RU2785621C1 - Codon-optimised haipl1-encoding nucleotide sequence and expression vector containing said sequence - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology and molecular biology, to genetic engineering. Described is a codon-optimised nucleic acid with a nucleotide sequence Seq Id No.: 2, encoding human aipl1. Presented is an expression vector containing said nucleic acid.

EFFECT: possibility of increasing the level of hAIPL1 mRNA by at least 15%, and the level of heterologous protein product by at least 29%; invention can be used for gene replacement therapy of type 4 Leber amaurosis.

9 cl, 10 dwg, 2 tbl, 5 ex

Description

Technical field

The invention relates to the field of biotechnology and molecular biology, in particular genetic engineering, namely to a codon-optimized hAIPL1 gene sequence.

State of the art

Monogenic diseases of the retina are a group of about 300 isolated or syndromic diseases that are heterogeneous in clinical manifestations and are united by similar development mechanisms - damage to one of the links of phototransduction or damage to the integrity of photoreceptor/auxiliary cells. The frequency of occurrence of monogenic retinal diseases ranges from 1:5000 (Stargardt's disease) to 1:100000 (Usher's syndrome, achromatopsia). To date, there is no pathogenetically directed effective treatment of this group of diseases in wide clinical practice, however, promising methods of treatment are at different stages of clinical and preclinical studies.

Leber Congenital Amaurosis (LCA), the fastest and most severe form of hereditary retinal dystrophy, is usually inherited in an autosomal recessive manner and is characterized by early vision loss, nystagmus, and an absent or severely reduced electroretinogram (ERG) [den Hollander AI, Roepman R, Koenekoop RK, Cremers FP. Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res. 2008 Jun; 27(4):391-419]. Occurs with a frequency of approximately 1:200,000 people. To date, mutations in 26 different genes encoding proteins that play an important role in the development and physiological function of the retina cause clinically different types of LCA [RetNet https://sph.uth.edu/retnet, accessed 06/17/2021]. Among them, mutations in the aryl hydrocarbon receptor interacting protein like 1 (AIPL1) gene are associated with LCA type IV (LCA4). Although it accounts for only 5-10% of LCA cases, the clinical phenotype caused by AIPL1 deficiency is on the spectrum of severe LCA. Such severe symptoms are caused by extensive and irreversible degeneration of rod and cone photoreceptors critical for visual phototransduction, in which AIPL1 plays an indirect but important role in maintaining functional integrity [Yadav RP, Artemyev NO. AIPL1: A specialized chaperone for the phototransduction effector. cell signal. Dec 2017; 40:183-189].

AIPL1 expression occurs exclusively in retinal and pineal photoreceptors [van der Spuy J, Chappie JP, Clark BJ, Luthert PJ, Sethi CS, Cheetham ME. The Leber congenital amaurosis gene product AIPL1 is localized exclusively in rod photoreceptors of the adult human retina. Hum Mol Genet. 2002 Apr 1; 11(7):823-31]. In the retina, AIPL1 acts as a specialized co-chaperone of the cyclic nucleotide phosphodiesterase of the sixth family (PDE6), an important enzyme effector in the phototransduction pathway [Sacristan-Reviriego A, van der Spuy J. The Leber Congenital Amaurosis-Linked Protein AIPL1 and Its Critical Role in Photoreceptors. Adv Exp Med Biol. 2018; 1074:381-386]. After stimulation with light, activated PDE6 hydrolyzes cyclic GMP (cGMP), triggering the closure of cGMP-dependent Ca ²⁺ ion channels and the propagation of a "light" electrical signal due to hyperpolarization of the plasma membrane [Yadav RP, Artemyev NO. AIPL1: A specialized chaperone for the phototransduction effector. cell signal. Dec 2017; 40:183-189]. Stabilization of PDE6 is provided by a chaperone heterocomplex, including HSP90 and the related PDE6-specific co-chaperone AIPL1 [Hidalgo-de-Quintana J, Evans RJ, Cheetham ME, van der Spuy J. The Leber congenital amaurosis protein AIPL1 functions as part of a chaperone heterocomplex. Invest Ophthalmol Vis Sci. 2008 Jun; 49(7):2878-87], which are required for stable assembly of the PDE6 holoenzyme [Kolandaivelu S, Huang J, Hurley JB, Ramamurthy V. AIPL1, a protein associated with childhood blindness, interacts with alpha-subunit of rod phosphodiesterase (PDE6) and is essential for its proper assembly. J Biol Chem. 2009 Nov 6; 284(45):30853-61].

AIPL1 mutations affect the functional domains of the translated protein, disrupting the interaction of AIPL1 with isoprenylated PDE6 or HSP90, which prevents the assembly of the PDE6 chaperone heterocomplex [Sacristan-Reviriego A, van der Spuy J. The Leber Congenital Amaurosis-Linked Protein AIPL1 and Its Critical Role in Photoreceptors. Adv Exp Med Biol. 2018; 1074:381-386]. In AIpl1 knockout mice and in hypomorphic mice, loss of AIPL1 function causes misassembly of the PDE6 holoenzyme, causing destabilization and rapid proteasome degradation of PDE6 subunits. Consequently, there is a rapid degeneration of rod photoreceptors due to an increase in intracellular cGMP, which leads to a prolonged opening of cyclic nucleotide-gated channels and an excessive influx of Ca ²⁺ [Wang T, Tsang SH, Chen J. Two pathways of rod photo cell death induced by elevated cGMP. Hum Mol Genet. 2017 Jun 15; 26(12):2299-2306].

Given the unique features of the eye (accessible location, small isolated structure, immune privilege), hereditary retinal dystrophies are one of the most attractive targets for gene therapy. In recent years, the number of clinical trials of gene therapy for hereditary retinal dystrophies has increased, culminating in the registration of the first gene therapy for the treatment - voretigene neparvovec-rzyl (Luxturna) - a recombinant adeno-associated virus (AAV) expressing the RPE65 gene for the treatment of Leber's amaurosis type II (LCA II ) [Auricchio F, Scavone C, Cimmaruta D, Di Mauro G, Capuano A, Sportiello L, Rafaniello C. Drugs approved for the treatment of multiple sclerosis: review of their safety profile. Expert Opin Drug Saf. Dec 2017; 16(12): 1359-1371]. AAVs are promising and most successful vectors for the delivery of therapeutic genes and the standard choice for photoreceptor transduction. Several naturally occurring or engineered AAV serotypes have been shown to efficiently transduce photoreceptors in animal models [Day TP, Byrne LC, Schaffer DV, Flannery JG. Advances in AAV vector development for gene therapy in the retina. Adv Exp Med Biol. 2014;801:687-93].

Patent application US 20030022165 A1 discloses a normal and mutant AIPL1 gene and its role in the development of LCA4, as well as a method of treating retinal disease, including, in particular, the introduction of an animal with an effective amount of a wild-type polynucleotide sequence of the AIPL1 gene.

In the article: Tan MH, Smith AJ, Pawlyk B, et al. Gene therapy for retinitis pigmentosa and Leber congenital amaurosis caused by defects in AIPL1: effective rescue of mouse models of partial and complete Aipl1 deficiency using AAV2/2 and AAV2/8 vectors. Hum Mol Genet. 2009; 18(12):2099-2114 disclose recombinant adeno-associated viruses AAV2/2 and AAV2/8 containing DNA sequences encoding murine Aipl1 (mAipl1) under the control of the cytomegalovirus (CMV) promoter. Gene therapy with subretinal injection of AAV2/2-CMV-mAIPL1 and AAV2/8-CMV-mAIPL1 reduces photoreceptor degeneration in hypomorphic AIPL11 ( ^hypo/hypo ) mice with slower retinal degeneration, PDE6 levels increase and localization is restored in outer segments of photoreceptors (OS). Subretinal injection of AAV2/8-CMV-mAipl1 at postnatal day 12 (P12) prevents photoreceptor degeneration in the most severe AIPL1 ^-/- knockout model, while maintaining the thickness of the outer nuclear layer of the retina (ONL) 3 months after injection . The wild-type murine AIPL11 gene was used in the work.

