genetic therapy for congenital adrenal cortex dysfunction is mediated by mutations of the hCYP21A2 gene, treatment can be carried out by exogenous expression of the hCYP21A2 gene from the genome of the vector that infected adrenal cells and is presented in the nuclei in the form of episomal DNA. In this case, a double-strand break is introduced into the genome of infected cells through the expression of a guide RNA specific to the sequence of the AAVS1 locus, adeno-associated virus integration site 1, PPP1R12C protein phosphatase 1 regulatory subunit 12C, NC_000019.10, as well as through the expression of the Cas9 protein , capable of introducing a double-strand break into DNA at the site of homologous binding of the guide RNA to the target. Integration occurs due to the presence of regions of homology to the AAVS1 locus in the AAV-hCYP21A2 vector. As a result of the processes of introducing a break, homologous repair and transgene expression, the hCYP21A2 gene is expressed and normal steroidogenesis is restored in the adrenal cortex.

Below is a more detailed description of the claimed invention. The present invention is subject to various changes and modifications that will become apparent to those skilled in the art based on reading this specification. Such changes do not limit the scope of the claims.

To obtain viral particles, we used plasmid constructs carrying the nucleotide sequences of the vector genomes, as well as the sequences of replication and selection of these plasmids in bacterial systems. The production of these plasmids was carried out using molecular cloning and gene synthesis methods.

Plasmids carrying the capsid genes of viral parts of AAV and helper genes were also used.

Viral vectors were assembled in an adherent or suspension cell/custom cell line system based on HEK293 cells.

EXAMPLE 1

Isolation and purification of plasmids

E.coli cells were thawed and stored at -80°C. Defrosting was carried out on a solid medium. To do this, frozen cells were added to 400 μl of liquid LB medium (at the tip of the tip), resuspended and transferred to solid LB medium containing carbenicillin at a concentration of 100 mg/l.

To obtain a suspension culture, 100 ml of LB medium was added to a 250 ml flask. 1 colony was added to the tip of the tip, E. coli was incubated for 16-20 hours at 37°C in a thermostatic shaker with constant stirring at 170 rpm.

Next, plasmid DNA was isolated using the Plasmid Midiprep 2.0 kit (Evrogen) according to the manufacturer’s protocol. The number of columns was taken at the rate of 1 column per 1 g of cell sediment (column capacity 500 μg of DNA). One column was eluted twice in 4 ml of water.

After isolation, the concentration of plasmids was measured and restriction analysis was performed using restriction enzymes according to Table 1.

For each sample, the following reactions were performed: restriction with each of the restriction enzymes indicated in Table 1 separately, for all restriction enzymes in one tube, K-reaction without restriction enzymes.

Prepared the mixture:

10XBuf - 2.5 µl;

restriction enzymes - 0.5 µl;

plasmid DNA - 600 ng;

water up to 25 µl.

Incubated for 1 hour at 37°C and visualized on a 1% agarose gel.

Table 1. Plasmid Restriction enzymes Fragment length Amount, mg р225 (15,419 bp) Helper NotI 6021 0.5-1 SalI 4930 PvuI 2727 1741 p226 (6,891 bp) GFP SalI 4893 0.5-1 SpeI 1998 p227 (7,700 bp) Cas9 AgeI 3433 0.5-1 NotI 2836 ApaI 1431

The number of viral genomes per cell is from 1 to 100.

Expression is from 10 ^-4 to 10 ^-1 relative to the control gene GAPDH.

EXAMPLE 2

Preparation of HEK293 cell culture

All volumes of media, solutions, etc. are given for one 15 cm culture dish.

Media for cell expansion (GM) and transfection (TM) were prepared. To prepare GM, 50 ml of FBS (Gibco), 5 ml x100 GlutaMax (Gibco) and 500 µl of antibiotic x 1000 Gentamicin (PanEco) were added to 450 ml of DMEM medium with a glucose content of 4.5 g/l. To prepare TM, 10 ml of FBS (Gibco), 5 ml x 100 GlutaMax (Gibco) and 500 µl of antibiotic x 1000 Gentamicin (PanEco) were added to 490 ml of DMEM medium with a glucose content of 4.5 g/l.

Solutions of Versene and Trypsin 0.25% were aliquoted into 50 ml. An aliquot of Versene solution was stored at room temperature, and aliquots of Trypsin solution 0.25% were stored at -20°C. Before starting work with cells, the trypsin solution was transferred to room temperature, and the GM/TM medium was transferred to a water bath at a temperature of +37°C.

The cell line was passaged. The passage of the cell line did not exceed 12.

HEK293 cell cultures were thawed. Before defrosting, the cryovials were heated with 25 ml of GM in a water bath at +37°C.

The cryovial was placed in a water bath at a temperature of +37°C until completely thawed. Immediately after thawing, the entire contents of the cryovial were transferred to a 15 ml tube, and 10 ml of heated GM was added. Centrifuged at 230 g for 3 minutes. The supernatant was discarded, the cell pellet was resuspended in 1 ml of GM, transferred to a T75 cell flask, and 14 ml of GM was added. The flask was incubated in a CO2 incubator, 5% CO2, +37°C until 70% cell confluence was achieved.

HEK293 cell culture was frozen.

Before freezing the cell bank, a test was carried out for the absence of mycoplasma in the cells according to the MycoReport protocol (Evrogen).

We took all the medium from the dish, added 5 ml of Versene solution, and incubated for 1 minute in a laminar flow hood. A Versene solution was taken, 3 ml of 0.25% Trypsin solution was added, and incubated for 3 minutes in a laminar flow hood. 3 ml of GM was added, the cells were carefully and thoroughly resuspended, and 10 μl of the cell suspension was collected into a 0.2 ml tube. The remaining cells were transferred to a 15 ml tube.

The number of cells was counted and their viability was determined using an automatic cell counter TC-20 (BioRad): 10 μl of a 0.4% trypan blue solution was added to 10 μl of a well-mixed cell suspension and mixed; 10 μl of the resulting suspension was applied to a slide for cell counting and measurements were taken.

Cells in a 15 ml tube were centrifuged at 230 g for 3 min.

We calculated the required number of cryovials so that one cryovial contained 5 million cells in 1 ml of freezing solution. Each cryovial was signed: line name, number of cells, date, operator.

A solution for freezing was prepared: one cryovial requires 700 μl of FBS (Gibco), 200 μl of Dmem with a glucose content of 4.5 g/l (PanEco), 100 μl of DMSO (PanEco).

The cell sediment from a 15 ml tube was resuspended in a freezing medium, centrifuged at 230 g for 3 min, and aliquoted in 1 ml portions into a cryovial. The cryovials were incubated for 1 day in a Kelvinator at -80°C, then transferred to liquid nitrogen at -196°C.

