KR20250135893A - Adeno-associated virus-based expression vectors - Google Patents

Abstract

This group of inventions relates to biotechnology and medicine, specifically to expression vectors for producing proteins in mammalian cells, combinations of said expression vectors for the treatment of congenital adrenal hyperplasia, as well as methods and medical uses contemplated for the use of said combinations, and in particular to a set of expression vectors based on adeno-associated virus (AAV) virions for the treatment of congenital adrenal hyperplasia.

Description

Adeno-associated virus-based expression vectors

The present invention relates to the fields of biotechnology and medicine, and more particularly to expression vectors for producing proteins in mammalian cells, combinations thereof for the treatment of congenital adrenal hyperplasia, and methods comprising the use of such combinations, as well as kits of expression vectors based on adeno-associated virus (AAV). These inventions can be applied to medicine, particularly to the treatment of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is a group of disorders associated with deficiencies of cortisol and/or aldosterone (steroid hormones). These deficiencies are caused by genetic mutations in the enzymes involved in the synthesis of cortisol and aldosterone from cholesterol. The enzymes may be completely absent or produced in insufficient amounts. The most well-known forms of CAH are 21-hydroxylase deficiency and 11β-hydroxylase deficiency. 21-hydroxylase (CYP21A2) deficiency can decrease cortisol and/or aldosterone levels and increase androgen levels. Excessive androgens cause virilization of the external genitalia in newborn females. Males with this genetic defect may not present with obvious clinical symptoms at birth, but they are prone to early puberty due to androgen excess. Affected individuals may experience premature growth of facial and body hair, acne, and menstrual irregularities, and it may also lead to infertility. 21-Hydroxylase deficiency is caused by mutations in the CYP21A2 gene. Patients may present with either a typical (more severe) or atypical form of the disease.

Typical forms may include:

1. Salt-wasting type - The most severe form, characterized by extremely low enzyme activity and resulting in both cortisol and aldosterone deficiency. Aldosterone deficiency causes hyponatremia and hyperkalemia. If left untreated, it can lead to acute adrenal insufficiency and shock.

2. Simple virilizing - enzyme activity is reduced, but sufficient to maintain normal or near-normal aldosterone synthesis.

The atypical form is the most common and presents as a mild disease variant. It occurs in approximately 1 in 1,500 newborns in the Caucasian population. In this form, enzyme activity is approximately 50%, avoiding significant sodium loss and serious complications. Androgen levels are only slightly elevated.

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

The treatment of choice for children with CAH is oral hydrocortisone tablets (evidence level 1+++). The use of hydrocortisone syrup and long-acting glucocorticoids is not recommended for children in active growth (evidence level 1++). All children with salt-wasting should be prescribed fludrocortisone, and infants and young children should additionally be prescribed dietary sodium chloride (evidence level 1++).

The cornerstone of treatment for all forms of congenital adrenal hyperplasia (CAH) is the administration of glucocorticoids. These drugs compensate for cortisol deficiency and thereby suppress excessive secretion of adrenocorticotropic hormone (ACTH). Consequently, by blocking specific enzymes, the adrenal glands reduce production of steroid hormones, which are synthesized in excess.

Several medications possess glucocorticoid activity, including prednisolone, cortisone, and dexamethasone. However, long-acting synthetic glucocorticoids (such as prednisolone and dexamethasone) negatively affect growth. For children whose growth plates have opened, especially in early childhood, hydrocortisone tablets are considered the most appropriate medication. The initial daily dose of hydrocortisone required to suppress ACTH secretion in infants and young children can reach up to 20 mg/m2. On average, for children over 1 year of age, the daily dose should be in the range of 10 to 15 mg/m2, administered in equal portions three times a day (e.g., at 7:00, 15:00 and 22:00).

In the salt-wasting form of CAH, mineralocorticoid therapy with fludrocortisone is typically required at a dose of 0.05 to 0.15 mg/day (given once or twice daily). In infants and young children, the need for mineralocorticoids is higher, and the dose can be up to 0.3 mg/day in three divided doses. In children without clinical signs of salt-wasting, subclinical mineralocorticoid deficiency can still be observed, criterion being elevated plasma renin levels. In these cases, fludrocortisone therapy is also indicated.

Gene therapy is considered a promising alternative to conservative treatment.

