Adeno-associated virus-based expression vector

Abstract

The set of inventions relates to biotechnology and medicine, and more particularly to expression vectors for producing proteins in mammalian cells, combinations of said expression vectors for the treatment of congenital adrenal hyperplasia and methods contemplating the use of said combinations, and sets of expression vectors based on adeno-associated virus (AAV) virions, it is useful for medical use, and in particular for the treatment of congenital adrenal hyperplasia.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, specifically to expression vectors for producing proteins in mammalian cells, combinations thereof for treating congenital adrenal hyperplasia, and methods for using said combinations and kits for expression vectors based on adeno-associated virus (AAV) particles. These inventions can be applied in medicine, particularly for the treatment of congenital adrenal hyperplasia. Background Technology

[0002] Congenital adrenal hyperplasia (CAH) is a group of conditions associated with a deficiency of cortisol and / or aldosterone (steroid hormones). This deficiency is caused by a mutation in a gene in an enzyme involved in the synthesis of cortisol and aldosterone from cholesterol. This enzyme may be completely absent or produced in insufficient quantities. The most well-known forms of congenital adrenal hyperplasia are 21-hydroxylase deficiency and 11β-hydroxylase deficiency. A deficiency of 21-hydroxylase (CYP21A2) can lead to decreased cortisol and / or aldosterone levels and increased androgen concentrations. Excess androgens cause masculinization of the external genitalia in female newborns. Males with this genetic defect may not have obvious clinical symptoms at birth but tend to exhibit precocious puberty due to excess androgens. Affected individuals may present with early growth of facial and body hair, acne, and irregular menstruation, and this condition can also lead to infertility. 21-hydroxylase deficiency is caused by a mutation in the CYP21A2 gene. Patients may have classic (more severe) or non-classical forms of the disease.

[0003] The classic form can be:

[0004] 1. Salt-wasting form – the most severe form, characterized by extremely low enzyme activity, leading to a deficiency of both cortisol and aldosterone. Aldosterone deficiency results in hyponatremia and hyperkalemia. If left untreated, it can lead to acute adrenal insufficiency and shock.

[0005] 2. Simple masculinization type – enzyme activity is reduced but remains sufficient to maintain normal or near-normal aldosterone synthesis.

[0006] The non-classical form is the most common and mildest variant of the disease. It is found in approximately 1 in 1500 newborns in Caucasian populations. In this form, enzyme activity is approximately 50%, avoiding critical sodium loss and serious complications. Androgen levels are only slightly elevated.

[0007] According to the Russian Federal Clinical Guidelines-Protocols for the Management of Patients with Congenital Adrenal Hyperplasia in Childhood, Problems of Endocrinology, 2014; 60(2):42-50, CAH should be managed conservatively.

[0008] The first-line treatment for children with CAH is oral hydrocortisone tablets (level of evidence 1+++). Hydrocortisone syrup and long-acting corticosteroids are not recommended during children's active growth period (level of evidence 1++). All children with salt-wasting CAH should be prescribed fludrocortisone, and infants should receive additional dietary sodium chloride (level of evidence 1++).

[0009] The basic principle of treatment for all forms of congenital adrenal hyperplasia (CAH) is the administration of glucocorticoids. These agents compensate for cortisol deficiency, thereby inhibiting the excessive secretion of adrenocorticotropic hormone (ACTH). As a result, the adrenal production of steroid hormones that were previously oversynthesized due to the blockade of specific enzymes is reduced.

[0010] Various medications possess glucocorticoid activity: prednisolone, cortisone, and dexamethasone. However, long-acting synthetic glucocorticoids (such as prednisolone and dexamethasone) have negative effects on growth. For children with open growth plates—especially in early childhood—tablet formulations of hydrocortisone are considered the most suitable. The initial daily dose required to inhibit ACTH secretion in infants can reach up to 20 mg / m². 2 On average, for children over one year old, the daily dose should range from 10-15 mg / m². 2 Apply in three equal portions daily (e.g., at 7:00, 15:00, and 22:00).

[0011] In the salt-wasting form of CAH, mineralocorticoid therapy with fludrocortisone is necessary, usually at a dose of 0.05–0.15 mg daily (administered once or twice daily). In infants, the need for mineralocorticoids is higher, reaching up to 0.3 mg / day, divided into three doses. Subclinical mineralocorticoid deficiency can still be observed in children without clinical signs of salt loss, characterized by elevated plasma renin levels. Fludrocortisone therapy is also indicated in these cases.

[0012] Gene therapy is considered a promising alternative to conservative treatments.

[0013] Adeno-associated virus (AAV) is a promising tool for human gene therapy. AAV can effectively infect both dividing and non-dividing human cells, and its genome can integrate into a single site within the host cell's chromosome. Importantly, the presence of AAV in humans is independent of any known pathology.