In the work: Sun X, Pawlyk V, Xu X, Liu X, Bulgakov OV, Adamian M, Sandberg MA, Khani SC, Tan MH, Smith AJ, Ali RR, Li T. Gene therapy with a promoter targeting both rods and cones rescues retinal degeneration caused by AIPL1 mutations. Gene Ther. Jan 2010; 17(1):117-31 discloses an AAV8 adeno-associated virus containing a DNA sequence encoding human Aipl1 (hAipl) under the control of a photoreceptor-specific rhodopsinkinase (RK) promoter. Injection of AAV8-RK-hAIPL1 into Aipl1 ^−/− mice at postnatal day 9 (P9) causes significant accumulation of rod-cone PDE6 in the outer photoreceptor segments, delaying photoreceptor degeneration for at least 5 months post-treatment. A formulation containing AAV8-RK-hAIPL1 is currently manufactured by MeiraGTx for compassionate use under license from MHRA Specials in the UK. We used the wild-type mouse and human Aipl1 gene, as well as the tissue-specific RK promoter, whose activity is controlled by transcription factors of the cell and can vary significantly depending on the physiological state of the cell.

In: Ku CA, Chiodo VA, Boye SL, Goldberg AF, Li T, Hauswirth WW, Ramamurthy V. Gene therapy using self-complementary Y733F capsid mutant AAV2/8 restores vision in a model of early onset Leber congenital amaurosis. Hum Mol Genet. 2011 Dec 1; 20(23):4569-81 disclosed an engineered scAAV2/8 mutated Y733F (scAAV-Y733) containing an hAIPL1 sequence that triggers early expression and upregulates hAIPL1 expression levels resulting in improved photoreceptor retention and retinal function in Aipl1 ^-/- mice. . Subretinal injection of Aipl1 ^−/− mice on the second day of postnatal development (P2) with the scAAV-Y733-RKhAIPL1 construct restores PDE6 expression in rods and cones within 2 months after treatment, improving photoreceptor ultrastructure. We used the wild-type human gene Aipl1, as well as the tissue-specific RK promoter, whose activity is controlled by transcription factors of the cell and can vary significantly depending on the physiological state of the cell. The authors use a self-complementary format of the AAV genome, which may have an advantage over the single-stranded AAV genome in terms of expression level, but imposes restrictions on the possibility of including additional regulatory elements, promoters in the AAV expression cassette, if such a need arises after obtaining the results of testing the drug on cells and animals. models.

The technical problem to be solved by the present invention is to solve at least one of the above problems in the prior art.

The essence of the invention

The technical solution is to use the features described in the claims.

One of the possible technical problems to which the present invention can be directed was the creation of a recombinant hAIPL1 gene, which makes it possible to increase gene expression and increase the level of the aipl1 protein in cells when introduced into an expression vector. The invention can be used in gene therapy of ophthalmic diseases.

The problem can be solved by optimizing codons aimed at increasing the stability of messenger RNA and increasing the level of the aipl1 protein in cells after the introduction of the gene in the expression vector. The technical result can be achieved by creating a codon-optimized hAIPL1opt gene sequence presented in Seq Id No: 2.

In one embodiment, the present invention provides a nucleotide sequence Seq Id No.: 2 encoding a human aipl1 protein codon-optimized based on the wild-type hAIPL1 gene sequence (NP_055151.3, Gene ID: 23746, Seq Id No.: 1).

The term "codon optimized" means a nucleotide sequence in which one or more codons have been replaced with synonymous ones without changing the sequence of the protein synthesized from the template of this sequence.

In another embodiment, the invention provides an expression vector containing the nucleotide sequence of Seq Id No.:2. The term "vector" refers to a nucleic acid molecule used to transfer genetic material into a cell. The term "expression vector" means a vector containing a promoter and other regulatory sequences that provide efficient transcription of a recombinant gene, followed by translation of mRNA and the formation of a recombinant protein. The plasmid and expression vectors used below are used by way of example and do not limit the scope of the present invention. The term "gene expression" refers to the transformation of hereditary information, encrypted in the nucleotide sequence of a gene, into a functional product of RNA or protein.

In another embodiment, the invention provides a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 2, the following items:

- origin of replication site ori;

- left inverted terminal repeat ITR;

- CMV enhancer of the cytomegalovirus promoter;

- cytomegalovirus promoter CMV promoter;

- the intron sequence of the human b-globin gene;

- sequence according to item 1 Seq Id No.: 2;

- hGH poly(A) signal polyadenylation signal sequence;

- right inverted terminal repeat ITR;

- f1 ori;

- ampicillin resistance gene promoter AmpR promoter;

- Ampicillin resistance gene AmpR.

In yet another embodiment, the invention provides a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 4A, the following elements:

- origin of replication site ori;

- left inverted terminal repeat ITR;

- CMV enhancer of the cytomegalovirus promoter;

- cytomegalovirus promoter CMV promoter;

- the intron sequence of the human b-globin gene;

- Kozak consensus sequence;

- sequence according to item 1 Seq Id No.: 2;

- hGH poly(A) signal polyadenylation signal sequence;

- right inverted terminal repeat ITR;

- f1 ori;

- ampicillin resistance gene promoter AmpR promoter;

- Ampicillin resistance gene AmpR.

In another embodiment, the invention provides a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 4B, the following elements:

- origin of replication site ori;

- left inverted terminal repeat ITR;

- CMV enhancer of the cytomegalovirus promoter;

- cytomegalovirus promoter CMV promotor;

- the intron sequence of the human b-globin gene;

- leader sequence L21;

- sequence according to item 1 Seq Id No.: 2;

- hGH poly(A) signal polyadenylation signal sequence;

- right inverted terminal repeat ITR;

- f1 ori;

- ampicillin resistance gene promoter AmpR promotor;

- Ampicillin resistance gene AmpR.

In another embodiment, the invention provides a viral expression vector containing the nucleotide sequence of Seq Id No.:2.

In another embodiment, the invention provides an adeno-associated virus containing the nucleotide sequence of Seq Id No.:2 as a viral expression vector.

In another embodiment, the invention provides adeno-associated virus serotype 2, 8 or 9, containing the nucleotide sequence of Seq Id No.:2, as a viral expression vector.

In another embodiment of the invention, an adeno-associated virus of serotype 2, 8 or 9 containing the nucleotide sequence of Seq Id No.: 2, produced in HEK293 suspension cell cultures, is proposed as a viral expression vector.

In another embodiment, the invention provides the use of an expression vector containing the nucleotide sequence of Seq Id No.:2 for the treatment of Leber type 4 amaurosis.

Thus, one of the technical problems of the present invention, for example, the creation of a recombinant hAIPL1 gene that allows to increase gene expression and increase the level of aipl1 protein in cells when introduced into an expression vector, can be solved using a codon-optimized nucleotide sequence of the present invention.

An increase in the expression of the hAIPL1 gene can lead to an increase in the effectiveness of gene therapy using DNA with the nucleotide sequence of the present invention and, consequently, to reduce the therapeutic dose of the drug containing the DNA with the nucleotide sequence of the present invention, and to reduce unwanted side effects.

figures

The invention is illustrated by the following graphics:

On FIG. 1 shows a pAAV-AIPL1_wt recombinant plasmid expression vector expressing the wild-type hAIPl1 gene. The hAIPLh1 gene was synthesized and cloned into the pAAV-MCS plasmid at the restriction sites (EcoRI-HindIII) under the control of the CMV promoter.

On FIG. 2 shows a recombinant plasmid expression vector pAAV-AIPL1_opt expressing a codon-optimized variant of the hAIPl1 gene. The hAIPLh1_opt gene was synthesized and cloned into the pAAV-MCS plasmid at the restriction sites (EcoRI-HindIII) under the control of the CMV promoter.

On FIG. 3 shows a recombinant plasmid expression vector pAAV-AIPL1_wt expressing the wild-type hAIPl1 gene, additionally containing the Kozak (A) and L21 (B) regulatory element.

On FIG. 4 shows a recombinant plasmid expression vector pAAV-AIPL1_opt expressing a codon-optimized variant of the hAIPl1 gene, additionally containing the Kozak regulatory element (A) and L21 (B).

On FIG. 5 shows transmission electron microscopy images of AAV capsids. A concentrated sample of AAV9-hAIPL1_wt (A) or AAV9-hAIPL1_opt (B) after affinity chromatography was fixed and stained with 1% PFA and applied to formvar-coated copper meshes. Magnification ×100000.