Adeno-associated virus (AAV) is a promising tool for human gene therapy. AAV can efficiently 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 not associated with any known pathology.

AAV vectors for gene therapy are known, and this addresses the challenge of developing means and methods for obtaining stable, high-yield AAV vectors in insect cells [see reference RU2457252 C2, July 27, 2012]. Therefore, currently known vectors are universal, i.e., suitable for use regardless of the specific disease. However, specific tasks often require specific solutions, necessitating the development of different optimal designs for each task.

AAV vectors containing gRNA and Cas9 are known, wherein the Cas9 molecule represents active or inactive Cas9 from Streptococcus pyogenes. These vectors are used for antitumor therapy, including for adrenal tumors [see reference RU2019133280 A, dated April 22, 2021]. However, this solution is not intended for the treatment of congenital adrenal cortex dysfunction. AAV vectors containing Cas9, NLS, and HA are also known [see reference KR1020150056539 A, dated May 26, 2015].

Furthermore, AAV vectors containing Cas9, SV40 NLS, HA [see document US20210054405 A, 25 February 2021] and AAV vectors containing guide RNA, ITR (including serotype 2) [see document WO2020082047 A1, 23 April 2020] are intended for adrenal gland therapy. However, none of these vectors address the treatment of congenital adrenocortical dysfunction.

Vectors known to be used to target the CYP21A2 gene in the human genome include guide RNA and Streptococcus pyogenes Cas9, NLS, and eukaryotic elongation factor EF1 alpha (see 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 not suitable for the treatment of congenital adrenocortical dysfunction.

AAV vector systems (combination) are known, wherein one of the vectors carries a guide RNA and the other carries a CAS. Simultaneously, the vectors also contain ITRs, promoters, and tags, and the CRISPR enzyme contains a C-terminal NLS and an N-terminal NLS.34.

The vectors are intended for use in ex vivo gene or genome editing, or in the composition of a therapeutic drug product for modifying a mammalian organism by manipulating a target sequence at a genomic locus of interest. The vectors may also be used in methods for treating or suppressing conditions in cells harboring defective nucleotide elements, trinucleotide repeats, or other repetitive nucleotide elements or nucleotide expansions [see document RU2016128069 A, January 17, 2018]. While this solution is closest to the present invention, it involves lentiviral vectors, which are not suitable for the treatment of congenital adrenocortical dysfunction.

The technical problem addressed by the group of claimed inventions lies in the development of genetic constructs comprising optimized sequences, including Streptococcus pyogenes Cas9, which ensure high levels of transgene expression, which can be used for the treatment of congenital adrenal hyperplasia.

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

1. Low immunogenicity;

2. Sufficiently easy to produce;

3. Nonspecific cleavage rate of 0.01%;

4. Extensive tropism toward different organizations;

5. High level of transgene expression: The number of viral genome copies per cell ranges from 1 to 100, expressed as 10 ^-4 to 10 ^-1 for expression of the GAPDH control gene.

The technical results are achieved as follows:

One invention of the present invention group provides a pAAV-nEFCas9 expression vector.

These vectors represent capsid proteins of viral particles of adeno-associated viruses, natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hul4, rh10, etc.) or serotypes obtained by synthetic biological methods (DJ/8, DJ, apc 32, etc.).

The genome packaged within the viral particles consists of a single-stranded DNA molecule flanked at both ends by AAV inverted terminal repeats (ITRs), preferably ITRs from serotype 2. These AAV ITRs have been well-studied, and their use provides reliable and expected results. However, since there is no prior art indicating that the use of ITRs from other AAV serotypes would produce inaccurate results, other AAV ITRs may be used. The ITRs within AAV are arranged sequentially: the promoter of the first eukaryotic elongation factor EF1 alpha. The selection of this particular promoter is the result of a series of experiments that have confirmed that it provides high levels of transgene expression in the vector construct according to the present invention. At the same time, this promoter is eukaryotic and not viral, and therefore is not methylated or repressed, which is an additional advantage of the present invention. Furthermore, this promoter is a constitutive promoter that functions in all cell types. Following the promoter is a histidine tag sequence (HA epitope tag (human influenza hemagglutinin epitope tag)), which facilitates detection of the transgene by Western blotting, 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 its proven efficiency in promoting nuclear import of proteins. Next is the codon-optimized sequence encoding the Cas9 enzyme from Streptococcus pyogenes.