HEK293 cell culture was passaged.

We took all the medium and added 5 ml of Versene solution to the cup. They were incubated for 1 min in a laminar flow hood, and the Versene solution was removed.

Trypsin 0.25% 3 ml per dish was added, incubated for 3 minutes in a laminar flow hood, an equal volume of GM medium was added, the cells were carefully resuspended, and transferred to a 15 ml falcon.

The number of cells was counted and their viability was determined using an automatic cell counter TC-20 (BioRad): 10 μl of a 0.4% trypan blue solution was added to 10 μl of a well-mixed cell suspension and mixed; 10 μl of the resulting suspension was applied to a slide for cell counting and measurements were taken.

6 million cells were transferred to a new dish, 24 ml of GM was added, and incubated at +37°C, 5% CO2 until the next passage.

EXAMPLE 3

Transfection of HEK293 cell culture

The transfection mixture was composed of lactate buffer, linear polyethylene amine and plasmid DNA.

Preparation of lactate buffer pH 4 (20 mM sodium lactate, 150 mM sodium chloride)

To prepare 300 ml of lactate buffer, 1.35 ml of 40% lactic acid was added to 290 ml of 150 mM sodium chloride. Using 1N sodium hydroxide solution, adjusted to pH 4. The resulting solution was adjusted to 300 ml with 150 mM sodium chloride. Sterilized through a 0.2 µm filter and stored at 4°C.

Preparation of a solution of polyethylenimine (l-PEI 25) 5 mg/ml

A sample of 1 g of dry linear polyethylenimine (25 kDa) was dissolved in 190 ml of 0.2 N hydrochloric acid solution with prolonged stirring. The volume was increased with hydrochloric acid to 200 ml. The final solution had a pH of 1-2. Aliquots of the solution were stored at minus 80°C.

Calculation of the required amount of plasmids and polyethyleneamine (PEI) for transfection

To transfect 1 15 cm dish, 38 μg of plasmid DNA was used, with a helper:packaging:transfer plasmid copy number ratio of 1:1:1.

One day before transfection, cells were transferred to new dishes at a rate of 10 million cells per dish. 24 ml of GM medium was added and incubated at +37°C, 5% CO2.

Transfection of HEK293 cell culture. Mixtures of DNA and PEI were prepared according to Table 2.

table 2
Scheme for preparing the transfection mixture for 40 cups Tube 1 (plasmid DNA) Test tube 2 (polymer) 0.53 mg target plasmid
0.53 mg of plasmid carrying the equivalent gene
nucleocapsid
0.53 mg helper plasmid 0.6 ml l-PEI 25 up to 40 ml lactate buffer 39.4 ml lactate buffer Total volume 40 ml Total volume 40 ml

Add 80 ml of DNA-l-PEI 25 mixture to 1 liter of TM medium and mix. All GM medium was collected from the plates. Added 27 ml of TM-DNA-PEI mixture per dish. Stir gently. The dishes were incubated in an incubator +37°C, 5% CO2, 72 hours.

Transfection efficiency was 95-98%.

Cell lysis

A lysis buffer was prepared containing: 150 mM NaCl, 50 mM Tris-HCl, 1 mM MgCl2, pH 8.5, 0.5% Triton X-100, benzonase 50U/ml. Sterilized by filtration rather than autoclaving. Stored at +4°C.

We took all the medium from the dish into a 25 ml serological pipette, and used it to wash off all the cells from the dish. The cell suspension was transferred to a 50 ml tube, and the iteration was repeated with all dishes. The tube with the cell suspension was centrifuged at 400 g for 5 minutes, the supernatant was collected as needed and stored at -80°C. The entire cell pellet from 40 dishes was resuspended in 60 ml of lysis buffer. Incubated at +37°C for 1 hour.

Centrifuged at 4000 g for 15 minutes, the clarified supernatant was transferred to a clean tube.

EXAMPLE 4

Isolation and purification of AAV

Buffers were prepared: 1M NaCl/PBS-MK buffer and 1x PBS-MK buffer.

To prepare 1M NaCl/PBS-MK buffer, dissolve 5.84 g NaCl, 26.3 mg MgCl2 (56.0 mg 6 H2O X MgCl2) and 14.91 mg KCl in 1× PBS to a final volume of 100 ml. Filtered through 0.22 µm and stored at +4°C.

To prepare 1x PBS-MK buffer, dissolve 26.3 mg MgCl2 (56.0 mg 6 H2O X MgCl2) and 14.91 mg KCl in 1x PBS to a final volume of 100 ml. Filtered through 0.22 microns and stored at +4°C.

For each ultracentrifugation, only iodixanol solutions prepared in accordance with Table 3 were used.

Table 3
Calculation for preparing iodixanol solutions for 2 Quick-Seal tubes (39 ml) Iodixanol concentration OptiPrep, ml 1 M NaCl/PBS-MK buffer, ml 1x PBS-MK buffer, ml Phenol Red, ml 15% 4.5 13.5 - - 25% 5.5 - 7.7 0.04 40% 8 - 4 - 60% 10.2 - - 0.04

Ultracentrifugation

Layers of iodixanol solutions were added to the centrifuge beaker in the order according to Table 4 using a 10 ml syringe and a Zeldiger needle.

Table 4 Iodixanol concentration, ml 39 ml
QuickSeal 1. Cell lysate 16 2. 15% 8 3. 25% 6 4. 40% 5 5. 60% 5

The gradient was applied from the top layers so as not to disturb the integrity of the gradient.

When filling a QuickSeal tube, fill the tube to the top with cell lysate, with no bubbles on top. The tubes were equilibrated with PBS to 0.0005 g on an analytical balance. Centrifuged at 360,000 g (59,000 rpm for a Ti 70 Beckman Coulter rotor) for 1 hour 30 minutes, +10°C.

After centrifuging the QuickSeal tubes, a separate needle was first pierced at the very top of the tube to allow air to enter, then the 40% fraction was taken with another needle and syringe, as shown in FIG. 1. The needle hole faces upward, the needle tip is located horizontally approximately 2 mm below 40-60% of the interphase. 40% of the gradient was aspirated, leaving the interphase of the 25% layer intact.

The selected fraction was transferred to a 50 ml falcon.

Ultrafiltration

The fraction of 40% iodixanol selected after ultracentrifugation was diluted 5 times with a solution of PBS/Pluronic F68 (0.001%) and filtered through a filter with a pore size of 0.22 μm. 3 ml of H2O was added to the Amicon Ultra-15 filter, centrifuged at 2000 g for 1 min, then all liquid was completely removed from the test tube and from the filter. 3 ml of PBS/Pluronic F68 solution (0.001%) was added to the filter, centrifuged at 2000 g for 1 min, and all liquid was completely removed from the test tube and filter. Add 15 ml of diluted virus to the Amicon Ultra-15 filter and centrifuge at 2500 g for 10 minutes in a swing-out rotor. Discard the liquid that passed through the column and fill the Amicon Ultra-15 tube until all the vector solution was used. Next, the virus solution was used to wash the membrane well with a 100-1000 µl pipette. Centrifuged at 2500 g for 10 minutes.