On FIG. 6 shows the result of a differential analysis of the relative expression of hAIPL1 gene variants in a HEK293T cell culture. Plasmid expression vectors containing the wild type hAIPL1 gene or DNA with the nucleotide sequence Seq Id No.: 2 were used to transfect the HEK293T cell line. The expression level was assessed by RT-PCR against the mRNA level of the human GAPDH and PPIA genes using the SybrGreen intercalating dye.

On FIG. 7 shows the results of hAIPL1_wt and hAIPL1_opt mRNA levels after transduction of the HEK293TN cell line with the recombinant AAV9 virus. MOI MAX - maximum multiplicity of infection, 100,000; MOI-1, MOI-2, MOI-3 - tenfold dilutions of AAV9.

On FIG. 8 shows the results of immunoblot detection of the hAIPL1 protein after transfection of the HEK293T cell line with plasmid expression vectors expressing hAIPL_wt and hAIPL-opt (denoted as hAIPL-CO in the figure). Antibodies to hAIPL-1 Mouse monoclonal antibody raised against a full-length recombinant AIPL1 (H00023746-M04) (1:1000). A) Immunoblotting; B) SDS-PAGE electrophoresis of HEK293T cell lysates after transfection.

On FIG. 9 shows the results of immunoblot detection of the hAIPL1 protein after transfection of the HEK293 cell line with plasmid expression vectors expressing hAIPL_wt-6xHIS and hAIPL-CO-6xHIS. Antibodies to HRP Anti-6x His tag antibody (ab1187) (1:5000).

On FIG. 10 shows a densitometric measurement analysis of hAIPl1 detection results by immunoblotting. A) hAIPL1 immunoblotting with antibodies to HRP Anti-6x His tag antibody (ab1187) (1:5000); B) Normalization by the amount of SDS-PAGE protein.

Detailed description of the invention

Unless otherwise indicated, all terms, designations and other scientific terms used in this application are intended to have the meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, definitions of terms with generally accepted meanings are provided herein for clarity and/or for quick reference and understanding, and the inclusion of such definitions in the present specification should not be construed as having a materially different meaning of the term from what is generally understood in the art.

In addition, unless the context requires otherwise, terms in the singular include terms in the plural, and terms in the plural include terms in the singular. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and widely used in this area. Enzymatic reactions and purification methods are carried out according to the manufacturer's instructions, as is commonly done in the art, or as described herein.

In one embodiment, the present invention provides a nucleotide sequence Seq Id No.: 2 encoding human aipl1. Based on the wild-type hAIPL1 gene sequence (NP_055151.3, Gene ID: 23746, Seq Id No.: 1), codon optimization was carried out in order to increase the stability of messenger RNA synthesized based on this gene and the level of expression of the heterologous AIPL1 gene when introduced into cells within a plasmid and/or viral expression vector.

The term "codon optimization" means an experimental approach to improve the codon composition of a recombinant gene based on various criteria without changing the amino acid sequence. Codon optimization is possible due to the degenerate genetic code, meaning that most amino acids are encoded by more than one codon. Most approaches to codon optimization are based on avoiding the use of rare codons. Also, the direction of codon optimization can be the identification of mRNA instability elements, mRNA secondary structures, sequence repeats, internal ribosome entry sites, promoter sequences, putative splicing sites, etc. Examples of such optimization technologies are described in the article [Gao, W. et al. (2004) UpGene: Application of a web-based DNA codon optimization algorithm. Biotechnol. Prog. 20, 443-448; Raab, D. et al. (2010) The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst. Synth. Biol. 4, 215-225; Gaspar, P. et al. (2012) EuGene: maximizing synthetic gene design for heterologous expression. Bioinformatics 28, 2683-2684; Fath, S. et al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PLoS ONE 6, e17596], the text of which is incorporated into the present description by reference. The frequencies of occurrence of synonymous codons vary in different organisms and even in different cells of the same organism. They are decoded by ribosomes at different rates, since the corresponding tRNAs are also present in different cells in different amounts, as shown in the article [Dana A., Tuller T. (2014) The effect of tRNA levels on decoding times of mRNA codons. Nucl. Acids Res. 42, 9171-9181], the text of which is incorporated into the present description by reference. Therefore, the level of translation of a recombinant protein can be significantly increased by replacing rare codons with those preferred by a given cell line or cells of target tissues.

The level of gene expression can be approximately predicted based on the frequency of codon usage using various methods and computer programs, examples of such programs are given in the article [Harraghy N., Gaussin A., Mermod N. (2008) Sustained transgene expression using MAR elements. Curr. Gene Ther. 8, 353-366]. The Codon usage database [http://www.kazusa.or.jp/codon/, accessed 11/25/2021] provides an opportunity to find out the frequency of use of synonymous codons and the percentage of occurrence of GC in the genetic code of more than 35,000 organisms. Approaches involving the replacement of rare codons by frequent ones, combined with a decrease in the total number of GCs, leading to the formation of a stable mRNA secondary structure, are often used by biotech companies and research groups to optimize the expression of heterologous proteins, in some cases, optimization of codon usage can significantly increase protein yield ( up to 1000 times). Codon optimization can be performed using bioinformatics software from various manufacturers (IDT Codon Optimization Tool, GenSmart™ Codon Optimization, ExpOptimizer, OptimumGene™ and many other bioinformatics tools).

Codon optimization in the creation of the present invention was carried out taking into account the secondary structure of RNA and the stability of the hairpins of the RNA molecule, and the optimization also took into account the structural features of the aipl1 protein, in particular the fact that the N-terminus of the aipl1 protein forms a globular structure, while the C- the end of the protein molecule is formed from unstructured elements. When creating the nucleotide sequence of the present invention, the EMBOSS Backtranseq software product (EMBL-EBI, UK) was used.

As a result of codon optimization, several variants of the nucleotide sequence encoding aipl1 were obtained, from which the nucleotide sequence Seq Id No.: 2 was selected with optimal properties predicted in silico. DNA with the nucleotide sequence of the present invention has been successfully synthesized and introduced into a plasmid expression vector based on pAAV and a viral expression vector based on adeno-associated virus. DNA with the nucleotide sequence of the present invention is stably expressed in both adherent culture cells and suspension culture cells at a significantly higher level than the wild-type heterologous hAIPL1 gene introduced into cells in a similar expression vector.

As a result of in silico selection from 9 designed variants, the hAIPL1opt nucleotide sequence (Seq Id No.: 2) was obtained. DNA with the claimed sequence can be obtained by any method known in the art, including, as non-limiting examples, recombinant methods such as cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning technology and PCR, and others, as well as chemical methods. synthesis.

The gene with a codon-optimized nucleotide sequence of the present invention can be introduced into a plasmid expression vector, for example, based on pAAV. Introduction of a plasmid expression vector containing a gene with a nucleotide sequence of the present invention into HEK293T cells by transfection leads to the expression of a heterologous hAIPL1opt gene, while the amount of target protein is at least 29% higher than when the wild-type hAIPL1 gene is introduced into a similar expression vector , the difference is statistically significant. Introduction of a plasmid expression vector containing a gene with a nucleotide sequence of the present invention together with a pHelper vector and a vector selected from pRC2/2, pRC2/8, pRC2/9 vectors into HEK293T cells results in the production of a viral expression vector. The introduction of the resulting viral expression vector into HEK293T cells by transduction leads to the expression of the heterologous hAIPL1opt gene, while the amount of hAIPL1opt mRNA is 15% higher than when the wild-type hAIPL1 gene is introduced into a similar expression vector, the difference is statistically significant.

In one embodiment, the invention provides an expression vector containing the nucleotide sequence Seq Id No.:2. DNA with the nucleotide sequence of the present invention can be introduced into any vector known from the prior art, including plasmid, viral, including those based on the SV40 virus, adenoviruses, herpesviruses, retroviruses, lentiviruses, adeno-associated and other viruses. The vectors into which DNA with the nucleotide sequence of the present invention can be introduced are not limited to this list. The choice of an expression vector is determined by the tasks for which the vector will be further used and does not limit the scope of the present invention, methods for obtaining vectors are known from the prior art, vectors containing DNA with the nucleotide sequence of the present invention can be obtained by experts in the field of genetic engineering. The use of plasmid expression vectors containing various additional regulatory sequences, such as the Kozak consensus sequence or the L21 leader sequence, has shown that they can be useful for solving certain technical problems, while other plasmid expression vector regulatory sequences can be used.