Cas9 is a relatively small effector capable of inducing double-stranded breaks at target loci within the genome. The CRISPR/Cas9-based genome editing system is easy to use and design, and has an off-target cleavage rate of approximately 0.01%, depending on the requirement for selecting specific, easily synthesized RNA guides. This is a key challenge in achieving this specificity. Optimal RNA guides have been developed to maximize the benefits of the CRISPR/Cas9-based genome editing system. Improperly selected RNA guides can lead to a decrease in the efficiency of the system. Codon optimization (SEQ ID NO: 5) has been performed to enable more efficient synthesis of bacterial proteins in human cells, contributing to the aforementioned beneficial effects. However, other variations of codon optimization are also possible, and the variation used in this development represents a major possibility for achieving the effects of the present invention by optimizing this sequence. Following the codon-optimized sequence is a second nuclear localization signal, the 3'-SV40 NLS. A synthetic polyA signal is used as a transcription terminator.

Another invention among the group relates to the expression vector AAV-hCYP21A2.

The viral genome packaged within the particles preferably comprises a single-stranded DNA molecule flanked by AAV serotype 2 inverted terminal repeats (ITRs). These ITRs are well characterized and are known to produce consistent and reliable results. However, the use of ITRs from other AAV serotypes is not ruled out, as prior art does not suggest that these ITRs would lead to inaccurate results.

Within the vector, a eukaryotic RNA polymerase III promoter, U6, is located. The U6 promoter has been shown to drive high levels of transgene transcription in the vector constructs described herein. The U6 promoter is eukaryotic, not viral, and thus may not be repressed in human cells, while its relatively small size allows the RNA guide transcription cassette to be packaged into the AAV genome. Following the U6 promoter is a guide RNA sequence in crRNA format. The guide RNA sequence is a unique 20 nucleotide sequence complementary to the target locus, fused to a trackRNA sequence (scaffold). A region homologous to the human genome locus AAVS1 (the left homologous arm) is adjacent to the guide RNA expression cassette. These loci were chosen based on experimental data confirming that integration at these sites does not negatively affect other genomic functions and thus provides a safe integration. Transgenes integrated into these loci are efficiently transcribed in any cell type. Transgene expression cassette: EFS-NS promoter. This promoter exhibits high levels of transgene expression. This promoter is eukaryotic, not viral, and therefore unlikely to be methylated or repressed. Furthermore, this promoter is a constitutive promoter that operates in all cell types. Under this promoter, the protein-coding codon-optimized sequence of the hCYP21A2 gene is located. Codon optimization can increase the efficiency of gene translation, and optimization was performed relative to the corresponding control sequence NG_007941.3. The polyA signal (human β-globin polyadenylation signal) of the human β-globin gene was used as a transcription terminator. The polyA signal is followed by a region homologous to the human genomic locus AAVS1 (right homologous arm).

Another invention within the present invention group is a combination of pAAV-nEFCas9 and AAV-hCYP21A2 expression vectors together with a pharmaceutically acceptable excipient. In this case, the combination includes a buffer and/or saline solution as the pharmaceutically acceptable excipient. Non-limiting examples of buffers that can be used in the present solution include the following: phosphate buffer, phosphate-citrate buffer, Tris buffer, carbonate buffer, bicarbonate buffer, borate buffer, acetate buffer, glycerol buffer, phosphate-glycerol buffer, Tris-glycerol buffer, and citrate buffer.

Another invention within the present invention group is a method for treating congenital adrenal hyperplasia, comprising administering to a mammalian subject an effective amount of a combination of the aforementioned expression vectors. Another invention within the present invention group provides a kit of expression vectors, wherein one of the vectors is a pAAV-nEFCas9 vector and the other vector is an AAV-hCYP21A2 vector. The kit may further comprise a buffer and/or physiological saline. Non-limiting examples of buffers that may be employed include: phosphate, phosphate-citrate, Tris, carbonate, bicarbonate, borate, acetate, glycerol, phosphate-glycerol, Tris-glycerol, and citrate buffers.