[0014] AAV vectors for gene therapy are known, and the problem of producing high-yield tools and methods that can potentially achieve stable and large-scale production of AAV vectors in insect cells has been solved [RU2457252 C2, 27.07.2012]. Therefore, the vectors currently known are general-purpose, suitable for use regardless of the specific disease. However, specific tasks often require specific solutions, thus necessitating the creation of different optimal designs for each task.

[0015] AAV vectors containing gRNA and Cas9 are known, where the Cas9 molecule represents the active or inactive Cas9 of *Streptococcus pyogenes*. This vector has been used for antitumor therapy, including adrenal tumors [RU2019133280 A, 22.04.2021]. However, this solution is not intended for the treatment of congenital adrenal insufficiency. AAV vectors containing Cas9, NLS, and HA are also known [KR1020150056539A, 26.05.2015].

[0016] In addition, there are AAV vectors containing Cas9, SV40 NLS, HA [US20210054405 A, 02 / 25 / 2021] and AAV vectors containing guide RNA and ITR, including serotype 2, which are intended for adrenal therapy [WO2020082047 A1, 04 / 23 / 2020]. However, none of these vectors have addressed the treatment of congenital adrenal insufficiency.

[0017] Vectors are known to target the CYP21A2 gene in the human genome, containing guide RNA and Cas9, NLS, and eukaryotic elongation factor EF1α from Streptococcus pyogenes [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]. However, this solution involves lentiviral vectors, which are unsuitable for treating congenital adrenocortical dysfunction.

[0018] A known AAV vector system (combination) consists of one vector carrying a guide RNA and a second vector carrying a CAS. The vectors also contain an ITR, a promoter, and a tag, and the CRISPR enzyme contains C-terminal NLS and N-terminal NLS. 34.

[0019] The vector is intended for use in compositions for manufacturing therapeutic agents for in vitro gene or genome editing, or for modifying mammalian organisms by manipulating target sequences at loci of interest in the genome. The vector can also be used in methods for treating or inhibiting conditions in cells with defective nucleotide elements, trinucleotide repeats, other repeating nucleotide elements, or nucleotide amplification [RU2016128069 A, 17.01.2018]. This solution is closest to the present invention but relates to a lentiviral vector, which is unsuitable for treating congenital adrenocortical dysfunction.

[0020] The technical problem solved by this invention group is to develop genetic constructs containing optimized sequences (including Cas9 of Streptococcus pyogenes) that ensure high levels of transgene expression and can be used to treat congenital adrenal hyperplasia. Summary of the Invention

[0021] After implementing the present invention, the following technical effects are achieved:

[0022] 1. Low immunogenicity;

[0023] 2. Easily manufactured;

[0024] 3. The nonspecific cutting rate is 0.01%;

[0025] 4. Broad tropism for different tissues;

[0026] 5. High transgene expression level: The viral genome copy number per cell ranges from 1 to 100, and its expression relative to the GAPDH reference gene expression ranges from 10. -4 Up to 10 -1 .

[0027] The technical effect is achieved through the following methods.

[0028] One invention of this group provides a pAAV-nEFCas9 expression vector.

[0029] This vector represents the viral particle of adeno-associated virus, the capsid protein of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hul4, rh10, etc.) or serotypes obtained through synthetic biology methods (DJ / 8, DJ, apc 32, etc.).

[0030] The genome packaged into the viral particle consists of a single-stranded DNA molecule, which is flanked at both ends by inverted terminal repeats (ITRs) of AAV, preferably serotype 2. These AAV ITRs have been well studied, and their use produces stable, expected results. Nevertheless, other AAV ITRs can also be used, as no prior art has shown that using ITRs from other AAV serotypes will produce incorrect results. Within the AAV ITR, the promoter of the first eukaryotic elongation factor EF1α is arranged in sequence. This particular promoter was selected as the result of a series of experiments showing that it produces high levels of transgene expression in the vector construct according to the invention. Furthermore, it is eukaryotic rather than viral, and therefore it is not methylated and repressed, which is another advantage of the invention. Moreover, it is a constitutive promoter, functioning in all cell types. Following the promoter is a histidine tag sequence (HA (human influenza hemagglutinin) epitope tag), which facilitates the detection of transgenes by Western blotting, flow cytometry, and immunohistochemistry. Following the histidine tag is the nuclear localization signal 5'-SV40 NLS. The advantages of this nuclear signal are its small size and proven efficiency in facilitating nuclear protein importation. Next is the codon-optimized sequence encoding the Cas9 enzyme from Streptococcus pyogenes.