After centrifugation, the vector suspension remained in the upper part of the concentrator (volume 500 μl); centrifugation was repeated for 3 minutes.

After passing through the entire solution of viral particles, the concentrated virus was washed with PBS/Pluronic F68 (0.001%) at room temperature 2 times. There should be about 500 µl of viral suspension left in the filter cassette.

The membranes were washed well, after which the virus was aliquoted in 20 μl increments into 0.2 ml tubes, leaving 5 μl for titer determination.

AAV-CYP21 - 7.80E+12 viral particles per 1 ml. AAV-Cas9 - 1.93E+12 viral particles per 1 ml.

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LIST OF SEQUENCES

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<INSDSeq_sequence>cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtc

gggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcac

taggggttcctgcggcctctagaaaggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacat

cgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcgg

ggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatata

agtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaa

tgtcgccgaagaaaaagcgcaaggtcgaagcgtccgacaaaaaatactccatcggtctggacattggtac

taactctgtaggctgggccgttatcacggacgagtacaaagtacctagcaaaaagttcaaagtattgggg

aatactgacaggcattccataaagaagaatctcataggagcattgctgtttgattccggagagacagcag

aagctactcgactcaagagaaccgcacgacgccgatataacgccgaaaaaaccgaatctgctacctgca

ggagatcttctccaatgaaatggctaaggtagacgacagcttctttcatcgacttgaagaatcttttctt

gttgaggaggataagaagcacgaacggcaccctattttcggcaatatcgtggatgaggtcgcgtatcatg

aaaagtatcccaccatctaccacttgcgaaagaagctggtagatagtaccgacaaggcggatttgagact

catataccttgcactggcccacatgataaaattcagagggcacttcctcatcgaaggtgatcttaatcca

gacaacagtgacgtcgataagttgttcattcagctcgtgcagacttacaatcaactgtttgaagaaaatc

ccataaatgcgtcaggcgtagatgcaaaggctatactctccgcgcgattgtcaaaatcccgacggcttga

gaatctcatagcccaacttcccggcgaaaagaaaaacggattgttcgggaaccttattgccttgtcactc

ggtttgactcctaattttaagagtaatttcgatctggctgaggatgcaaaactgcagctgagtaaagata

catacgacgacgacctggacaatctcctggcccagattggagaccagtacgccgacctgttcttggctgc

caaaaacctctcagacgctattctcctgtcagacattttgcgcgtgaatactgagattacgaaggccccc

ctgtctgcctccatgattaagcgctatgatgagcaccatcaagacttgacccttcttaaagcgctggtac

ggcagcagctccctgaaaaatataaagaaatcttcttcgaccagtccaaaaatggctacgcgggctacat

cgacggaggtgcaagccaggaggagttctataagtttataaagccgatccttgagaagatggatgggacc

gaagaactcctggtgaaacttaacagggaggatctgctgcgaaagcagcgcacatttgataatgggtcca

ttccacatcaaattcatcttggagaattgcacgccattttgagaagacaggaggacttctatccgtttct

taaggacaatcgggaaaaaatagaaaagatacttacattccgaattccttactatgtcgggcccttggcg

cgagggaactctcggttcgcttggatgactcgaaaaagtgaggagactataactccttggaattttgaag

aagtcgtagacaaaggtgcttccgcgcaatcttttatagagagaatgactaatttcgacaaaaacctccc

aaacgagaaggttctgccaaagcacagccttctctacgagtattttactgtctataacgaactcactaag

gtgaaatacgtaactgaagggatgaggaagcctgcctttttgagtggcgagcagaagaaagcgatagtag

atttgctttttaaaaccaaccggaaggtcactgtgaaacaacttaaagaagactacttcaagaagataga

gtgtttcgacagcgtagaaatatctggcgtagaggaccgcttcaatgcaagcttgggcacttaccatgac

ctcctcaagataatcaaagataaagatttcttgataatgaagagaatgaggatatccttgaagacatag

tgttgactctcacgttgtttgaggatagggagatgatagaagagcgcttgaagacctatgctcatctctt

cgatgataaagtaatgaagcaactcaagcggagaaggtatactggttggggtcggctttctaggaaactc

atcaacggtattcgggataaacagtccggcaaaactatattggatttccttaagagtgatggcttcgcga

accgaaactttatgcaacttatacatgatgattcacttacatttaaggaagacattcaaaaagcgcaagt

atctggccagggcgactctctgcacgaacacatagcaaaccttgctggatcccctgcgataaagaaaggt

atactgcagacagtaaaagttgtggacgaactcgtcaaggttatgggtaggcataagcccgaaaatatag

tgattgaaatggcacgcgagaaccaaactacacaaaaaggtcaaaagaacagcagagaacggatgaaacg

aatagaggaggggatcaaggaacttggatcccaaatactgaaagagcaccccgtagaaaatacccagctt

caaaacgagaagctttatctgtactacctgcaaaacggcagagatatgtacgtagaccaggaactggaca

tcaacagattgtctgattatgatgtagatcacattgtgccgcagagcttcctcaaagatgattccataga

taacaaagtactcactcgaagtgacaaaaacagagggaaatccgataatgttcccagtgaggaagtcgtc

aagaagatgaagaattattggcggcaacttctcaacgcgaaactcatcacccaaagaaaattcgataacc

tcactaaagctgaacgcggaggactgtcagagcttgataaagcaggttttatcaaaaggcaactcgtaga

aacgagacaaataacaaaacatgtcgctcaaattttggactctcgcatgaatacaaagtatgatgaaaat

gacaagctgattcgcgaggtcaaagtaataacgctcaaaagtaaacttgtctccgatttccgaaaggact

ttcagttttacaaagtcagagagataaataattaccaccacgcccatgacgcctacctcaatgctgtggt

tggtacggctcttatcaagaaatatccgaagctcgagagcgagtttgtctatggcgactacaaggtttac

gatgtcaggaaaatgatagcgaagtctgagcaagagataggtaaggccacggctaaatactttttctatt

caaatatcatgaattttttcaaaaccgagataacgttggcgaatggcgaaatcaggaaaaggcctcttat

agagactaacggagaaacaggcgagattgtctgggataagggtagggatttcgctactgtgcgaaaggta

ctgagcatgccccaggttaacattgtcaaaaagactgaggtccaaacgggtggcttttctaaggagagta

tcctgccaaaacgcaattcagataagctcatagcgagaaaaaaggattgggacccaaagaaatatggcgg