The present invention is illustrated with the following exemplary materials.
Figure 1 is a schematic diagram of the layered arrangement of an iodixanol solution in a centrifuge beaker. A 40% fraction is aspirated from the QuickSeal tubes after centrifugation by first puncturing the top of the tube with a separate needle to allow air to pass through, and then the desired fraction is aspirated with another syringe needle with the needle hole pointing upward and the needle tip positioned horizontally approximately 2 mm below the 40-60% interphase. In this manner, a 40% gradient is aspirated without disturbing the interphase of the 25% layer.
Figure 2 is a schematic diagram of AAV-hCYP21A2.
Figure 3 is a schematic diagram of pAAV-nEFCas9.
Figure 4 is a schematic diagram to confirm restoration of steroidogenesis when integrating a normal copy of the CYP21 gene into the genome of an organism mutant for this gene.
Figure 5 is a photograph of a sample after immunofluorescent staining of EGFP in the adrenal glands of CYP21A1-/- mice 2 weeks after vector administration.
Figure 6 shows the results of studies confirming donor integration into the Rosa26 target locus in the adrenal genome of mice. A) Electrophoresis of target amplicons in an agarose gel ("C+" positive control, "C-" negative control). B) Chromatograms of Sanger sequencing of amplicons from (A).
Figure 7 is a diagram showing the viral load and transgene expression levels in target organs.
Figures 8a to 8d are charts showing body weight gain and steroid hormone levels in experimental animals.
Figure 9 shows representative images of uninjected CYP21A1+/+ mice (control) and CYP21A1-/- mice injected with AAVDJ-CYP21A1-01, AAVDJ-SpCas9 + AAVDJ-CYP21A1-01, and PBS 16 weeks after vector injection.

One invention of the present group provides an expression vector based on adeno-associated viruses, comprising capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 8, 9hul4, rhl0, etc.) or serotypes obtained by synthetic biological methods (DJ/8, DJ, anc32, etc.), wherein the following elements are sequentially arranged: a eukaryotic elongation factor EF1 alpha promoter, a histidine tag (HA epitope tag from human influenza hemagglutinin), a 5'-SV40 nuclear localization signal (NLS), a codon-optimized sequence of the Cas9 gene from Streptococcus pyogenes, a 3'-SV40 NLS, and a synthetic polyA signal as a transcription terminator. One exemplary embodiment of the present invention is SEQ ID NO: 1.

Another invention of the present invention group provides expression vectors based on adeno-associated viruses comprising capsid proteins of natural serotypes (1, 2, 3, 4, 5, 7, 7, 8, 9hul4, rhl0, etc.) or serotypes obtained by synthetic biological methods (DJ/8, DJ, anc32, etc.).

The genome packaged within the vector is a single-stranded DNA molecule flanked by ITRs of AAV serotype 2, in which the following elements are arranged sequentially: a eukaryotic RNA polymerase III promoter (U6), a guide RNA in the form of a crRNA (a unique 20-nucleotide sequence complementary to the target locus and fused to a trackRNA (scaffold), a region homologous to the AAVS1 locus of the human genome following the guide RNA (left homology arm), and a transgene expression cassette comprising the EFS-NS promoter (under the control of the protein-coding sequence of the hCYP21A2 gene corresponding to the reference sequence NG_007941.3). The human β-globin polyadenylation signal was used as a transcription terminator, followed by a right homology arm to AAVS1. One exemplary embodiment is SEQ ID NO: 2.

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

SEQ ID NO: 4 represents a variant optimization sequence of SEQ ID NO: 3.

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

The present invention also relates to a method for treating congenital adrenal hyperplasia, comprising administering to a mammal an effective amount of a combination of the above vectors.

Within the scope of the present invention, the term "therapeutically effective amount" refers to an amount of a vector combination that has a therapeutic effect on CAH, or that alleviates, improves, or eliminates one or more symptoms of CAH. The term "therapeutically effective amount" refers to a quantitative content of a vector combination sufficient to provide the desired therapeutic effect. Therefore, when preparing a pharmaceutical composition in the form of a unit dose, an effective amount is considered. In this case, the dosage of the drug to a patient may be adjusted according to the therapeutic efficacy and bioavailability of the active ingredients in the body, their metabolic rate, and the rate of excretion from the body, as well as age, sex, and stage of the disease.

The recommended therapeutic dose is a single administration of 2 Х 10 11 to 2 Х 10 14 viral genomes per kg body weight, equivalent to 2 ml/kg, diluted with injection buffer or 9 mg/ ^ml (0.9 ^% ) sodium chloride solution and administered via intravenous infusion.