[0031] Cas9 is an effector with a relatively small size, capable of inducing double-strand breaks at target loci within the genome. CRISPR / Cas9-based genome editing systems are easy to use and design, with an off-target cleavage rate of approximately 0.01%, and require the selection of readily synthesizable specific RNA guides. This is a key challenge upon which achieving such specificity depends. Optimal RNA guides have been developed that maximize the advantages of CRISPR / Cas9-based genome editing systems. Incorrectly selected RNA guides will lead to reduced effectiveness of such systems. Codon optimization (sequence SEQ ID NO: 5) was performed to more efficiently synthesize bacterial proteins in human cells, contributing to the aforementioned beneficial effects. However, other codon-optimized variants are also possible, and therefore the variant used in this development demonstrates the primary possibility of achieving the effects according to the invention by optimizing this sequence. Following the codon-optimized sequence is a second nuclear localization signal, 3'-SV40 NLS. The synthesized polyA signal serves as a transcription terminator.

[0032] Another invention in this group relates to the expression vector AAV-hCYP21A2.

[0033] The viral genome packaged into the particle contains a single-stranded DNA molecule flanked by AAVITR (inverted terminal repeat) serotype 2. These ITRs are well characterized and are known to produce consistent and reliable results. Nevertheless, the use of other AAV serotype ITRs is not excluded, as existing techniques have not shown that they would produce incorrect results.

[0034] The eukaryotic RNA polymerase III promoter U6 is located within this vector. The U6 promoter has been shown to produce high levels of transgene transcription in the vector construct described herein. It is eukaryotic, not viral, and therefore not repressed in human cells, while its relatively small size allows for the packaging of the RNA guide transcription cassette into the AAV genome. Following the U6 promoter is a guide RNA sequence in crRNA format. This is a unique 20-nucleotide sequence complementary to the target locus and fused to the trackRNA sequence (backbone). Adjacent to the guide RNA expression cassette is a region homologous to the human genomic locus AAVS1 (left homologous arm). This locus was selected based on experimental data confirming that integration at this site does not adversely affect the function of other genomes, thus providing safe integration. Transgenes integrated into this locus are efficiently transcribed in any cell type. Transgenic expression cassette: The EFS-NS promoter exhibits high levels of transgenic expression. It is eukaryotic, not viral, and therefore not subject to methylation or repression. It is also a constitutive promoter, meaning it functions in all cell types, with the protein-coding codon optimized under its control. Codon optimization allows for increased gene translation efficiency, performed relative to the corresponding reference sequence NG_007941.3. The polyA signal (human β-globin polyadenylation signal) of the human β-globin gene serves as a transcription terminator. Following the polyA signal is a region homologous to the human genomic locus AAVS1 (right homologous arm).

[0035] Another invention of this group is a combination of pAAV-nEFCas9 and AAV-hCYP21A2 expression vectors with pharmaceutically acceptable excipients. In this case, the combination contains buffers and / or saline solutions as pharmaceutically acceptable excipients. Non-limiting examples of buffers that can be used in this solution are: phosphate buffers, phosphate-citrate buffers, Tris buffers, carbonate buffers, bicarbonate buffers, borate buffers, acetate buffers, glycerol buffers, phosphate-glycerol buffers, Tris-glycerol buffers, and citrate buffers.

[0036] Another invention is a method for treating congenital adrenal hyperplasia, comprising administering an effective amount of the aforementioned combination of expression vectors to a mammalian subject. Another invention in this group provides a kit for expression vectors, one of which is a pAAV-nEFCas9 vector and the other is an AAV-hCYP21A2 vector. The kit may also contain buffers and / or physiological saline. Non-limiting examples of buffers that may be used include: phosphates, phosphate-citrate, Tris, carbonates, bicarbonates, borates, acetates, glycerol, phosphate-glycerol, Tris-glycerol, and citrate buffers.

[0037] Brief description of the attached figures

[0038] This invention is explained by the following illustrative materials.

[0039] Figure 1 This is a schematic diagram of the layer arrangement of the iodixanol solution in a centrifuge beaker. After centrifuging the QuickSeal tube, air is first introduced by piercing the top of the tube with a separate needle, and then the desired fraction is aspirated using another syringe needle. 40% of the fraction is aspirated, with the needle opening facing upwards and the needle tip positioned horizontally approximately 2 mm below the 40-60% phase interface. This aspirates 40% of the gradient without adversely affecting the 25% phase interface.

[0040] Figure 2 - This is a schematic diagram of AAV-hCYP21A2.

[0041] Figure 3 - This is a schematic diagram of pAAV-nEFCas9.

[0042] Figure 4 - This is a schematic diagram used to verify the restoration of steroid production when a normal copy of the CYP21 gene is integrated into the genome of a mutant organism of the gene.

[0043] Figure 5 - This is a photograph of an adrenal gland sample from a CYP21A1- / - mouse after immunofluorescence staining with EGFP two weeks after administration of the vector.

[0044] Figure 6 The study results confirmed that the donor integrated into the Rosa26 target locus of the mouse adrenal genome. A) Electrophoresis of the target amplicon in an agarose gel (“C+” positive control, “C-” negative control). B) Chromatogram of Sanger sequencing of the amplicon from (A).