cttcgactctccgacggtcgcatatagtgtgctggtggttgccaaagttgaaaagggcaagtctaaaaaa

ctgaaatcagtgaaggagcttctcggaataactattatggagcgcagctcattttgaaaagaatccaatcg

atttcttggaagcaaaaggatataaagaagtcaagaaggacctgatcatcaaacttccaaagtactcact

gttcgaacttgagaacggacggaagcggatgcttgcgagtgctggagaacttcaaaaaggtaacgagctc

gcccttccatcaaagtacgtaaattttttgtacttggcatcacactatgagaagctcaaaggctcccccg

aggataatgagcaaaagcagcttttcgtggaacaacataaacattaccttgatgaaatcatcgaacaaat

aagcgaattctctaaacgagttattcttgcagacgcgaatctggacaaggtgcttagcgcatacaacaag

catcgggataagccgattcgagaacaagccgaaaatataatccatcttttcactctgacgaacttgggtg

cgccggcagccttcaagtacttcgataccactattgataggaaacggtatacgagtacaaaagaagtgct

ggacgctacgcttatccatcagtccatcactgggctctacgagacgaggattgaccttagtcagttgggc

ggggacagccccaagaagaagaaaggtggaggccagctaagaattcaataaaagatctttattttcat

tagatctgtgtgttggttttttgtgtgcggccgcaggaacccctagtgatggagttggccactccctctc

tgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggc

ctcagtgagcgagcgagcgcgcagctgcctgcagg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>3119</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..3119</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q5">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtc

gggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcac

taggggttcctgcggcctctagagagggcctatttcccatgattccttcatatttgcatatacgatacaa

ggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgt

agaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgctta

ccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgggggcca

ctagggacaggatgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaa

agtggcaccgagtcggtgcttttttgaattccaattggacaccccgttctcctgtggattcgggtcacct

ctcactcctttcatttggggcagctccctaccccccttacctctctagtctgtgctagctcttccagccc

cctgtcatggcatcttccaggggtccgagagctcagctagtcttcttcctccaacccgggcccctatgtc

cacttcaggacagcatgtttgctgcctccagggatcctgtgtccccgagctgggaccaccttatattccc

agggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtggggccacta

gggacaggattaagcttgctaggtcttgaaaggagtgggaattggctccggtgcccgtcagtgggcagag

cgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagagaaggt

ggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgaggtgggggagaacc

gtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggaccgg

tgtctcgccatgctgctcctgggcctgctgctgctgctgcccctgctggctggcgcccgcctgctgtgga

actggtggaagctccggagcctccacctcccgcctcttgccccgggcttcttgcacctgctgcagcccga

cctccccatctatctgcttggcctgactcagaaattcgggcccatctacaggctccaccttgggctgcaa

gatgtggtggtgctgaactccaagaggaccattgaggaagccatggtcaaaaagtgggcagactttgctg

gcagacctgagccacttacctacaagctggtgtctaggaactacccggacctgtccttgggagactactc

cctgctctggaaagcccacaagaagctcacccgctcagccctgctgctgggcatccgtgactccatggag

ccagtggtggagcagctgacccaggagttctgtgagcgcatgagagcccagcccggcacccctgtggcca

ttgaggaggaattctctctcctcacctgcagcatcatctgttacctcaccttcggagacaagatcaagga

cgacaacttaatgcctgcctattacaaatgtatccaggaggtgttaaaaacctggagccactggtccatc

caaattgtggacgtgattccctttctcaggttcttccccaatccaggtctccggaggctgaagcaggcca

tagagaagagggatcacatcgtggagatgcagctgaggcagcacaagggagagcctcgtggcaggccagtg

gagggacatgatggactacatgctccaaggggtggcgcagccgagcatggaagagggctctggacagctc

ctggaagggcacgtgcacatggctgcagtggacctcctgatcggtggcactgagaccacagcaaacaccc

tctcctgggccgtggttttttgcttcaccaccctgagattcagcagcgactgcaggaggagctagacca

cgaactgggccctggtgcctccagctcccggggtcccctacaaggaccgtgcacggctgcccttgctcaat

gccaccatcgccgaggtgctgcgcctgcggcccgttgtgcccttagccttgccccaccgcaccacacggc

ccagcagcatctccggctacgacatccctgagggcacagtcatcattccgaacctccaaggcgcccacct

ggatgagacggtctgggagaggccacatgagttctggcctgatcgcttcctggagccaggcaagaactcc

agagctctggccttcggctgcggtgcccgcgtgtgcctgggcgagccgctggcgcgcctggagctcttcg

tggtgctgacccgactgctgcaggccttcacgctgctgccctccggggacgccctgccctccctgcagcc

cctgccccactgcagtgtcatcctcaagatgcagcctttccaagtgcggctgcagccccgggggatgggg

gccccacagcccggggccagagccagtaaaataaaagatctttattttcattagatctgtgtgttggttttt

tgtgtgctcgagtgacagaaaagccccatccttaggcctcctccttcctagtctcctgatattgggtcta

acccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctcc

ttgccagaacctctaaggtttgcttacgatggagccagagaggatcctgggaggagagcttggcagggg

gtgggagggaagggggggatgcgtgacctgcccggttctcagtggccaccctgcgctaccctctcccaga

acctgagctgctctgacgcggccgtctggtgcgtttcactgatcctggtgcgcggccgcaggaaccccta

gtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc

gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>10338</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..10338</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>genomic DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q7">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Homo sapiens</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggagaacttacaggtcctgccacaatcctaatgcaaggatggagctgca