The recommended route of administration is a single intravenous infusion.

Since gene therapy for congenital adrenal hyperplasia is mediated by mutations in the hCYP21A2 gene, treatment can be achieved by exogenous expression of the hCYP21A2 gene from a vector genome that infects adrenal cells and persists as episomal DNA in the nucleus. In this case, double-strand breaks are achieved in the genomes of infected cells through expression of an RNA guide specific for the sequences of the AAVS1 locus, adeno-associated virus integration site 1, PPPIR12C protein phosphatase 1 regulatory subunit 12C, NC_000019.10, and through expression of the Cas9 protein capable of introducing double-strand breaks in DNA at the site of homologous binding of the RNA guide to the target. Integration occurs due to the presence of a region of homology to the AAVS1 locus in the AAV-hCYP21A2 vector. As a result of cleavage, homologous recombination, and transgene expression, hCYP21A2 gene expression is achieved and steroidogenesis in the adrenal cortex is restored.

A more detailed description of the claimed invention is provided below.

The present invention can be modified in various ways that will be apparent to those skilled in the art from this description. Such modifications do not limit the scope of the claims.

Viral particles were generated using plasmid constructs carrying nucleotide sequences of vector genomes as well as sequences necessary for plasmid replication and selection in bacterial systems. These plasmids were obtained by molecular cloning and gene synthesis methods.

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

Viral vectors were assembled in specialized cell lines based on adherent or suspension cell systems/HEK 293 cells.

Example 1

Plasmid extraction and cleaning

E. coli cells were thawed and stored at -80°C.

Thawing was performed on solid media. For this, frozen cells were added to 400 μl of liquid LB medium (pipetted 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 placed in a 250 ml flask. A single colony was inoculated onto the tip of a pipette, and the E. coli suspension was cultured at 37°C in a thermostated shaker with constant agitation at 170 rpm for 16 to 20 hours.

Next, plasmid DNA was isolated using the Plasmid Midiprep 2.0 kit (Eurogen) according to the manufacturer's protocol. The number of columns was set at a ratio of one column per 1 g of cell pellet (column capacity 500 μg of DNA). Each column was eluted twice with 4 ml of water.

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

The following reactions were performed for each sample: digestion with each of the restriction enzymes listed in Table 1 individually, digestion with all restriction enzymes combined in one tube, and a control reaction (K-) involving no restriction enzymes.

Preparation of the mixture:

10XBuf - 2.5 μl;

Restriction enzymes - 0.5 μl each;

Plasmid DNA - 600 ng;

Up to 25 μl of water.

Incubate at 37°C for 1 hour and visualize on a 1% agarose gel.

[Table 1]

Number of viral genomes per cell - 1 to 100.

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

Example 2

Preparation of HEK 293 cell culture

All volumes of media, solutions, etc. were provided per 15 cm culture dish.

Culture media for cell mass expansion (GM) and transfection (TM) were prepared.

To prepare GM, 50 ml of FBS (Gibco), 5 ml of 100 x GlutaMax (Gibco) and 500 μl of 1000 x gentamicin antibiotic (PanEco) were added to 450 ml of DMEM medium containing 4.5 g/L glucose.

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

Versene and 0.25% trypsin solutions were dispensed in 50 ml aliquots. Versene aliquots were stored at room temperature, while 0.25% trypsin aliquots were stored at -20°C. Before starting experiments on cells, the trypsin solution was brought to room temperature, and the GM/TM medium was placed in a water bath at +37°C.

Cell line passaging was performed. The number of passages of the cell line did not exceed 12.

HEK 293 cell cultures were thawed. Before thawing the cryovials, 25 ml of GM were pre-warmed in a +37°C water bath. The cryovials were kept in a +37°C water bath until completely thawed. Immediately after thawing, the entire contents of the cryovials were transferred to a 15-ml tube, and 10 ml of pre-warmed GM was added. The suspension was centrifuged at 230 g for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 1 ml of GM, transferred to a T75 culture flask, and supplemented with 14 ml of GM. The flasks were cultured in a _CO2 incubator at 5% _CO2 and 37°C until the cells reached 70% confluency.

HEK 293 cell cultures were frozen.

Prior to freezing the cell bank, cells were tested for mycoplasma contamination using the MycoReport protocol (Evrogen).