[0045] Figure 7 - A schematic diagram showing viral load and transgene expression levels in target organs.

[0046] Figure 8AD-chart showing the curves of weight gain and steroid hormone levels in experimental animals.

[0047] Figure 9 Representative photographs of uninjected CYP21A1+ / + mice (control) and CYP21A1- / - mice injected with AAVDJ-CYP21A1-01, AAVDJ-SpCas9+AAVDJ-CYP21A1-01 and PBS at 16 weeks post-vector injection. Detailed Implementation

[0048] One invention of this group provides an adeno-associated virus-based expression vector comprising capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hul4, rhl0, etc.) or serotypes obtained through synthetic biology methods (DJ / 8, DJ, anc32, etc.), wherein the following elements are arranged in sequence: a eukaryotic elongation factor EF1α promoter, a histidine tag (HA epitope tag from human influenza hemagglutinin), a 5'-SV40 nuclear localization signal (NLS), a codon-optimized sequence from the Cas9 gene of Streptococcus pyogenes, a 3'-SV40 NLS, and a synthetic polyA signal as a transcription terminator. An exemplary embodiment of the invention is SEQ ID NO:1.

[0049] Another invention of this group provides an adeno-associated virus-based expression vector containing capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 7, 8, 9hul4, rh10, etc.) or serotypes (DJ / 8, DJ, ans32, etc.) obtained by synthetic biology methods.

[0050] The genome packaged in the vector is a single-stranded DNA molecule side-joined by the ITR of AAV serotype 2, wherein the following elements are arranged in sequence: a eukaryotic RNA polymerase III promoter (U6), a guide RNA in crRNA format (a unique 20-nucleotide sequence complementary to the target locus and fused to the track RNA (backbone), followed by a region homologous to the AAVS1 locus in the human genome (left homologous arm), a transgene expression cassette containing the EFS-NS promoter, under which is the protein-coding sequence of the hCYP21A2 gene corresponding to the reference sequence NG_007941.3. Human β-globin polyadenylation signal is used as a transcription terminator, followed by the right homologous arm targeting AAVS1. An exemplary embodiment is SEQ ID NO:2.

[0051] SEQ ID NO:3 represents the sequence of the wild-type CYP21A2 gene NG_007941.3.

[0052] SEQ ID NO:4 represents an optimized variant sequence of SEQ ID NO:3.

[0053] SEQ NO:5 represents the codon-optimized sequence of the Cas9 gene of Streptococcus pyogenes.

[0054] The present invention also relates to a method for treating congenital adrenal hyperplasia, comprising administering an effective amount of the combination of said carriers to a mammal.

[0055] Within the scope of this invention, the term "therapeutic effective amount" refers to the amount of a combination of carriers that has a therapeutic effect on CAH or reduces, improves, or eliminates one or more symptoms of the disease. The term "therapeutic effective amount" refers to a quantitative content of a combination of carriers sufficient to provide the desired therapeutic effect. Therefore, when preparing a pharmaceutical composition in unit dose form, the effective dose is considered. In this case, the dosage of the agent in the patient can be adjusted based on the therapeutic efficacy and bioavailability of the active ingredients in vivo, their metabolism and excretion rates from the body, and factors such as age, sex, and stage of the disease.

[0056] The recommended treatment dose is 2 × 10⁻⁶ per kilogram of body weight. 11 Up to 2×10 14 A single administration of one viral genome, equivalent to 2 ml / kg, is administered via intravenous infusion after dilution in buffer or 9 mg / ml (0.9%) sodium chloride injection.

[0057] The recommended administration method is a single intravenous infusion.

[0058] Since gene therapy for congenital adrenal hyperplasia is mediated by the hCYP21A2 gene mutation, treatment can be achieved by infecting adrenal cells and exogenously expressing the hCYP21A2 gene as episome DNA persistently present in the cell nucleus via a vector genome. In this case, double-strand breaks are introduced into the genome of the infected cell through RNA-guided expression specific to the AAVS1 locus (i.e., adeno-associated virus integration site 1), the PPPIR12C protein phosphatase 1 regulatory subunit 12C, and the NC_000019.10 sequence, as well as through the expression of Cas9 protein, which can introduce double-strand breaks into DNA at the RNA-guided homologous binding site to the target. Integration occurs because of the presence of a homologous region to the AAVS1 locus in the AAV-hCYP21A2 vector. As a result of cleavage, homologous recombination, and transgenic expression, hCYP21A2 gene expression is achieved, and steroid production is restored in the adrenal cortex.

[0059] A more detailed description of the invention is provided below.

[0060] This invention can be modified in various ways that will be obvious to those skilled in the art from this description. Such modifications do not limit the scope of the claims.