agttcagtttgggaatcatcagcctggattggtttggtggaagccagggagtggttgagacccccacagg

ggagctctgaggaaggaagttccgaaggagggaacgtaagaaatgaccaggtcagaaccaagggtggtcc

agaagctaacccttagcttagggacagtttcacagagaacacgtccatgatgcaagactctgctgagggc

ctggagcagtgaagactggggcaaggtcaccctctgggaagtgaagtcaccagagaccttgcggagcagc

tttgagagttctctgagtaggaaggtaacagaatgtgaaggacactggagagaaggccaataggaagcaa

acaaaaacaggccaaggaaacccagtacagggggctgcagggcccagggagtgggtccctcatctctcct

ccccacgcttggccaggtccccacctcccgggagtgcgtgggctttgaggctgtgcaggaagtgccggtg

gggctggtgcagccggccagcgcaaccctgtacgactactacaaccccggtgagcactgcaggacaccct

gaaattcaggagaactttggcataggtgccctcctatgggacaatggacaccggggtagtgaggggggcag

agagccctggggctccctgggactgaggaggcagaatggaggggcctgtgccctaactcctctctgttct

ccagagcgcagatgttctgtgttttacggggcaccaagtaagagcagactcttggccaccttgtgttctg

ctgaagtctgccagtgtgctgagggtgagactgagggcctggggcggggcagtggaggcgggatggccgg

ggccccccccacactgtctgatgggttccccaacttcagggaagtgccctcgccagcgtcgcgccctgga

gcggggtctgcaggacgaggatggctacaggatgaagtttgcctgctactacccccgtgtggagtacggt

cagtcttcccaccgaggccctggcctgaccctccctcggggaccggccgttttggtctctctgggtgtag

cctgctcctcttacaggtcatgcacgcagcctgtttgctctgacaccaacttcctaccctctcagcctca

aagtaactcaccttcccccttctcctcaccccctcttaggcttccaggttaaggttctccgagaagaca

gcagagctgctttccgcctctttgagaccaagatcacccaagtcctgcacttcagtatgaagcaaaccgg

agaggcgggcagggctggggggagacagggaggctgaggtgtggccgaggacctgaccatctggaagtgt

gaaaatccccttgggctgtcagaagccttgggcttggccataaatagggaggcagtggcacctctccatg

ggggtggcgaaggtggaatgagaggatctacacagagtccccagcctgggctcaccctgcaccttctctt

cccctctgaccacttttgcgcacgtcatccccgcagccaaggatgtcaaggccgctgctaatcagatgcg

caacttcctggttcgagcctcctgccgccttcgcttggaacctgggaaagaatatttgatcatgggtctg

gatggggccacctatgacctcgagggacagtgagtcatctggtcccctcagtctcttgtcctccccatgc

ctcgccacctaggccttgcccctcagaagccagatgcctgtgctctccgtttccacctgccatcctcccg

agccctgctgactgcccctttgccccctgcagcccccagtacctgctggactcgaatagctggatcgagg

agatgccctctgaacgcctgtgccggagcacccgccagcgggcagcctgtgcccagctcaacgacttcct

ccaggagtatggcactcaggggtgccaggtgtgagggctgccctcccacctccgctgggaggaacctgaa

cctgggaaccatgaagctggaagcactgctgtgtccgctttcatgaacacagcctgggaccagggcatat

taaaggcttttggcagcaaagtgtcagtgttggcagtgaagtgtcagtgtgtgttgctagggctgagagc

agtgcccctgcccgatgcagttctgggcaggccaggttgacataaccttagactctctgagccctgatga

cccttgggctgttcagctctgctagaacctcccagatgacccgctaggagtctagtgcttcacaggacca

ccccgagcagaactgggacccaagagcctgcaccccaaggaccagagtccatgccaagaccacccttcag

cttccaaggccctccactgcccggctgtcgccagtcaccacggcctcagacagggcttgtgctcagctga

cacctgtgacacagctcttctgcctcatgagctgttgtccagctacacctccccgactctgtcctcgtgc

tgctggcggttctgaggtctgcagattttagctgagttccgggctgttgaaagcctgctgacgcttggtt

ctgttatcagtggaatgaggtgactttcccggagttgtgcaatcctcaggtccggcagtgtcttcttcca

gttactggtttcaaacaagccaaaagtctgactttggtgtgtttgtgaatcctctgaggaagccgctgtt

ctcctggggtctccccttcccaccggacctgcctaactttcccccatttagtggcacacctggggtcttc

agagatgactccgcgtctgtccaaagaagtttggtgagatcagtttccgtagaggtcatgacagttcagc

agcctgccatccagtcattcgacagaaattcgggaatctttcacttcatgccatgccctgtgccaggtgc

cagagatacagctgctcactccagggctcatcgctggggagacagataagaggacgggcagtccccaccc

tctgtgaaagatgtgatgtcagggagcagtgtggtcctgtggggcatctaaccaagtcaggggcattgcc

aggcagggacagggaaggcttcctggagcaggtggcctccaagtggggctctgaagactgagaaggagcc

aggaaaagagcaggggtagatgagggcatctggggcagaaggagaatatacaaaggcccagaggccgggg

gcaggacagggtacctttggggacattgcatgtaattgaccacattcggagtttggatttggaagtggtg

gaagagatggagatggtgagacaagtagtaagcacgtcagccttccaggtgcgctcctttccgatgagca

ctgtcttatcccacgtaactttgagaagtttgggcctttcccactgtggcagaggtttcctgaggctctt

gcatacatggccctatggttgctcatcagatctttctcccagtagctgctcagcatggtggtggcataag

cccattttccggagccagggattcagttgcagcaagacatggcccggtctgggaggtcaaccatgaagaa

ggcagtagctgtcattgcccaaccccagaaatcccaatcctgttttctccctctcagtcctgatcatgga

ttcagcagcagcgaactcgccaatgtagtgggtggcacagccagggtcttgactctggctctgcagtagc

acagtctggaaaagctctgaggggagagagacccccactggtccgagggtctggcacagagccagaaatg

gggggggaaggtatggggctgggtcgcctctgacctctcaggtaccatccaggaggccctggcctctcact

gaacccggccactcctctttggcatggcctcttcccaaatccccaaactgcctccttacccacaaaagtg

gtctctgagtgtcagtccagtgggacccccaccccttatggcttcagttccccaaatagggctggaccct

tgatcctgatccagctgtggctatccagccccttcctggggactttggactttgaggggggcatgcccag

ttgtgctgggaatccatactttccctggctggagtagaacctgtggactgtagtcctgagggcagtcatg