All media was removed from the dish, 5 ml of Versene solution was added, and the dish was incubated for 1 minute in a laminar flow hood. The Versene solution was removed, 3 ml of 0.25% trypsin solution was added, and the dish was incubated for 3 minutes in a laminar flow hood. Then, 3 ml of GM was added, and the cells were carefully and thoroughly resuspended. A 10 μl sample of the cell suspension was transferred to a 0.2 ml tube. The remaining cells were transferred to a 15 ml tube.

Cell counts 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, mixed, and 10 μl of the resulting suspension was applied to a counting slide for measurement.

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

The required number of cryovials was counted so that each cryovial contained 5,000,000 cells in 1 ml of freezing solution. Each cryovial was signed: cell line name, number of cells, date, and operator.

For one cryovial, the freezing solution was prepared as follows: 700 μl of FBS (Gibco), 200 μl of DMEM (PanEco) containing 4.5 g/L glucose and 100 μl of DMSO (PanEco) were used.

The cell pellet from a 15-mL tube centrifuged at 230 × g for 3 min was resuspended in freezing medium. The suspension was dispensed into 1-mL cryovials. The cryovials were incubated in a -80°C freezer (Kelvinator) for 24 h and then transferred to liquid nitrogen at -196°C for long-term storage.

Subculture of HEK 293 cells was performed.

The entire medium was removed, and 5 ml of Versene solution was added to the dish. This was incubated in a laminar flow hood for 1 minute, and then the Versene solution was removed.

Next, 3 ml of 0.25% trypsin was added to the dish and 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 tube.

Cell counts and viability were determined using a TC-20 automated cell counter (BioRad): 10 μl of well-mixed cell suspension was mixed 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.

6,000,000 cells were transferred to a new plate, 24 ml of GM was added, and culture was performed at +37°C, 5% _CO2 until the next subculture.

Example 3

Transfection of HEK293 cell cultures.

The transfection mixture consisted of lactate buffer, linear polyethyleneimine (PEI), 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. The pH was adjusted to 4 with 1 N sodium hydroxide solution. The resulting solution was diluted to 300 ml with 150 mM sodium chloride, sterilized through a 0.2 μm filter, and stored at 4°C.

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

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

Calculating the amount of plasmids and polyethyleneimine (PEI) required for transfection:

For transfection of one 15 cm dish, 38 μg of plasmid DNA was used with a plasmid copy ratio of helper:packaging:transfer = 1:1:1.

Cell preparation for transfection:

One day before transfection, cells were reseeded onto new plates at a density of 10,000,000 cells per plate. 24 mL of GM medium was added, and cells were cultured at +37°C, 5% CO ₂ .

Transfection of HEK293 cell cultures:

DNA and PEI mixtures were prepared according to [Table 2].

[Table 2]

Plan for preparing the calculated transfection mixture for 40 culture dishes

80 ml of the DNA-1-PEI 25 mixture was added to 1 L of TM medium and mixed thoroughly. All GM medium was removed from the culture dishes. Then, 27 ml of the TM-DNA-PEI mixture was added to each dish. The contents were mixed carefully. The dishes were incubated at +37°C, 5% CO ₂ for 72 h.

Transfection efficiency reached 95 to 98%.

cell lysis

A lysis buffer was prepared containing the following: 150 mM NaCl, 50 mM Tris-HCl, 1 mM MgCl ₂ , pH 8.5, 0.5% Triton X-100, and 50 U/mL Benzonase. The solution was sterilized by filtration rather than autoclaving and stored at +4°C.

A 25-mL serological pipette was used to collect all the medium from each dish, which was also used to wash the cells from the surface. The resulting cell suspension was transferred to a 50-mL tube, and this process was repeated for all dishes. The tubes containing the cell suspensions were centrifuged at 400 × g for 5 minutes. The supernatant was collected as needed and stored at -80°C.

Whole cell pellets from 40 plates were resuspended in 60 ml of lysis buffer and incubated at +37°C for 1 h. The suspension was then centrifuged at 4000 × g for 15 min, and the clarified supernatant was transferred to a clean tube.

Example 4

AAV isolation and purification

Preparation of buffers: 1 M NaCl/PBS-MK buffer and 1× PBS-MK buffer

To prepare 1 M NaCl/PBS-MK buffer, 5.84 g of NaCl, 26.3 mg of MgCl ₂ (corresponding to 56.0 mg of MgCl ₂ ㆍ6H ₂ O), and 14.91 mg of 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.