[0061] Viral particles are generated using plasmid constructs that carry the nucleotide sequence of a vector genome and plasmids that replicate and select the desired sequence in a bacterial system. These plasmids are obtained through molecular cloning and gene synthesis methods.

[0062] It also uses plasmids carrying AAV viral capsid genes and helper genes.

[0063] Assemble viral vectors in adherent or suspension cell systems / dedicated cell lines based on HEK 293 cells.

[0064] Example 1

[0065] Plasmid extraction and purification (cleaning)

[0066] The E. coli cells were thawed and stored at -80°C.

[0067] Thawing was performed on solid culture medium. For this purpose, frozen cells were added to 400 μl of liquid LB medium (on a pipette tip), resuspended, and transferred to solid LB medium containing 100 mg / L carbenicillin.

[0068] To obtain a suspension culture, add 100 ml of LB medium to a 250 ml culture flask. Inoculate one colony onto a pipette tip, and incubate the E. coli suspension in a constant temperature shaker at 37°C for 16–20 hours with constant stirring at a speed of 170 rpm.

[0069] Next, plasmid DNA was isolated using the Plasmid Midiprep 2.0 kit (Eurogen) according to the manufacturer's instructions. Columns were prepared at a rate of one column per 1g of cell pellet (column capacity 500μg DNA). Each column was eluted twice with 4ml of water.

[0070] After isolation, the plasmid concentration was measured and restriction analysis was performed using restriction enzymes according to Table 1.

[0071] For each sample, the following reactions were performed: digestion with each restriction enzyme listed in Table 1 individually, digestion with all restriction enzymes in a single tube, and a control reaction (K-) without any restriction enzymes.

[0072] Preparation of the mixture:

[0073] 10XBuf-2.5μl;

[0074] Restriction enzymes - 0.5 μl each;

[0075] Plasmid DNA - 600 ng;

[0076] Add water to 25 μl.

[0077] Incubate at 37°C for 1 hour and visualize in 1% agarose gel.

[0078] Table 1

[0079]

[0080] Number of viral genomes per cell - from 1 to 100.

[0081] Compared to the reference GAPDH gene, the expression range is from 10 -4 Up to 10 -1 .

[0082] Example 2

[0083] Preparation of HEK293 cell culture

[0084] The volumes of all culture media, solutions, etc., are given per 15cm petri dish.

[0085] Prepare culture media for cytospheric expansion (GM) and transfection (TM). To prepare GM, add 50 ml FBS (Gibco), 5 ml 100×GlutaMax (Gibco), and 500 μl 1000×Gentamicin antibiotic (PanEco) to 450 ml DMEM medium containing 4.5 g / L glucose.

[0086] To prepare TM, 10 ml FBS (Gibco), 5 ml 100×GlutaMax (Gibco), and 500 μl 1000×Gentamicin antibiotic (PanEco) were added to 490 ml DMEM medium containing 4.5 g / L glucose.

[0087] Aliquot the ethylenediaminetetraacetic acid (Versene) and 0.25% trypsin solution into 50 ml portions. Store the Versene aliquot at room temperature, while storing the 0.25% trypsin aliquot at -20°C. Before using the cells, keep the trypsin solution at room temperature and the GM / TM medium in a +37°C water bath.

[0088] The cell line was passaged. The number of passages for the cell line did not exceed 12.

[0089] Thaw HEK293 cell culture. Before thawing the cryovials, preheat 25 ml of GM in a +37°C water bath. Place the cryovials in a +37°C water bath until completely thawed. Immediately after thawing, transfer all contents of the cryovials to a 15 ml tube and add 10 ml of preheated GM. Centrifuge the suspension at 230 g for 3 minutes. Discard the supernatant and resuspend the cell pellet in 1 ml of GM, transfer to a T75 culture flask, and add 14 ml of GM. Incubate the culture flask in a 5% CO2 incubator at +37°C until the cells reach 70% confluence.

[0090] Frozen HEK293 cell culture.

[0091] Before freezing the cell bank, the MycoReport protocol (Evrogen) was used to test the cells for mycoplasma contamination.

[0092] Remove all culture medium from the culture dish, add 5 ml of EDTA solution, and incubate the dish in a laminar flow hood for 1 minute. Remove the EDTA solution, add 3 ml of 0.25% trypsin solution, and incubate the dish in a laminar flow hood for 3 minutes. Then, add 3 ml of GM and gently and thoroughly resuspend the cells. Transfer 10 μl of the cell suspension to a 0.2 ml tube. Transfer the remaining cells to a 15 ml tube.

[0093] Cell count and viability were determined using a TC-20 automated cell counter (Bio-Rad): 10 μl of well-mixed cell suspension was combined with 10 μl of 0.4% trypan blue solution, and the resulting suspension was applied to a counting slide for measurement.