ttctgcctgtgcctggaaacacaagaaacttgactgcagagagaagaaagaggagagaggaacagagcga

ggaaaccgcccgtctccggggctttttctgttccctatccttgactttctaagaccagtggggtcccctc

ctctgcttctttttcctgagttctgtgaaattccccaattcttattttttatctcaaaccagctcaaggt

gggctgttttcctttcaaccaaagaaaggtgctcctggtggctaaaggtacatattcgacagctagattt

ccaggctggaatcctgccctccacaacatgcgaacaatacccgtgttgcatatagagcatggctgtgaag

agttgagtgagtgcccacaaagcacttagagcagtgtctggtacatgctattactccgcagcgggaaacc

acttcctcctttgtcttctgggcacttttgtgagtgaaaggaggcactaataacaatcacactgggatac

ctgtatatactggaatgccccaggcaaaccaggcttaaactgtattactctatctgtagcttaaactaac

aaacaacccacacaaatcacattttgttcttcaggcgattcaggaaggcctattaggcagggactgccat

tttctctctgagacaaacatcatgccagtaaactggcccacggtggggtggcagaggggagagggcccagg

tggggggcggacactattgcctgcacagttgatgtggaaccagaaagctgactctggatgcaggaaaaagg

tcagggttgcatttcccttccttgcttcttgatgggtgatcaatttttttgaaatacggacgtcccaagg

ccaatgagactggtgtcattccagaaaagggccactctgtgggcgggtcggtgggaggtacctgaaggt

ggggtcaagggaggccccaaaacagtctacacagcaggagggatggctggggctcttgagctataagtgg

cacctcagggccctgacgggcgtctcgccatgctgctcctgggcctgctgctgctgctgcccctgctggc

tggcgcccgcctgctgtggaactggtggaagctccggagcctccacctcccgcctcttgccccgggcttc

ttgcacctgctgcagcccgacctccccatctatctgcttggcctgactcagaaattcgggcccatctaca

ggctccaccttgggctgcaaggtgagaggctgatctcgctctggccctcaccataggagggggcggaggt

gacggagagggtcctctctccgctgacgctgctttggctgtctcccagatgtggtggtgctgaactccaa

gaggaccattgaggaagccatggtcaaaaagtgggcagactttgctggcagacctgagccacttacctgt

aagggccgggggcattttttctttcttaaaaaaatttttttttaagagatgggttcttgctatgctgccc

aggctggtcttaaattcctagtctcaaatgatcctcccacctcagcctcaagtgtgagccacctttgggg

catccccaatccaggtccctggaagctcttgggggggcatatctggtggggagaaagcaggggttgggga

ggccgaagaaggtcaggccctcagctgccttcatcagttcccaccctccagcccccacctcctcctgcag

acaagctggtgtctaggaactacccggacctgtccttgggagactactccctgctctggaaagcccacaa

gaagctcacccgctcagccctgctgctgggcatccgtgactccatggagccagtggtggagcagctgacc

caggagttctgtgaggtaaggctgggctcctgaggccacctcgggtcagcctcgcctctcacagtagccc

ccgccctgcccgctgcacagcggcctgctgaactcacactgtttctccacagcgcatgagagcccagccc

ggcacccctgtggccattgaggaggaattctctctcctcacctgcagcatcatctgttacctcaccttcg

gagacaagatcaaggtgcctcacagcccctcaggcccacccccagcccctccctgagcctctccttgtcc

tgaactgaaagtactccctccttttctggcaggacgacaacttaatgcctgcctattacaaatgtatcca

ggaggtgttaaaaacctggagccactggtccatccaaattgtggacgtgattccctttctcagggtgagg

acctggagcctagacacccctgggttgtaggggagaggctggggtggagggagaggctccttcccacagc

tgcattctcatgcttcctgccgcagttcttccccaatccaggtctccggaggctgaagcaggccatagag

aagagggatcacatcgtggagatgcagctgaggcagcacaaggtggggactgtacgtggacggcctcccc

tcggcccacagccagtgatgctaccggcctcagcattgctatgaggcgggttcttttgcataccccagtt

atgggcctgttgccactctgtactcctctccccaggccagccgctcagcccgctcctttcaccctctgca

ggagagcctcgtggcaggccagtggagggacatgatggactacatgctccaaggggtggcgcagccgagc

atggaagagggctctggacagctcctggaagggcacgtgcacatggctgcagtggacctcctgatcggtg

gcactgagaccacagcaaacaccctctcctgggccgtggtttttttgcttcaccaccctgaggtgcgtcc

tggggacaagcaaaaggctccttcccagcaacctggccagggcggtgggcaccctcactcagctctgagc

actgtgcggctggggctgtgcttgcctcaccggcactcaggctcactgggttgctgagggagcggctgga

ggctgggcagctgtgggctgctggggcaggactccacccgatcattccccagattcagcagcgactgcag

gagggagctagaccacgaactgggccctggtgcctccagctcccgggtcccctacaaggaccgtgcacggc

tgcccttgctcaatgccaccatcgccgaggtgctgcgcctgcggcccgttgtgcccttagccttgcccca

ccgcaccacacggcccagcaggtgactcccgagggttggggatgagtgaggaaagcccgagcccagggag

gtcctggccagcctctaactccagcccccttcagcatctccggctacgacatccctgagggcacagtcat

cattccgaacctccaaggcgcccacctggatgagacggtctgggagaggccacatgagttctggcctggt

atgtggggggccggggggcctgccgtgaaaatgtggtggaggctggtccccgctgccgctgaacgcctccc

cacccacctgtccacccgcccgcagatcgcttcctggagccaggcaagaactccagagctctggccttcg

gctgcggtgcccgcgtgtgcctgggcgagccgctggcgcgcctggagctcttcgtggtgctgacccgact

gctgcaggccttcacgctgctgccctccggggacgccctgccctccctgcagcccctgccccactgcagt

gtcatcctcaagatgcagcctttccaagtgcggctgcagccccgggggatgggggcccacagcccgggcc

agagccagtgatggggcaggaccgatgccagccgggtacctcagtttctcctttattgctcccgtacgaa

cccctcccctcccccctgtaaacacagtgctgcgagatcgctggcagagaaggcttcctccagcggctgg

gtggtgaaggaccctggctcttctctcggggcgacccctcagtgctcggcagtcatactggggtgcgaga

gaggtgggcagcagctcagcctccccccgctggggagcgaaagtttcttggtctcagcttcatttccgtg

aagggcaccgagaactcgaagcccttccagtggtaccagctcactccctgggaaaggggttgtcaagaga

gagtcaaagccggatgtcccatctgctcttcccgttcccttaaggaggtagctcccagcactcaaccaa