In some embodiments, the invention provides plasmid expression vectors containing elements according to the physical and genetic map shown in FIG. 2, Fig. 4A, Fig. 4B. The term "promoter" as used herein specifically refers to the DNA nucleotide sequence recognized by RNA polymerase at which transcription is initiated at the element to which the promoter is operably linked. The promoter may also be accompanied by an "enhancer". Enhancers increase the activity of the promoter and stimulate the transcription process. To obtain a large amount of protein in eukaryotic cells and, in particular, in human cells, it is advisable to use strong promoters active in target cells. Strong constitutive promoters capable of initiating the expression of a recombinant gene in different cell types are well known in the art. In one embodiment, the cytomegalovirus (CMV) promoter is used as the promoter and the cytomegalovirus enhancer is used as the enhancer.

In some embodiments, the plasmid expression vector includes the following elements in the 5' to 3' direction:

- origin of replication site ori;

- left inverted terminal repeat ITR;

- CMV enhancer of the cytomegalovirus promoter;

- cytomegalovirus promoter CMV promotor;

- the sequence of the intron of the human b-globin gene (intron of the hBG1 gene - gamma-1 hemoglobin subunits);

- sequence according to item 1 Seq Id No.: 2;

- hGH poly(A) signal polyadenylation signal sequence (hGH poly(A) signal, polyadenylation signal of the human growth hormone gene);

- right inverted terminal repeat ITR;

- f1 ori;

- ampicillin resistance gene promoter AmpR promotor;

- Ampicillin resistance gene AmpR.

In one embodiment, the plasmid expression vector contains a Kozak consensus sequence (Kozak consensus sequence). The Kozak consensus sequence is a sequence of nucleotides in the mRNA molecule of eukaryotes, as well as the corresponding sequence in their genes, surrounding the start codon and important for the initiation of translation.

Y. Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence. FEBS Lett. 2002 Dec 4;532(1-2):143-6].In one embodiment, the plasmid expression vector contains the L21 leader sequence. L21 - sequence element 21 bp long. is derived from the 5'-untranslated cDNA leader sequence of lobster tropomyosin L21. L21 contains both a Kozak sequence and an A-rich sequence. In some model systems, an enhancing effect of L21 on gene expression and protein synthesis has been shown [Sano K, Maeda K, Oki M,

Y. Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence. FEBS Lett. 2002 Dec 4;532(1-2):143-6].

Transfection of cells with plasmid expression vectors containing DNA with the nucleotide sequence of Seq Id No.: 2 leads to stable expression of the AIPL1 gene, while the amount of mRNA when cells are transfected with a plasmid expression vector containing the nucleotide sequence of Seq Id No.: 2 of the present invention is maintained at the same level as with the introduction of the wild-type gene in a similar vector, and the amount of the heterologous aipl1 protein in cells increases by 29% compared to cells transfected with a similar vector containing wild-type hAIPL1 DNA, the difference is statistically significant.

In one embodiment, the invention provides a viral expression vector containing the nucleotide sequence of Seq Id No.: 2. The vector can be an adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus, or a lentiviral vector based on human immunodeficiency virus and other viruses. AAV and lentiviruses can provide long term expression while adenovirus can provide transient expression.

In one embodiment, the invention provides an adeno-associated virus containing the nucleotide sequence of Seq Id No.: 2 as a viral expression vector. Examples of such AAV-based vectors can be found in: Peters, C.W., Maguire, C.A., & Hanlon, K.S. (2021). Delivering AAV to the Central Nervous and Sensory Systems. Trends in Pharmacological Sciences, 42(6), 461-474. AAV vectors are found to be effective for gene therapy of neural tissue derivatives, including tissues of sensory organs such as the eye. AAV is a small, self-replicating, non-enveloped virus about 20 nm in size. Many different AAV serotypes have been described in humans and primates. The genome of all known AAV serotypes is similarly organized: it is a linear single-stranded DNA molecule less than about 5000 nucleotides (nt). Inverted terminal repeats (ITR) limit within themselves unique nucleotide sequences encoding capsid proteins (Cap) and replication proteins (Rep). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. Upon formation of a recombinant AAV vector, an expression cassette containing no Rep genes and restricted to ITR is placed in the AAV capsid. Recombinant AAV is incapable of replication and is currently recognized as one of the safest and most widely used viral expression vectors for in vivo gene transfer. AAV-based vectors can enter cells of various tissues, providing stable expression of a heterologous gene. These viruses are non-pathogenic and have low immunogenicity [High KA, Aubourg P. rAAV human trial experience. Methods Mol Biol. 2011; 807:429-57].

In one embodiment, the invention provides an adeno-associated virus serotype 2, 8 or 9, containing the nucleotide sequence of Seq Id No.: 2, as a viral expression vector. All known adeno-associated virus serotypes can infect cells in many tissue types. Tissue specificity is determined by the serotype of the capsid proteins, so adeno-associated virus-based vectors are constructed by specifying the required serotype. Viral expression vectors based on AAV serotypes 2, 8 and 9 containing DNA with the nucleotide sequence of the present invention lead to stable expression of heterologous DNA in the cells transduced by them, while the level of mRNA synthesized from heterologous DNA with the nucleotide sequence Seq Id No.: 2, 15% higher than the level of mRNA in cells transduced with the same vector containing the wild-type hAIPL1 gene, the difference is statistically significant. The data obtained may allow one to expect an increased amount of heterologous aipl1 protein in cells transduced with viral vectors containing DNA with the nucleotide sequence of the present invention, even compared to cells transfected with plasmid expression vectors carrying DNA with the same nucleotide sequence.

In one of the embodiments of the invention, an adeno-associated virus of serotype 2, 8 or 9 containing the nucleotide sequence of Seq Id No.: 2, produced in HEK293 suspension cell cultures, is proposed as a viral expression vector. Introduction of a plasmid expression vector containing a gene with a nucleotide sequence of the present invention, together with the pHelper vector and a vector selected from the pRC2/2, pRC2/8, pRC2/9 vectors, into cells of a HEK293 suspension cell culture results in the production of viral expression vectors, which are adeno-associated virus 2, 8 or 9 serotype. It was shown that when cells are transduced with the obtained AAV vectors containing the hAIPL1opt gene according to the present invention, the level of AIPL1 mRNA increases when using the virus at a lower concentration (multiplicity of infection) than when using the vector with the wild-type construct, which may allow to reduce the therapeutic doses of the viral a vector containing a gene with a nucleotide sequence of the present invention, with further use for the treatment of Leber type IV amaurosis.

In one embodiment, the invention provides for the use of an expression vector containing the nucleotide sequence of Seq Id No.:2 for the treatment of Leber type 4 amaurosis. The expression vectors of the present invention demonstrate stable and high expression of the AIPL1 gene in cells. This testifies in favor of the fact that the introduction of any of the claimed expression vectors of the present invention into the tissues of the eye can lead to stable expression of the AIPL1 gene with the sequence Seq Id No.: 2 and replenish the deficiency of the aipl1 protein in the cells and tissues of the eye, and, consequently, restore its function and vision stabilization in Leber type IV amaurosis.

The essence and industrial applicability of the invention are illustrated by the following examples:

Example 1. Creation of plasmid expression vectors.

Example 1.1. Codon optimization of the nucleotide sequence encoding the hAIPL1 gene.

Annotation of pAIPL1 domains was performed using the NCBI/CCD service. Nucleotide sequences were aligned using the UniPro UGENE tool [Okonechnikov K, Golosova O, Fursov M, the UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics, 2012, 28:1166-1167. doi:10.1093/bioinformatics/bts091]. The translation of the nucleotide sequence into an amino acid was performed using the https://web.expasy.org/translate/ tool [accessed 11/26/2021]. Analysis of the stability of aipl1 protein domains was performed in IUPred2A [https://iupred2a.elte.hu/, accessed 11/26/2021]. As a result of the analysis, the globular domain of aipl1 and the unorganized region of the protein were identified, and each separately was used to optimize codons. Codon optimization was performed using the EMBOSS Backtranseq bioinformatics software (EMBL-EBI, UK). Additionally, the sequence of recognition sites for restriction enzymes involved in cloning was taken into account. The RNA fold program [http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, accessed November 26, 2021] was used to study transcript stability.