[0094] Centrifuge the cells in a 15ml tube at 230×g for 3 minutes.

[0095] Calculate the required number of cryovials so that each cryovial contains 5 million cells in 1 ml of cryopreservation solution. Label each cryovial with the following information: cell line name, number of cells, date, and operator.

[0096] The following is a sample of cryosol preparation for a cryovial: 700 μl FBS (Gibco), 200 μl DMEM (PanEco) containing 4.5 g / L glucose, and 100 μl DMSO (PanEco).

[0097] Resuspend the cell pellet centrifuged at 230×g for 3 minutes in a 15 ml tube in cryopreservation medium. Aliquot the suspension into 1 ml portions for each cryopreservation tube. Incubate the cryopreservation tubes in a Kelvinator at -80°C for 24 hours, then transfer them to liquid nitrogen at -196°C for long-term storage.

[0098] HEK293 cell cultures were passaged.

[0099] Remove all culture medium and add 5 ml of ethylenediaminetetraacetic acid (EDTA) solution to the petri dish. Incubate the petri dish in a laminar flow hood for 1 minute, then remove the EDTA solution.

[0100] Next, add 3 ml of 0.25% trypsin to the culture dish and incubate in a laminar flow hood for 3 minutes. Add an equal volume of GM medium, gently resuspend the cells, and transfer to a 15 ml Falcon tube.

[0101] Cell count and viability were determined using a TC-20 automated cell counter (BioRad): 10 μl of thoroughly mixed cell suspension was combined with 10 μl of 0.4% trypan blue solution, mixed again, and 10 μl of the resulting suspension was applied to a counting slide for measurement.

[0102] Transfer six million cells to a new culture dish, add 24 ml of GM, and incubate at +37°C and 5% CO2 until the next passage.

[0103] Example 3

[0104] HEK293 cell culture transfection

[0105] The transfection mixture consists of lactate buffer, linear polyethyleneimine (PEI), and plasmid DNA.

[0106] Preparation of lactate buffer (pH 4) (20 mM sodium lactate, 150 mM sodium chloride):

[0107] To prepare a 300 ml lactate buffer, 1.35 ml of 40% lactate was added to 290 ml of 150 mM sodium chloride. The pH was adjusted to 4 using 1 N sodium hydroxide solution. The resulting solution was brought to a final volume of 300 ml with 150 mM sodium chloride, sterilized through a 0.2 μm filter, and stored at 4 °C.

[0108] Preparation of a 5 mg / ml linear polyethyleneimine solution (l-PEI 25):

[0109] A total of 1 g of dry linear polyethyleneimine (25 kDa) was dissolved in 190 mL of 0.2 N hydrochloric acid with continuous stirring. The volume was adjusted to 200 mL with hydrochloric acid. The final pH of the solution was 1–2. Aliquots of this solution were stored at -80 °C.

[0110] Calculation of the required amounts of plasmid and polyethyleneimine (PEI) for transfection:

[0111] For transfection of a 15cm culture dish, use 38μg of plasmid DNA with a plasmid copy ratio of helper:packaging:transfer = 1:1:1.

[0112] Cell preparation for transfection:

[0113] One day before transfection, cells were reseeded onto new culture dishes at a density of 10 million cells per dish. 24 ml of GM medium was added, and the cells were incubated at +37°C and 5% CO2.

[0114] Transfection of HEK293 cell culture:

[0115] Prepare a mixture of DNA and PEI according to Table 2.

[0116] Table 2 shows the preparation schemes for the transfection mixture calculated for forty culture dishes.

[0117]

[0118] Add 80 ml of the DNA-l-PEI 25 mixture to 1 L of TM medium and mix thoroughly. Remove all GM medium from all culture dishes. Then, add 27 ml of the TM-DNA-PEI mixture to each culture dish. Gently mix the contents. Incubate the culture dishes at +37°C and 5% CO2 for 72 hours.

[0119] The transfection efficiency reaches 95-98%.

[0120] Cell lysis

[0121] Prepare a lysis buffer containing: 150 mM NaCl, 50 mM Tris-HCl, 1 mM MgCl2, pH 8.5, 0.5% Triton X-100, and 50 U / ml Benzosinase. Sterilize the solution by filtration rather than autoclaving and store at +4°C.

[0122] Collect all culture medium from each culture dish using a 25 ml serum pipette, which also serves to wash cells off the surface. Transfer the resulting cell suspension to a 50 ml tube and repeat the process for all culture dishes. Centrifuge the tube containing the cell suspension at 400 × g for 5 minutes. Collect the supernatant as needed and store at -80°C.

[0123] All cell pellets from 40 culture dishes were resuspended in 60 ml of lysis buffer and incubated at +37°C for 1 hour. The suspension was then centrifuged at 4000 × g for 15 minutes, and the clear supernatant was transferred to a clean tube.