cctccccgcagagctcccttcctgaccctccgctgcagaggattgaggcttaattctgagctggcccttt

ccagccaataaatcaactccagctccctctgcgaggctggcatgattgttccatttcacccagccactca

gtcccttgcctgttacactgtggggctgaaacctaggcaggccgagccccagccaccccagctctgagcc

gcctccccacccctcacctgatggtccactgtgctcccgtagagcccgttgaggttggcgtagtggcagt

tcctgtaccaccaggcccctcggtaggagacagcgcaggagatgagcaagctgttggggtcccgatcacg

ggcagagaagacactgccgctgtggtagctcatggagtcccctgggcagggtggaggaaggagccatgag

ggcctcccctcccagcctcaccctcccagcctcacagcctctgcttacctgcggtgccgtggtagccctc

caagtggaggcggtagtactccgcagccgagtctacgtggaaggagtcgtactgggcgaacacagcctcg

tccccagcccgcaggtccacgcgcatggagtagtcacctgcctgtgtcaggctgtgcagggcctcattgc

ctggggggtgggatacgtgccctcatcagggtcctggtgtccacagggcccccatccccatccgtagttcc

ccagtccctgtgaggcactgacccagccagaactctccagagatgttcccaaaaccatgggcatagtcct

cccagtccctccagaagtctgtctgtccatccatgcggcgctggaacacctgggaagcaagtgggggcac

catcagcctctggctcccggggcaacagaccctgccctgcacagacccctgggcttcccaatgccaccca

ccagccagccgcccccatcagtctccatgtcgcaaaacacgttcaggggccgctcgcggttgccgttgag

gaagatggtgctggtcctggaggcaccggctccgttctgcatctcctccccgcagtccctggggaagggg

atccgcagcccacctgggagaggagagcaggggccagtccttttccaagccttaggccctggctgcccac

ccagcccccggccccgggcccgtgcgtccaggtacccgtggtgaaagaggtggacacgggcggcaggagg

ctctggccccacatggcctggagccgtgcattgtaggaggtggagggaaagaggccaaggagctggtgag

atgtgatccctcctgggagcaggatctcctgtgggacagacaagggggggtcaggggagagggaggtgga

gaccctccgggaggggccagaggcagcacctcctggaatcacccagggaggggagttgggtcagtggggcc

ggggcacctggttctgtccaccaggggtgtggaagctgagcaggtagcctgcgggccggactgggggctc

agtccaagtgagcagggcggtgcggggggtcacttccttggcctccaagtcccgaggggcctctagccct

aggaggaaagcaggaagaggagatggggatgaggcccaacctggctccctctacctcctctccctgtcc

cacacaccccacagaccctacctgtggtgaaggtgatgctggctggggaagtgaggttggggccccgcag

gccacgcactgtggcggtgtagttggtgtggaggacaaggtcatgcagggggtagtccaccgcgctgcct

ggggtctccgcctgcagaggcggggctgggagtgtagagaggggcatcaaggcctgccccctccatcctc

ggccagagtccagcctcccccctgcaatccccaccctgaacaagtcccctccagaggcctcaggcctgct

cacccccaggggctgtgacctggacgtcataggtgtccacaggattctgggggggcttccagtgcagcac

ggcgaatccctcggtcaagttcagtgcacgcaactgtgtgggaccgtcaggaactgggggaaggggaggg

gctcagaagggtccccgcggctctctctactccgtgcctccccagactccactggcctcccgtccgcaa<

/INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>1485</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1485</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q9">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgctcttgctcggtctgttgttgttgctcccattgcttgcaggagccc

gcctgttgtggaactggtggaagctccggagcctgcatctcccccccttggcccctggctttcttcatct

tttgcagcccgacctgcctatatctgctgggtctcactcagaagtttggacctatctaccgcctccac

ctggggctgcaggatgtggttgtgctcaattcaaaacgcacaattgaggaggcgatggttaagaaatggg

ccgactttgccggaaggcctgaaccactgacatataagctcgttagccgcaattacccagacctttccct

gggcgattactccctgctctggaaagcccataagaagctcacacgatcagcactgctgctgggaattcgc

gacagcatggagcctgtcgtagaacagctgactcaggaattctgtgagcgcatgcgggcccaacctggca

cacctgttgccatcgaggaggagttttctttgcttacatgctccattatctgctacctcacattcggcga

caagattaaagacgacaatctgatgcccgcctactacaagtgcatccaagaggtgctgaagacatggagt

cactggagcatccaaatcgtcgatgtgatcccatttctgagattcttccccaaccccgggcttaggagac

ttaagcaggctatcgaaaagagggatcacatagtcgaaatgcaacttagacagcacaaagagtctctggt

ggccggccagtggcgcgacatgatggactatatgctgcagggcgtggctcagccatccatggaagaaggc

agcggacaactccttgaggggcatgttcatatggccgccgttgacttgctgattggcggaaccgaaacaa

ctgctaatacactgtcctgggctgtggtattcctgctgcatcaccctgagatacagcaacggctccaaga

ggagctcgatcatgagctggggcccggtgcctcttcaagccgggtcccttacaaggaccgggcccgcctt

cctctgttgaatgcaaccatagctgaagtgctcagattgcgcccggtcgtaccactggccctccctcata

gaaccaccaggcccagcagtatctccggatatgatatcccagaggggactgtgattatcccaaacctgca

gggcgcccatctggacgagactgtgtgggagcggccacatgaattctggcctgacagattcctcgagcct

gggaagaactctcgggctctggcctttggttgcggggcaagggtatgcctgggggaacccttggcccggc

tggagcttttcgtagtgctgacacggctcctccaggcctttacgctgctgccaagcggcgacgctctgcc

tagcctgcaaccactgcctcattgctcagtcattctcaaaatgcagccttttcaagtgaggctgcagcca

cgaggtatgggcgcacattctcctggccagagccag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>4101</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..4101</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q11">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gacaaaaaatactccatcggtctggacattggtactaactctgtaggctgg