Example 1.2. Synthesis of nucleotide sequences encoding the hAIPL1wt and hAIPL1opt genes and preparation of plasmid expression vectors.

A nucleic acid with a wild-type AIPL1 gene sequence (hAIPL1wt, Seq Id No: 1) and with an optimized nucleotide sequence of the present invention (hAIPL1opt, Seq Id No: 2) was synthesized to order using the TopGenetech service (Canada). To further obtain viral vectors, expression plasmids were used, as well as commercially available helper plasmid (pHelper) and packaging plasmid pRC2/8/9 for different virus serotypes: AAV2, AAV8, AAV9, respectively (Cell Biolabs Inc, USA).

To obtain viral vectors, the expression plasmid AAV-2 was used, into which the nucleotide sequence of the hAIPL1wt or hAIPL1opt gene was delivered by cloning at the BamHI and HindIII restriction sites. Plasmid expression vectors with AAV expression cassettes with wild-type AIPL genes (Fig. 1) and optimized variants, including hAIPL1opt (Fig. 2), His-tag plasmids necessary for translation level assays, as well as plasmid expression vectors additionally carrying the Kozak consensus sequence or leader peptide L21 (FIGS. 3A, 4A) and L21 (FIGS. 3B, 4B).

Example 1.3. Isolation of plasmid expression vectors

The preparative amount of plasmid DNA for transfection was obtained using Qiagen Plasmid Mini Kit and Qiagen Plasmid Maxi Kit (Qiagen, Germany). The correspondence of the resulting plasmids to the maps was checked by restriction analysis followed by agarose gel electrophoresis and Sanger sequencing.

Example 1.4. Transfection of HEK293T cells with plasmid expression vectors.

The HEK293T adhesion cell line was used to assess the expression level after transfection. Thawing and cultivation of the cell line was carried out according to standard methods. Transfection of cells was performed using polyethyleneamine (PEI) in a ratio of 5:1 to plasmid DNA (μg/μg) and 6-well plates.

Example 1.5. AIPL1 expression level after transfection of cells with the obtained plasmid transcription vectors.

HEK293T cells transfected with plasmid expression vectors as described in Example 1.4 were grown to confluence according to standard cell culture techniques, after which the cells were harvested and mRNA was isolated from them using the RNeasy Kit (Qiagen, Germany) according to the manufacturer's recommendations.

To assess the level of expression after transfection, 3 genes (GAPDH, TBP, PPIA) were selected as housekeeping genes for a relative assessment of the level of expression, as the most stable genes for cancer and non-cancer lines.

The forward and reverse primers for real-time PCR (RT-PCR) were designed to be complementary to both the wild-type AIPL1wt nucleotide sequence and the optimized AIPL1opt nucleotide sequence.

AIPL-common-for GTGGCTGAAGCTGGAGAAG

AIPL-common-rev CTCCAGCTCCAGCACTTTC

The expected length of the mRNA fragment/ AIPL1-WT/ AIPL1-OPT is 220 bp.

>ENST00000570466.5_AIPL1:645+864 220 b.p.

Expected length of genomic DNA fragment=1004bp

>chr17:6425733-6426736 1004bp

Specific probes were selected for AIPL1opt and AIPL1wt.

Overlay of primer sequences on the sequence of the AIPL1 gene is marked with double underlining, probes with wavy underlining

AIPL1-WT-probe 56-ROXN/ AGTGCCTGCTGAAGAAGGAG /3BHQ_2/

AIPL1-OPT-probe /56-ROXN/ CAACACCCTGATCCTGAAC /3BHQ 2/

AIPL1opt

AIPL1wt

Primer sequences for housekeeping genes were taken from Lemma et al. [Lemma, S., Avnet, S., Meade, M.J., Chano, T., & Baldini, N. (2018). Validation of suitable housekeeping genes for the normalization of mRNA expression for studying tumor acidosis. International journal of molecular sciences, 19(10), 2930]. Three genes were selected as housekeeping genes for a relative assessment of the expression level: GAPDH, TBP, PPIA, as the most stable genes for cancer and non-cancer cell lines. Probe sequences for housekeeping genes were selected independently (highlighted in bold underline.

>ENST00000396856.5_GAPDH:42+107 66bp

>ENST00000636632.1_TBP:1098+1237 140bp

>ENST00000446829.1_TBP:53+153 101bp

>ENST00000677107.1_PPIA:451+547 97bp

Oligonucleotide sequences were verified using the OligoAnalyzer™ Tool [https://www.idtdna.com/pages/tools/oligoanalyzer, accessed 11/29/2021]. The product length was estimated by alignment to GRch38/hg38 genomic DNA using the Genome Browser tool [https://genome.ucsc.edu/, accessed 11/29/2021].

The level of heterologous mRNA encoding aipl1 does not differ in cells transfected with plasmid expression vectors containing the wild-type hAIPL1 gene and DNA with the nucleotide sequence of the present invention (Fig. 6). This indicates that the performed codon optimization does not lead to a deterioration in the heterologous expression of the hAIPL1 gene.

Example 1.6. Evaluation of the amount of aipl1 protein after transfection of cells with the obtained plasmid transcription vectors.

HEK293T cells transfected with plasmid expression vectors as described in Example 1.4. were grown to confluency according to standard cell culture methods, after which the cells were collected, protein was isolated from them according to the standard method, the total amount of protein was determined by the Lowry method [Lowry O.N., Rosebrough N.J., Farr A.L., Randall R.J. (1951) Protein measurement with the folin phenol reagent. J Biochem Sci, 193(1):265-275].

Electrophoresis was carried out under denaturing conditions according to the standard method [U. K. Laemmli. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 1970; V.227, P.680-685]. A sample was applied to the PAAG gel in an amount of 2.5 to 10 μg of protein. Transfer to a nitrocellulose membrane was carried out semi-dry using a Trans-blot turbo automated semi-dry immunoblotting system (BioRad, USA). For membrane staining, specific mouse monoclonal antibodies against the full-length recombinant aipl1 protein at a dilution of 1:1000 (2 B Scientific Ltd, UK, Cat. No. H00023746-M04) and rabbit polyclonal antibodies against His6-tag conjugated with horseradish peroxidase at a dilution of 1 :5000 (HRP Anti-6x His tag antibody ab1187, Abcam, UK). Changes in the expression level by protein concentration after membrane staining with antibodies were assessed using the ChemiDoc gel-documentation system with ImageLab software (BioRad, USA).

The results of electrophoresis and immunoblotting showed that the level of aipl1 protein is 29% higher after transfection of cells with a vector containing DNA with the nucleotide sequence of the present invention compared to the wild-type hAIPl1 gene (Fig. 8-10). The differences are statistically significant.

Example 2: Creation of viral expression vectors.

Example 2.1. Cell cultures and transfection

The HEK293 line, adapted to growth on a suspension medium, was used to generate vectors. The adhesion cell line HEK293T was used to assess the level of expression after transfection and the efficiency of transduction. Thawing and cultivation of the cell line was carried out according to standard methods. Transfection of cells was performed using polyethyleneamine (PEI) in a ratio of 5:1 to plasmid DNA (μg/μg) and 6-well plates. To obtain viral vectors used expression plasmid AAV-2, which is described in example 1.2. Helper plasmid (pHelper) and packaging plasmid (pRC2/8/9) for different virus serotypes AAV-2, AAV-8, AAV-9 were used to assemble viral expression vectors commercially available, for example, from Cell Biolabs, USA.

Example 2.2. Production of viral expression vectors.

The thawing and cultivation of the HEK293T cell line was performed according to standard operating procedures. At the time of receipt, the estimated density of the cell culture was 10 ⁶ cells/ml, the volume of the cell culture was 30 ml.