[0124] Example 4

[0125] AAV isolation and purification

[0126] Preparation of buffer solutions: 1M NaCl / PBS-MK buffer and 1×PBS-MK buffer

[0127] To prepare a 1M NaCl / PBS-MK buffer, 5.84 g NaCl, 26.3 mg MgCl2 (equivalent to 56.0 mg MgCl2·6H2O), and 14.91 mg KCl were dissolved in 1×PBS to a final volume of 100 mL. The solution was filtered through a 0.22 μm membrane and stored at +4 °C.

[0128] To prepare 1×PBS-MK buffer, 26.3 mg MgCl2 (56.0 mg MgCl2·6H2O) and 14.91 mg KCl were dissolved in 1×PBS, resulting in a final volume of 100 mL. The solution was filtered through a 0.22 μm membrane and stored at +4 °C.

[0129] For each ultracentrifugation step, only the iodixanol solution prepared according to Table 3 was used.

[0130] Table 3 shows the calculations for preparing iodixanol solution for two Quick-Seal tubes (39 ml each).

[0131]

[0132] Ultracentrifugation

[0133] Using a 10ml syringe and a Seldinger needle, add the layer of iodixanol solution to the centrifuge tube in the order specified in Table 4.

[0134] Table 4

[0135]

[0136] Apply a gradient from the upper layer to avoid compromising its integrity.

[0137] When filling QuickSeal tubes, completely fill the tubes with cell lysate, ensuring no air bubbles are left at the top. Equilibrate the tubes to within 0.0005 g using an analytical balance with PBS. Ultracentrifuge at +10°C at 360,000 × g (59,000 rpm using a Beckman Coulter Ti 70 rotor) for 1 hour and 30 minutes.

[0138] After centrifugation, first puncture the top of the QuickSeal tube with a separate needle to allow air to enter. Then, use a second needle attached to the syringe, such as... Figure 1Carefully collect the 40% iodixanol fraction as shown. Position the needle with the opening facing upwards and the tip horizontally approximately 2 mm below the 40-60% interface. Aspirate the 40% gradient layer without disturbing the 25% layer interface.

[0139] Transfer the collected fractions to 50 ml Falcon tubes.

[0140] Ultrafiltration

[0141] The 40% iodixanol fraction collected after ultracentrifugation was diluted five-fold with PBS / Pluronic F68 (0.001%) solution and filtered through a 0.22 μm membrane filter. The Amicon Ultra-15 filter unit was pre-rinsed by adding 3 ml of H2O and then centrifuging at 2,000 × g for 1 minute. All residual liquid was then removed from the tube and filter unit. Next, 3 ml of PBS / Pluronic F68 (0.001%) solution was added, and the mixture was centrifuged again at 2,000 × g for 1 minute to completely remove the liquid.

[0142] Next, add 15 ml of diluted virus solution to the Amicon Ultra-15 unit and centrifuge at 2,500 × g for 10 minutes using a basket rotor. Discard the flow-through and refill the filter with the remaining virus vector solution until the entire volume has been processed. Thoroughly rinse the membrane with 100–1000 μl pipettes of virus solution. Repeat centrifugation at 2,500 × g for 10 minutes.

[0143] After centrifugation, the viral vector suspension (approximately 500 μl) was retained in the upper chamber of the concentrator. Centrifugation was repeated for another 3 minutes. Once the entire viral solution had passed through the membrane, the concentrated virus was washed twice with room temperature PBS / Pluronic F68 (0.001%). Approximately 500 μl of the viral suspension was retained in the filter cartridge.

[0144] Rinse the membrane thoroughly and divide the virus into 20 μl portions, which are then placed into 0.2 ml tubes. Reserve 5 μl of the virus for titration.

[0145] AAV-CYP21: 7.80 × 10⁻⁶ per ml 12 A virus particle

[0146] AAV-Cas9: 1.93 × 10⁻⁶ per ml 12 A virus particle

[0147] Example 5

[0148] according to Figure 4The protocol described evaluates the restoration of steroid production by integrating a copy of the wild-type CYP21 gene into the genome of an organism carrying a mutation of that gene. The dose was administered intravenously at a rate of 3 × 10⁻⁶ to 5-week-old CD-1-C57Bl / 10SnSlc-H-2aw18 mice via the tail vein. 11 Two recombinant adeno-associated virus vectors, AAV-Cas9 and AAV-Cyp, of the DJ serotype were used to generate one viral particle. Animals injected once with phosphate-buffered saline (PBS) served as the control group. Mice were maintained under standard feeding conditions for 2, 4, 8, 16, and 30 weeks after injection. After the designated time points, the animals were euthanized and target organs and tissues were collected for analysis.