gccgttatcacggacgagtacaaagtacctagcaaaaagttcaaagtattggggaatactgacaggcatt

ccataaagaagaatctcataggagcattgctgtttgattccggagagacagcagaagctactcgactcaa

gagaaccgcacgacgccgatataacgccgaaaaaaccgaatctgctacctgcaggagatcttctccaat

gaaatggctaaggtagacgacagcttctttcatcgacttgaagaatcttttcttgttgaggaggataaga

agcacgaacggcaccctattttcggcaatatcgtggatgaggtcgcgtatcatgaaaagtatcccaccat

ctaccacttgcgaaagaagctggtagatagtaccgacaaggcggatttgagactcatataccttgcactg

gcccacatgataaaattcagagggcacttcctcatcgaaggtgatcttaatccagacaacagtgacgtcg

ataagttgttcattcagctcgtgcagacttacaatcaactgtttgaagaaaatcccataaatgcgtcagg

cgtagatgcaaaggctatactctccgcgcgattgtcaaaatcccgacggcttgagaatctcatagcccaa

cttcccggcgaaaagaaaaacggattgttcgggaaccttattgccttgtcactcggtttgactcctaatt

ttaagagtaatttcgatctggctgaggatgcaaaactgcagctgagtaaagatacatacgacgacgacct

ggacaatctcctggcccagattggagaccagtacgccgacctgttcttggctgccaaaaacctctcagac

gctattctcctgtcagacattttgcgcgtgaatactgagattacgaaggcccccctgtctgcctccatga

ttaagcgctatgatgagcaccatcaagacttgacccttcttaaagcgctggtacggcagcagctccctga

aaaatataaagaaatcttcttcgaccagtccaaaaatggctacgcgggctacatcgacggaggtgcaagc

caggagagttctataagtttataaagccgatccttgagaagatggatgggaccgaagaactcctggtga

aacttaacagggaggatctgctgcgaaagcagcgcacatttgataatgggtccattccacatcaaattca

tcttggagaattgcacgccattttgagaagacaggaggacttctatccgtttcttaaggacaatcgggaa

aaaatagaaaagatacttacattccgaattccttactatgtcgggcccttggcgcgagggaactctcggt

tcgcttggatgactcgaaaaagtgaggagactataactccttggaattttgaagaagtcgtagacaaagg

tgcttccgcgcaatcttttatagagaatgactaatttcgacaaaaacctcccaaacgagaaggttctg

ccaaagcacagccttctctacgagtattttactgtctataacgaactcactaaggtgaaatacgtaactg

aagggatgaggaagcctgccttttgagtggcgagcagaagaaagcgatagtagatttgctttttaaaac

caaccggaaggtcactgtgaaacaacttaaagaagactacttcaagaagatagagtgtttcgacagcgta

gaaatatctggcgtagaggaccgcttcaatgcaagcttgggcacttaccatgacctcctcaagataatca

aagataaagattttcttgataatgaagagaatgaggatatccttgaagacatagtgttgactctcacgtt

gtttgaggatagggagatgatagaagagcgcttgaagacctatgctcatctcttcgatgataaagtaatg

aagcaactcaagcggagaaggtatactggttggggtcggctttctaggaaactcatcaacggtattcggg

ataaacagtccggcaaaactatattggatttccttaagagtgatggcttcgcgaaccgaaactttatgca

acttatacatgatgattcacttacatttaaggaagacattcaaaaagcgcaagtatctggccagggcgac

tctctgcacgaacacatagcaaaccttgctggatcccctgcgataaagaaaggtatactgcagacagtaa

aagttgtggacgaactcgtcaaggttatgggtaggcataagcccgaaaatatagtgattgaaatggcacg

cgagaaccaaactacacaaaaaggtcaaaagaacagcagagaacggatgaaacgaatagaggaggggatc

aaggaacttggatcccaaatactgaaagagcaccccgtagaaaatacccagcttcaaaacgagaagcttt

atctgtactacctgcaaaacggcagagatatgtacgtagaccaggaactggacatcaacagattgtctga

ttatgatgtagatcacattgtgccgcagagcttcctcaaagatgattccatagataacaaagtactcact

cgaagtgacaaaaacagagggaaatccgataatgttcccagtgaggaagtcgtcaagaagatgaagaatt

attggcggcaacttctcaacgcgaaactcatcacccaaagaaaattcgataacctcactaaagctgaacg

cggaggactgtcagagcttgataaagcaggttttatcaaaaggcaactcgtagaaacgagacaaataaca

aaacatgtcgctcaaattttggactctcgcatgaatacaaagtatgatgaaaatgacaagctgattcgcg

aggtcaaagtaataacgctcaaaagtaaacttgtctccgatttccgaaaggactttcagttttacaaagt

cagagagataaataattaccaccacgcccatgacgcctacctcaatgctgtggttggtacggctcttatc

aagaaatatccgaagctcgagagcgagtttgtctatggcgactacaaggtttacgatgtcaggaaaatga

tagcgaagtctgagcaagagataggtaaggccacggctaaatactttttctattcaaatatcatgaattt

tttcaaaaccgagataacgttggcgaatggcgaaatcaggaaaaggcctcttatagagactaacggagaa

acaggcgagattgtctgggataagggtagggatttcgctactgtgcgaaaggtactgagcatgccccagg

ttaacattgtcaaaaagactgaggtccaaacgggtggcttttctaagggagagtatcctgccaaaacgcaa

ttcagataagctcatagcgagaaaaaaggattgggacccaaagaaatatggcggcttcgactctccgacg

gtcgcatatagtgtgctggtggttgccaaagttgaaaagggcaagtctaaaaaactgaaatcagtgaagg

agcttctcggaataactattatggagcgcagctcatttgaaaagaatccaatcgatttcttggaagcaaa

aggatataaagaagtcaagaaggacctgatcatcaaacttccaaagtactcactgttcgaacttgagaac

ggacggaagcggatgcttgcgagtgctggagaacttcaaaaaggtaacgagctcgcccttccatcaaagt

acgtaaattttttgtacttggcatcacactatgagaagctcaaaggctcccccgaggataatgagcaaaa

gcagcttttcgtggaacaacataaacattaccttgatgaaatcatcgaacaaataagcgaattctctaaa

cgagttattcttgcagacgcgaatctggacaaggtgcttagcgcatacaacaagcatcgggataagccga

ttcgagaacaagccgaaaatataatccatcttttcactctgacgaacttgggtgcgccggcagccttcaa

gtacttcgataccactattgataggaaacggtatacgagtacaaaagaagtgctggacgctacgcttatc

catcagtccatcactgggctctacgagacgaggattgaccttagtcagttgggcggggac</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (9)

1. An expression vector, which is a viral particle of an adeno-associated virus (AAV) having AAV capsid proteins, in which a single-stranded DNA molecule is packaged as a genome, at the ends of which the AAV ITR sequences are located, within the AAV ITR the promoter of the first eukaryotic elongation factor EF1 alpha is located sequentially , histidine tag sequence, intracellular nuclear localization signal 5'-SV40 NLS, codon-optimized sequence of the Cas9 gene of Streptococcus pyogenes, another intracellular nuclear localization signal 3'-SV40 NLS, synthetic polyA signal was used as a transcription terminator. 2. The expression vector according to claim 1, characterized in that the AAV ITR is an AAV serotype 2 ITR. 3. The expression vector according to claim 1, characterized in that the codon-optimized sequence of the Streptococcus pyogenes Cas9 gene is SEQ ID NO:5. 4. The expression vector according to claim 1, characterized in that the vector is the sequence of SEQ ID NO: 1. 5. An expression vector, which is a viral particle of an adeno-associated virus (AAV) having AAV capsid proteins, in which a single-stranded DNA molecule is packaged as a genome, at the ends of which the AAV ITR sequences are located, within the AAV ITR the eukaryotic polymerase III promoter - U6 is sequentially located, after which there is a guide RNA expression cassette, including a guide RNA sequence in crRNA format, complementary to the targeted locus, linked to the trackRNA sequence, a region of homology with the human genome locus AAVS1 - the left homologous arm, a transgene expression cassette: the EFS-NS promoter, under the control of which the protein-coding sequence of the hCYP21A2 gene is 78-99% identical to the sequence of SEQ ID NO: 3, the transcription terminator is a polyA signal, the region of homology with the AAVS1 locus of the human genome is the right homologous arm. 6. The expression vector according to claim 5, characterized in that the guide RNA sequence in crRNA format is a sequence of 20 nucleotides. 7. The expression vector according to claim 5, characterized in that the AAV ITR is an AAV serotype 2 ITR. 8. The expression vector according to claim 5, characterized in that the polyA signal is the polyA signal of the human β-globin gene. 9. The expression vector according to claim 5, characterized in that the vector is the sequence of SEQ ID NO: 2.