Transfection was carried out in Erlenmeyer flasks (125 ml), working volume 30 ml. Transfection conditions: cell concentration during transfection 10 ⁶ cells/ml, viability 85-90%, DNA mass 1.5 μg/1 million cell biomass. The ratio of DNA:PEI 1:5, the volume of the transfection mixture is 5% of the volume of the cell culture. Conditions for cultivation of the transfected cell culture: 37°C, 5% CO ₂ , 70% humidity, 92 rpm, platform diameter 50 mm. The cultivation time of the transfected mixture is 96-120 hours.

The preparation of transfection mixtures was carried out by sequentially adding pAAV-AIPL1wt or pAAV-AIPL1opt, pHelper, pRC2/8/9 with a total mass of 45 µg, PEI 1 µg/ml (225 µl for a ratio of 5:1, 135 µl for 3:1 ratio) and serum-free BalanCD® HEK293 cell culture medium up to 1000 µl or 1500 µl. After adding the medium, the contents of the tubes were mixed by vortexing and kept at room temperature for 10 minutes.

For transfection, a pre-prepared transfection mixture was added to 30 ml of HEK293T cell culture with a concentration of plasmid DNA (pDNA) of 1.5 μg/million cell culture and the following pDNA:PEI ratios. Ready transfection mixtures were added directly to the flasks with a single-channel dispenser. Thereafter, the flasks were transferred to a culture incubator and maintained at 37.0° C., humidity 70%, CO ₂ content 5%, agitation 120 rpm.

96-120 hours after transfection, a cell suspension in a volume of 30 ml was transferred into sterile 50 ml centrifuge tubes, the cell suspension was chemically lysed with 10% Tween 20 for 2.5-3 hours at 37.0°C and 130-330 rpm After lysis, samples were frozen at 20°C. After 14 hours, the lysates were thawed and treated with benzonase (fermentolysis, DNA hydrolysis). To do this, 35 μl of a 1M MgCl ₂ solution was added to the lysate, stirred for 30 minutes at 37.0°C and 330 rpm, then benzonase was added at a rate of 30 units/ml. and kept for 90 minutes at 37.0° C. and 130,330 rpm. After that, the resulting lysates were clarified by adding diatomaceous earth (0.01 g/ml) and keeping for 5 min at 37.0°C and 330 rpm, followed by sterilizing filtration of the clarified lysates through a filter with a pore diameter of 0.22 μm.

Next, the virus particles were purified using affinity chromatography on a CaptureSelect AAVX sorbent under the conditions recommended by the manufacturer.

A chromatographic column packed with 5 ml of CaptureSelect AAVX affinity sorbent was equilibrated with a 5 CV volume of equilibration buffer, then the filtered sample was applied to the sorbent in a volume of ≈1400 ml (until the sorbent was contaminated with coagulating proteins formed during sedimentation of the filtered sample during loading). Contact time 2 min, application rate 2.5 ml/min. Thereafter

- washed the column with equilibration buffer, washing buffer in a volume of 5CV each;

- elute the first part of AAV-hAIPL with elution buffer;

- titrated the eluate to the target pH of 7.0-8.0 1 M Tris-OH;

- filter the resulting solution through a sterilizing filtration system (PES, 0.22 µm);

The eluates obtained during chromatographic purification were concentrated and dialyzed using Vivaspin 500 PES centrifuge concentrators (Sartorius, Germany) with a nominal molecular weight cutoff of 100 kDa (MWCO 100 kDa) under the conditions recommended by the manufacturer, and sterilizing filtration was performed using centrifuge filters with pore diameter 0.22 µm. Sterile preparations of viral vectors were further used to analyze the concentration of viral particles and cell transduction.

In the case of AAV2 production, instead of affinity chromatography, ultracentrifugation was used using a discontinuous iodixanol gradient according to the known method [Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999 Jun; 6(6):973-85. doi: 10.1038/sj.gt.3300938. PMID: 10455399].

Example 2.3. Visualization of AAV samples by transmission electron microscopy (TEM)

To fix the sample, 12–15 µl of the studied solution with viral particles were taken, the concentration of samples for analysis was 10 ¹³ vg/ml, applied to copper grids for electron microscopy coated with formvar, fixed with 1% FVA in MiliQ H ₂ O (pH 7.0 -7.5). After fixation, the samples were washed with distilled water and dried. The preparations thus obtained were analyzed on a Jeol 110 ultramicroscope (Toshiba, Japan) at ×100,000 magnification.

The number of complete capsids ~50%, viral capsids do not aggregate under the given conditions, have the correct shape and size (Fig. 5).

Example 2.4. Determination of the number of viral particles

The number of viral genomes (vg) of AAV vectors in the culture liquid and lysates was determined by RT-PCR using the OneTube RT-PCR TaqMan reagent kit using Hot Start Taq DNA Polymerase (NEB, New England Biolabs, USA) cat# M0495L. The probe sequences were self-selected. The assessment methodology was verified (Table 1).

The productivity of cells after transfection and the total yield of AAV of three different serotypes (in vg per 1 liter of culture fluid) was assessed.

AAV-AIPL productivity results are presented in Table 2.

Modification of the expression cassette (sequence optimization) does not affect the efficiency of AAV assembly, as the productivity is in the range acceptable for operation (10 ¹² -10 ¹³ vg/l for subsequent purification, concentration and use, taking into account the approximate dose of the product for subretinal injection to the patient - 10 ¹¹ vg).

Any of the developed serotypes is suitable for assessing the level of transduction and further use.

Example 3 Transduction of cells with viral expression vectors containing the AIPL1 gene.

Example 3.1. Cell cultures and transduction

The adhesion cell line HEK293T was used to assess the level of expression after transduction. Thawing and cultivation of the cell line was carried out according to standard methods. Cell transduction was carried out according to known from the prior art and standard methods. The viral vector was suspended in cell culture medium and added to cells in culture grown to about 80% confluency. Cells were incubated with virus-containing medium for 6-12 hours.

Example 3.2. AIPL1 expression level after cell transduction with the obtained viral expression vectors.

HEK293T cells transduced with viral expression vectors were grown to confluency according to standard methods, after which the cells were collected, mRNA was isolated from them using the RNeasy Kit (Qiagen, Germany) according to the manufacturer's recommendations.

To assess the level of expression after transduction, 3 genes (GAPDH, TBP, PPIA) were selected as housekeeping genes for a relative assessment of the level of expression, as the most stable genes for cancer and non-cancer lines.

Forward and reverse primers for RT PCR were chosen to be complementary to both -WT and AIPL1-OPT. Oligonucleotide sequences were verified by the OligoAnalyzer™ Tool https://www.idtdna.com/pages/tools/oligoanalyzer. Product length estimated by alignment to GRch38/hg38 genomic DNA using the Genome Browser https://genome.ucsc.edu/

The level of heterologous mRNA encoding aipl1 in cells transduced with a viral expression vector containing DNA with the nucleotide sequence of the present invention is 15% higher compared to cells in which the wild-type hAIPL1 gene was introduced (Fig. 7). The difference is statistically significant. The data obtained suggest an increased amount of heterologous aipl1 protein in cells transduced with viral vectors containing DNA with the nucleotide sequence of the present invention, even compared to cells transfected with plasmid expression vectors carrying DNA with the same nucleotide sequence. When analyzing cell transduction with the viral expression vector of the present invention, an unexpected effect was found: it was shown that when transducing cells with AAV vectors containing the hAIPL1opt gene of the present invention, the level of AIPL1 mRNA increases when using the virus at a lower concentration (multiplicity of infection) than when using a vector with the wild-type hAIPL1 gene, which will reduce the therapeutic doses of the viral vector containing the gene with the nucleotide sequence of the present invention, with further use for the treatment of Leber type IV amaurosis.

All publications, patents and patent applications are incorporated herein by reference. While this invention has been described in the foregoing description with respect to certain preferred embodiments thereof, and many of the details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is capable of further embodiments and that some of the details described herein are may vary significantly without deviating from the essence of the invention.