To prepare 1× PBS-MK buffer, 26.3 mg of MgCl ₂ (equivalent to 56.0 mg of MgCl ₂ ㆍ6H ₂ O) and 14.91 mg of 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.

In each ultracentrifugation step, only iodixanol solutions prepared according to [Table 3] were used.

[Table 3]

Calculation for the preparation of iodixanol for two Two Quick-Seal tubes (39 ml each)

Ultracentrifugation

Layers of iodixanol solutions were added to centrifuge tubes in the order specified in [Table 4] using a 10 ml syringe and a Seldinger-type needle.

[Table 4]

The gradient was applied from the upper layers to avoid disturbing the integrity of the layers.

When filling QuickSeal tubes, completely fill the tubes with cell lysate, leaving no air bubbles at the top. The tubes were equilibrated with PBS to within 0.0005 g using an analytical balance. Ultracentrifugation was performed at 360,000 × g (59,000 rpm in a Beckman Coulter Ti 70 rotor) for 1 h 30 min at +10°C.

After centrifugation, the QuickSeal tube was first punctured at the top with a separate needle to allow air to enter. Then, as shown in Figure 1, a second needle connected to a syringe was used to carefully withdraw the 40% iodixanol fraction. The needle hole was pointed upward, and the needle tip was positioned horizontally approximately 2 mm below the 40% to 60% interface. The 40% gradient layer was aspirated without disturbing the 25% layer interface.

The collected fractions were transferred to 50 ml Falcon tubes.

Ultrafiltration

The 40% iodixanol fraction collected after ultracentrifugation was diluted with a five-fold volume of PBS/Pluronic F68 (0.001%) solution and filtered through a 0.22 μm membrane filter. After pre-rinsing by adding 3 ml of H ₂ O to an Amicon Ultra-15 filter unit, centrifugation was performed at 2,000 × g for 1 min. All residual liquid was then removed from both the tube and the filter unit. Next, 3 ml of PBS/Pluronic F68 (0.001%) solution was added and centrifuged again at 2,000 × g for 1 min to completely remove the liquid.

Then, 15 ml of the diluted virus solution was placed in an Amicon Ultra-15 unit and centrifuged at 2,500 × g for 10 min using a swinging-bucket rotor. The flow-through was discarded, and the filter was refilled with the remaining viral vector solution until the entire volume was processed. The membrane was thoroughly washed with the virus solution using a 100-1000 μl pipette. Centrifugation was repeated at 2,500 × g for 10 min.

After centrifugation, the viral vector suspension (approximately 500 μl) remained in the upper compartment of the concentrator. Centrifugation was repeated for an additional 3 minutes. Once the entire virus solution passed through the membrane, the concentrated virus was washed twice with room temperature PBS/Pluronic F68 (0.001%). Approximately 500 μl of the virus suspension remained in the filter cassette.

The membrane was rinsed thoroughly, and the virus was dispensed into 0.2 ml tubes in 20 μl aliquots, with 5 μl reserved for titer determination.

AAV-CYP21: 7.80 × 10 ¹² virus particles per ml

AAV-Cas9: 1.93 × 10 ¹² virus particles per ml

Example 5

Restoration of steroidogenesis by integration of a wild-type copy of the CYP21 gene into the genome of an organism carrying a mutation in this gene was assessed according to the scheme shown in Figure 4. Five-week-old CD-1-C57Bl/10SnSlc-H-2aw18 mice were injected intravenously via the tail vein with 3 × 10 ¹¹ viral particles per dose of two recombinant adeno-associated virus vectors of the DJ serotype, AAV-Cas9 and AAV-Cyp. Animals injected once with phosphate-buffered saline (PBS) served as controls. Mice were maintained for 2, 4, 8, 16, and 30 weeks post-injection under standard housing conditions. After the indicated time points, animals were euthanized, and target organs and tissues were harvested for analysis.

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 administered vectors. Figure 5 shows staining for GFP protein in organs of treated mice.