[0149] Target samples included blood, adrenal glands, liver, brain, spleen, gonads, kidneys, thymus, and lungs. Immunofluorescence analysis of adrenal tissue sections confirmed the presence and expression of the applied vector. Figure 5 The image shows GFP protein staining in organs of treated mice.

[0150] DNA was also extracted from adrenal gland samples of laboratory animals and analyzed to verify transgene integration into the target genomic locus Rosa26. Specific primers were designed to amplify the DNA fragment only if transgene integration into the desired locus was successful. Previously characterized cell lines with integrated inserts were used as positive controls. PCR of the experimental samples produced three bands of the expected length, which were subsequently re-amplified and sequenced using direct Sanger sequencing. Sequencing confirmed the presence of the target sequence, thus supporting the conclusion of successful transgene integration into the Rosa26 locus (see [link to original text]). Figure 6 ).

[0151] In addition, viral load and transgene expression levels were assessed in target organs. Figure 7 ).

[0152] The efficacy of the treatment was evaluated by analyzing the hormone profiles and weight gain of the experimental animals. Serum collected post-mortem from the heart was analyzed by UPLC-MS / MS. Figure 8 The figure shows the ratio of 11-deoxycorticosterone (DOC, the product) to progesterone (Prog, the precursor). Elevated DOC levels in the viral treatment group indicate increased 21-hydroxylase activity, an enzyme encoded by the Cyp21a1 gene.

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Claims

1. An expression vector comprising an AAV viral particle containing an adeno-associated virus (AAV) capsid protein, wherein the viral particle encapsulates its genome into a single-stranded DNA molecule, with AAVITR sequences located at both ends therebetween, and within the AAV ITR sequentially locating a eukaryotic elongation factor 1α (EF1α) promoter, a histidine tag sequence, a nuclear localization signal 5'-SV40 NLS, a codon-optimized sequence of the Cas9 gene sequence of Streptococcus pyogenes, and a nuclear localization signal 3'-SV40 NLS, wherein a synthesized polyA signal is used as a transcription terminator.

2. The expression vector according to claim 1, characterized in that, AAV ITR is AAV ITR serotype 2.

3. The expression vector according to claim 1, characterized in that, The optimized codon sequence of the Cas9 gene of Streptococcus pyogenes 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 comprising an AAV viral particle containing an AAV capsid protein, wherein the viral particle encapsulates its genome into a single-stranded DNA molecule, with AAV ITR sequences located at both ends therebetween, a eukaryotic polymerase III promoter U6 sequentially located within the AAV ITR, followed by an RNA guide expression cassette comprising an RNA guide sequence in crRNA format fused to a trackRNA sequence and complementary to a target locus, a region homologous to the human genomic locus AAVS1, i.e., a left homologous arm, a transgenic expression cassette operatively linked to an EFS-NS promoter of a protein-coding sequence of the hCYP21A2 gene that is 78-99% identical to the sequence of SEQ ID NO:3, a transcription terminator, followed by a polyA signal, and a region homologous to the human genomic locus AAVS1, i.e., a right homologous arm.

6. The expression vector according to claim 5, characterized in that, The crRNA guide sequence is a 20-nucleotide sequence.

7. The expression vector according to claim 5, characterized in that, AAV ITR is AAV ITR serotype 2.

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.

10. A combination for treating congenital adrenal hyperplasia, comprising the expression vector according to claim 1 and the expression vector according to claim 5.

11. The combination according to claim 10, characterized in that, It contains pharmaceutically acceptable excipients.

12. The combination according to claim 11, characterized in that, Pharmaceutically acceptable excipients are buffer solutions and / or saline solutions.

13. The combination according to claim 11, characterized in that, It represents a pharmaceutical composition.

14. The combination according to claim 11, characterized in that, It represents a pharmaceutical composition.

15. A method for treating congenital adrenal hyperplasia, comprising administering an effective amount of the combination of claim 10 to a mammal.

16. A kit for producing proteins in mammalian cells, comprising (i) the expression vector according to claim 1, and (ii) the expression vector according to claim 5.

17. The kit according to claim 16, characterized in that, AAV ITR is AAVITR serotype 2.

18. The kit according to claim 16, characterized in that, The optimized codon sequence of the Cas9 gene of Streptococcus pyogenes is SEQ ID NO:

5.

19. The kit according to claim 16, characterized in that, One of the vectors contains a sequence of SEQ ID NO:1 or SEQ ID NO:

2.

20. The kit according to claim 16, characterized in that, One of the vectors contains the sequence of SEQ ID NO:1, while the other contains the sequence of SEQ ID NO:

2.

21. The kit according to claim 16, characterized in that, The polyA signal is the polyA signal of the human β-globin gene.

22. The reagent kit according to claim 16, characterized in that, The crRNA guide RNA sequence is a 20-nucleotide sequence.

23. The reagent kit according to claim 16, characterized in that, It also contains buffer solutions and / or saline solutions.

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