The use of terms in the singular in the context of the description of the invention should be construed as covering both the singular and the plural, unless otherwise specified in this document or clearly contrary to the context. The terms "consisting of", "having", "including" and "comprising" should be construed as non-limiting terms, i.e. meaning "including but not limited to" unless otherwise noted. The listing of ranges of values in this document is merely intended to be used as a shorthand way of individually referring to each individual value falling within that range, unless otherwise noted here, and each individual value is included in the specification as if it were set out separately in this document. . All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise expressly contradicted by the context. The use of any and all examples or illustrative language (eg, "such as") provided herein is merely intended to better describe the invention and is not meant to limit the scope of the invention unless otherwise stated. Nothing in the specification should be construed as indicating any unclaimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best method known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those skilled in the art upon reading the foregoing description. The authors expect those skilled in the art to use such variations as the case may be, and the authors expect the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the features set forth in the appended claims, as permitted by applicable law. Moreover, any combination of the above features in all their possible variations is covered by the invention, unless otherwise specified in this document or otherwise clearly contrary to the context.

The Applicant asks to consider the submitted materials of the application "Codon-optimized nucleotide sequence encoding hAIPL1 and its containing expression vector" in order to grant a patent for the invention.

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Sequence listing

<110> Autonomous non-profit educational organization

higher education "Scientific and technological university "Sirius"

Sirius University of Science and Technology

<120> Codon-optimized nucleotide sequence,

encoding hAIPL1 and its containing expression vector

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<212> DNA

<213> Homo sapiens

<220>

<222> (1)…(1158)

<223> Human AIPL1 gene AIPL1wt

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ATG GAT GCC GCT CTG CTC CTG AAC GTG GAA GGG GTC AAG AAA ACC ATT 48

1 5 10 15

CTG CAC GGG GGC ACG GGC GAG CTC CCA AAC TTC ATC ACC GGA TCC CGA 96

20 25 30

GTG ATC TTT CAT TTC CGC ACC ATG AAA TGT GAT GAG GAG CGG ACA GTC 144

35 40 45

ATT GAC GAC AGT CGG CAG GTG GGC CAG CCC ATG CAC ATC ATC ATC GGA 192

50 55 60

AAC ATG TTC AAG CTC GAG GTC TGG GAG ATC CTG CTT ACC TCC ATG CGG 240

65 70 75 80

GTG CAC GAG GTG GCC GAG TTC TGG TGC GAC ACC ATC CAC ACG GGG GTC 288

85 90 95

TAC CCC ATC CTA TCC CGG AGC CTG AGG CAG ATG GCC CAG GGC AAG GAC 336

100 105 110

CCC ACA GAG TGG CAC GTG CAC ACG TGC GGG CTG GCC AAC ATG TTC GCC 384

115 120 125

TAC CAC ACG CTG GGC TAC GAG GAC CTG GAC GAG CTG CAG AAG GAG CCT 432

130 135 140

CAG CCT CTG GTC TTT GTG ATC GAG CTG CTG CAG GTT GAT GCC CCG AGT 480

145 150 155 160

GAT TAC CAG AGG GAG ACC TGG AAC CTG AGC AAT CAT GAG AAG ATG AAG 528

165 170 175

GCG GTG CCC GTC CTC CAC GGA GAG GGA AAT CGG CTC TTC AAG CTG GGC 576

180 185 190

CGC TAC GAG GAG GCC TCT TCC AAG TAC CAG GAG GCC ATC ATC TGC CTA 624

195 200 205

AGG AAC CTG CAG ACC AAG GAG AAG CCA TGG GAG GTG CAG TGG CTG AAG 672

210 215 220

CTG GAG AAG ATG ATC AAT ACT CTG ATC CTC AAC TAC TGC CAG TGC CTG 720

225 230 235 240

CTG AAG AAG GAG GAG TAC TAT GAG GTG CTG GAG CAC ACC AGT GAT ATT 768

245 250 255

CTC CGG CAC CAC CCA GGC ATC GTG AAG GCC TAC TAC GTG CGT GCC CGG 816

260 265 270

GCT CAC GCA GAG GTG TGG AAT GAG GCC GAG GCC AAG GCG GAC CTC CAG 864

275 280 285

AAA GTG CTG GAG CTG GAG CCG TCC ATG CAG AAG GCG GTG CGC AGG GAG 912

290 295 300

CTG AGG CTG CTG GAG AAC CGC ATG GCG GAG AAG CAG GAG GAG GAG CGG 960

305 310 315 320

CTG CGC TGC CGG AAC ATG CTG AGC CAG GGT GCC ACG CAG CCT CCC GCA 1008

325 330 335

GAG CCA CCC ACA GAG CCA CCC GCA CAG TCA TCC ACA GAG CCA CCT GCA 1056

340 345 350

GAG CCA CCC ACA GCA CCA TCT GCA GAG CTG TCC GCA GGG CCC CCT GCA 1104

355 360 365

GAG CCA GCC ACA GAG CCA CCC CCG TCC CCA GGG CAC TCG CTG CAG CAC 1152

370 375 380

TAA TGA 1158

385 386

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<211> 1158

<212> DNA

<213> Artificial sequence

<220>

<222> (1)…(1158)