DNA was also extracted from adrenal samples of experimental animals and analyzed to confirm integration of the transgene into the Rosa26 target genomic locus. Specific primers were designed to amplify DNA fragments only in cases of successful transgene integration into the desired locus. A previously identified cell line with integrated inserts was used as a positive control. PCR of the experimental samples yielded 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, supporting successful integration of the transgene into the Rosa26 locus (see Figure 6).

Additionally, viral load and transgene expression levels in target organs were assessed (Fig. 7).

Therapeutic efficacy was assessed by analyzing the hormonal profiles and body weight gain of experimental animals. Postmortem serum collected from the heart was analyzed by UPLC-MS/MS. The ratio of 11-deoxycorticosterone (DOC, product) to progesterone (Prog, precursor) is shown in Figure 8. The increased DOC levels in the virus-treated group indicate enhanced activity of 21-hydroxylase, an enzyme encoded by the Cyp21a1 gene.

Attach an electronic file of the sequence list

Claims (23)

An expression vector, which is an adeno-associated virus (AAV) viral particle comprising AAV capsid proteins, wherein the viral particle encapsulates a single-stranded DNA molecule as its genome, and AAV ITR sequences are located at both ends thereof, and within the AAV ITR, a eukaryotic elongation factor 1 alpha (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, a nuclear localization signal 3'-SV40 NLS, and a synthetic polyA signal used as a transcription terminator are sequentially arranged. An expression vector according to claim 1, characterized in that the AAV ITR is AAV ITR serotype 2. An expression vector according to claim 1, characterized in that the codon-optimized sequence of the Cas9 gene of Streptococcus pyogenes is SEQ ID NO: 5. An expression vector according to claim 1, characterized in that the vector has a sequence of SEQ ID NO: 1. An expression vector, which is an AAV viral particle comprising AAV capsid proteins, wherein the viral particle encapsidates a single-stranded DNA molecule as its genome, wherein AAV ITR sequences are located at both ends thereof, and sequentially within the AAV ITR, a eukaryotic polymerase III promoter U6 is located, followed by an RNA guide expression cassette comprising an RNA guide sequence in the form of a crRNA, which is complementary to a target locus and is fused to a trackRNA sequence, and wherein the expression vector comprises: a region homologous to the human genomic locus AAVS1 - left homology arm, a transgene expression cassette: an EFS-NS promoter operably linked to a protein-coding sequence of the hCYP21A2 gene, which is 78 to 99% identical to the sequence of SEQ ID NO: 3, a polyA signal transcription terminator, and a region homologous to the locus of the human genomic locus AAVS1 - right homology arm. An expression vector according to claim 5, characterized in that the RNA guide sequence in the form of crRNA is a sequence of 20 nucleotides. An expression vector according to claim 5, characterized in that the AAV ITR is AAV ITR serotype 2. An expression vector according to claim 5, characterized in that the polyA signal is a polyA signal of a human β-globin gene. An expression vector according to claim 5, characterized in that the vector has a sequence of SEQ ID NO: 2. A combination for the treatment of congenital adrenal hyperplasia, comprising an expression vector according to claim 1 and an expression vector according to claim 5. A combination according to claim 10, characterized in that it comprises a pharmaceutically acceptable excipient. A combination according to claim 11, characterized in that the pharmaceutically acceptable excipients are a buffer and/or physiological saline. In claim 11, a combination characterized in that it represents a pharmaceutical composition. In claim 11, a combination characterized in that it represents a pharmaceutical composition. A method for treating congenital adrenal hyperplasia, comprising administering to a mammal an effective amount of a combination according to claim 10. A kit for producing a protein in mammalian cells, comprising (i) an expression vector according to claim 1 and (ii) an expression vector according to claim 5. A kit according to claim 16, characterized in that the AAV ITR is AAV ITR serotype 2. A kit according to claim 16, characterized in that the codon-optimized sequence of the Cas9 gene of Streptococcus pyogenes is SEQ ID NO: 5. A kit according to claim 16, characterized in that one of the vectors comprises a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A kit according to claim 16, characterized in that one of the vectors comprises the sequence of SEQ ID NO: 1 and the other vector comprises the sequence of SEQ ID NO: 2. A kit according to claim 16, characterized in that the polyA signal is a polyA signal of the human β-globin gene. A kit according to claim 16, characterized in that the crRNA guide RNA sequence is a 20 nucleotide sequence. A kit characterized in that, in claim 16, it additionally comprises a buffer solution and/or physiological saline solution.