<223> Optimized AIPL1opt gene sequence

<400> 2

ATG GAC GCC GCT CTG CTG CTG AAT GTG GAA GGC GTG AAA AAG ACC ATC 48

Met Asp Ala Ala Leu Leu Leu Asn Val Glu Gly Val Lys Lys Thr Ile

1 5 10 15

CTG CAC GGT GGA ACA GGC GAG CTG CCT AAC TTC ATC ACC GGC TCC AGA 96

Leu His Gly Gly Thr Gly Glu Leu Pro Asn Phe Ile Thr Gly Ser Arg

20 25 30

GTG ATC TTC CAC TTC CGG ACC ATG AAG TGC GAC GAA GAG AGA ACC GTG 144

Val Ile Phe His Phe Arg Thr Met Lys Cys Asp Glu Glu Arg Thr Val

35 40 45

ATT GAC GAT AGC AGA CAG GTG GGC CAG CCT ATG CAC ATC ATC ATC GGC 192

Ile Asp Asp Ser Arg Gln Val Gly Gln Pro Met His Ile Ile Ile Gly

50 55 60

AAC ATG TTT AAG CTG GAG GTC TGG GAA ATC CTG CTG ACC AGC ATG AGA 240

Asn Met Phe Lys Leu Glu Val Trp Glu Ile Leu Leu Thr Ser Met Arg

65 70 75 80

GTG CAT GAA GTT GCC GAG TTC TGG TGC GAT ACA ATC CAC ACA GGA GTG 288

Val His Glu Val Ala Glu Phe Trp Cys Asp Thr Ile His Thr Gly Val

85 90 95

TAC CCT ATC CTG TCT AGA AGC CTG AGA CAG ATG GCC CAG GGA AAG GAC 336

Tyr Pro Ile Leu Ser Arg Ser Leu Arg Gln Met Ala Gln Gly Lys Asp

100 105 110

CCC ACA GAA TGG CAC GTG CAT ACC TGC GGC CTG GCC AAT ATG TTC GCT 384

Pro Thr Glu Trp His Val His Thr Cys Gly Leu Ala Asn Met Phe Ala

115 120 125

TAC CAC ACC CTG GGA TAC GAG GAC CTG GAT GAG CTT CAG AAG GAG CCA 432

Tyr His Thr Leu Gly Tyr Glu Asp Leu Asp Glu Leu Gln Lys Glu Pro

130 135 140

CAG CCT CTG GTC TTT GTG ATC GAG CTG CTT CAA GTG GAC GCC CCT AGC 480

Gln Pro Leu Val Phe Val Ile Glu Leu Leu Gln Val Asp Ala Pro Ser

145 150 155 160

GAC TAC CAG CGG GAA ACC TGG AAC CTG AGC AAC CAC GAA AAG ATG AAA 528

Asp Tyr Gln Arg Glu Thr Trp Asn Leu Ser Asn His Glu Lys Met Lys

165 170 175

GCT GTG CCT GTG CTG CAC GGC GAA GGC AAT CGG CTG TTC AAA CTG GGC 576

Ala Val Pro Val Leu His Gly Glu Gly Asn Arg Leu Phe Lys Leu Gly

180 185 190

AGA TAC GAG GAG GCC AGC AGC AAG TAC CAG GAG GCC ATC ATT TGT TTG 624

Arg Tyr Glu Glu Ala Ser Ser Lys Tyr Gln Glu Ala Ile Ile Cys Leu

195 200 205

CGC AAC CTG CAG ACC AAG GAA AAA CCT TGG GAG GTG CAG TGG CTG AAG 672

Arg Asn Leu Gln Thr Lys Glu Lys Pro Trp Glu Val Gln Trp Leu Lys

210 215 220

CTG GAG AAG ATG ATC AAC ACC CTG ATC CTG AAC TAC TGC CAG TGT CTG 720

Leu Glu Lys Met Ile Asn Thr Leu Ile Leu Asn Tyr Cys Gln Cys Leu

225 230 235 240

CTG AAG AAA GAG GAG TAC TAC GAG GTC CTG GAG CAC ACA TCT GAC ATC 768

Leu Lys Lys Glu Glu Tyr Tyr Glu Val Leu Glu His Thr Ser Asp Ile

245 250 255

CTG AGA CAC CAC CCC GGC ATC GTG AAG GCC TAT TAC GTG CGG GCC CGG 816

Leu Arg His His Pro Gly Ile Val Lys Ala Tyr Tyr Val Arg Ala Arg

260 265 270

GCC CAC GCC GAA GTG TGG AAC GAG GCC GAG GCC AAG GCG GAC CTC CAG 864

Ala His Ala Glu Val Trp Asn Glu Ala Glu Ala Lys Ala Asp Leu Gln

275 280 285

AAA GTG CTG GAG CTG GAG CCG TCC ATG CAG AAG GCG GTG CGC AGG GAG 912

Lys Val Leu Glu Leu Glu Pro Ser Met Gln Lys Ala Val Arg Arg Glu

290 295 300

CTG AGG CTG CTG GAG AAC CGC ATG GCG GAG AAG CAG GAG GAG GAG CGG 960

Leu Arg Leu Leu Glu Asn Arg Met Ala Glu Lys Gln Glu Glu Glu Arg

305 310 315 320

CTG CGC TGC CGG AAC ATG CTG AGC CAG GGT GCC ACG CAG CCT CCC GCA 1008

Leu Arg Cys Arg Asn Met Leu Ser Gln Gly Ala Thr Gln Pro Pro Ala

325 330 335

GAG CCA CCC ACA GAG CCA CCC GCA CAG TCA TCC ACA GAG CCA CCT GCA 1056

Glu Pro Pro Thr Glu Pro Pro Ala Gln Ser Ser Thr Glu Pro Pro Ala

340 345 350

GAG CCA CCC ACA GCA CCA TCT GCA GAG CTG TCC GCA GGG CCC CCT GCA 1104

Glu Pro Pro Thr Ala Pro Ser Ala Glu Leu Ser Ala Gly Pro Pro Ala

355 360 365

GAG CCA GCC ACA GAG CCA CCC CCG TCC CCA GGG CAC TCG CTG CAG CAC 1152

370 375 380

TAA TGA 1158

385 386

<210> 3

<211> 384

<212> protein

<213> Homo sapiens

<220>

<222> (1)…(384)

<223> AIPL1 protein

<400> 3

Met Asp Ala Ala Leu Leu Leu Asn Val Glu Gly Val Lys Lys Thr Ile

1 5 10 15

Leu His Gly Gly Thr Gly Glu Leu Pro Asn Phe Ile Thr Gly Ser Arg

20 25 30

Val Ile Phe His Phe Arg Thr Met Lys Cys Asp Glu Glu Arg Thr Val

35 40 45

Ile Asp Asp Ser Arg Gln Val Gly Gln Pro Met His Ile Ile Ile Gly

50 55 60

Asn Met Phe Lys Leu Glu Val Trp Glu Ile Leu Leu Thr Ser Met Arg

65 70 75 80

Val His Glu Val Ala Glu Phe Trp Cys Asp Thr Ile His Thr Gly Val

85 90 95

Tyr Pro Ile Leu Ser Arg Ser Leu Arg Gln Met Ala Gln Gly Lys Asp

100 105 110

Pro Thr Glu Trp His Val His Thr Cys Gly Leu Ala Asn Met Phe Ala

115 120 125

Tyr His Thr Leu Gly Tyr Glu Asp Leu Asp Glu Leu Gln Lys Glu Pro

130 135 140

Gln Pro Leu Val Phe Val Ile Glu Leu Leu Gln Val Asp Ala Pro Ser

145 150 155 160

Asp Tyr Gln Arg Glu Thr Trp Asn Leu Ser Asn His Glu Lys Met Lys

165 170 175

Ala Val Pro Val Leu His Gly Glu Gly Asn Arg Leu Phe Lys Leu Gly

180 185 190

Arg Tyr Glu Glu Ala Ser Ser Lys Tyr Gln Glu Ala Ile Ile Cys Leu

195 200 205

Arg Asn Leu Gln Thr Lys Glu Lys Pro Trp Glu Val Gln Trp Leu Lys

210 215 220

Leu Glu Lys Met Ile Asn Thr Leu Ile Leu Asn Tyr Cys Gln Cys Leu

225 230 235 240

Leu Lys Lys Glu Glu Tyr Tyr Glu Val Leu Glu His Thr Ser Asp Ile

245 250 255

Leu Arg His His Pro Gly Ile Val Lys Ala Tyr Tyr Val Arg Ala Arg

260 265 270

Ala His Ala Glu Val Trp Asn Glu Ala Glu Ala Lys Ala Asp Leu Gln

275 280 285

Lys Val Leu Glu Leu Glu Pro Ser Met Gln Lys Ala Val Arg Arg Glu

290 295 300

Leu Arg Leu Leu Glu Asn Arg Met Ala Glu Lys Gln Glu Glu Glu Arg

305 310 315 320

Leu Arg Cys Arg Asn Met Leu Ser Gln Gly Ala Thr Gln Pro Pro Ala

325 330 335

Glu Pro Pro Thr Glu Pro Pro Ala Gln Ser Ser Thr Glu Pro Pro Ala

340 345 350

Glu Pro Pro Thr Ala Pro Ser Ala Glu Leu Ser Ala Gly Pro Pro Ala

355 360 365

Glu Pro Ala Thr Glu Pro Pro Pro Ser Pro Gly His Ser Leu Gln His

370 375 380

*** ***

385 386

<---

Claims (44)

1. Nucleic acid with nucleotide sequence Seq Id No.: 2 encoding human aipl1 . 2. An expression vector containing the nucleic acid according to claim 1. 3. The vector according to claim 2, which is a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 2, the following items: — site of origin of replication ori; - left inverted terminal repeat ITR; — CMV enhancer of the cytomegalovirus promoter; — cytomegalovirus promoter CMV promotor; — the intron sequence of the human b-globin gene; - sequence according to item 1 Seq Id No.: 2; — polyadenylation signal sequence hGH poly(A) signal; - right inverted terminal repeat ITR; - f1 ori; — ampicillin resistance gene promoter AmpR promotor; Ampicillin resistance gene AmpR. 4. The vector according to claim 2, which is a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 4A, the following elements: — site of origin of replication ori; - left inverted terminal repeat ITR; — CMV enhancer of the cytomegalovirus promoter; — cytomegalovirus promoter CMV promotor; — the intron sequence of the human b-globin gene; — Kozak consensus sequence; - sequence according to item 1 Seq Id No.: 2; — polyadenylation signal sequence hGH poly(A) signal; - right inverted terminal repeat ITR; - f1 ori; — ampicillin resistance gene promoter AmpR promotor; Ampicillin resistance gene AmpR. 5. The vector according to claim 2, which is a plasmid expression vector containing, in accordance with the physical and genetic map shown in FIG. 4B, the following elements: — site of origin of replication ori; - left inverted terminal repeat ITR; — CMV enhancer of the cytomegalovirus promoter; — cytomegalovirus promoter CMV promotor; — the intron sequence of the human b-globin gene; — leader sequence L21; - sequence according to item 1 Seq Id No.: 2; — polyadenylation signal sequence hGH poly(A) signal; - right inverted terminal repeat ITR; - f1 ori; — ampicillin resistance gene promoter AmpR promotor; Ampicillin resistance gene AmpR. 6. The vector according to claim 2, which is a viral expression vector. 7. The vector according to claim 6, which is an adeno-associated virus. 8. The vector according to claim 7, which is an adeno-associated virus 2, or 8, or 9 serotype. 9. Vector according to claim 8, produced in HEK293 suspension cell cultures.

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