USH2a miniprotein for gene therapy of usher syndrome type ii

Abstract

The present invention relates to the field of biotechnology, namely to the USH2A miniprotein which, due to a small size enabling its delivery in viral vectors, retains LamGL, LamNT, EGF Lam, FN3 domains and PDZ-binding motif of a full-length protein, which are required for the USH2A protein function. Also disclosed is a nucleic acid encoding the USH2A miniprotein, an expression vector, and the use of said vector in the gene therapy of Usher syndrome type II. The invention can be effectively used for stable and high expression of the USH2A miniprotein.

Description

[0001] USH2A MINIPROTEIN FOR GENE THERAPY OF USHER SYNDROME TYPE II

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of biotechnology and molecular biology, in particular gene engineering, namely to a USH2A miniprotein, a nucleic acid encoding the USH2A miniprotein, and an expression vector comprising the same, and can find a use in gene therapy of Usher syndrome type II.

[0004] BACKGROUND OF THE INVENTION

[0005] Usher syndrome is characterized by congenital bilateral sensorineural hearing loss and progressive retinitis pigmentosa, and follows an autosomal recessive inheritance pattern (Hereditary and congenital diseases of the retina and optic nerve. A guide for clinicians. Ed. Shamshinova A.M. M.: Medicine; 2001). Based on the data provided by a number of authors, the prevalence of Usher syndrome is 4.4 cases per 100 thousand of individuals and 4-5 cases per 100 thousand newborns. (Boughman JA, Vernon M, Shaver KA. Usher syndrome: definition and estimate of prevalence from two high-risk populations. Journal of Chronic Diseases. 1983;36(8):595-603).

[0006] The main challenge in studying and developing treatment for Usher syndrome lies in its significant clinical and genetic heterogeneity, i.e., existence of distinct clinical forms and a large number of genes responsible for this syndrome (Eenarduzzi S., Vozzi D., Morgan A., Rubinato E., D’Eustacchio A., Osland T.M., Rossi C., Graziano C., Castorina P., Ambrosetti U., Morgutti M., Girotto G. Usher syndrome: an effective sequencing approach to establish a genetic and clinical diagnosis. Hearing Research. 2015;320: 18-23). In-depth clinical trials, along with attempts to discover candidate genes based on clinical differences, have identified four types of Usher syndrome.

[0007] Type II, associated with the USH2A gene, is the most common of these four identified types. A child with Usher syndrome type II has partial or total hearing loss from early childhood, and sometimes developmental delays. There is a progressive vision loss, narrowing of visual fields, and developing night blindness. The primary clinical signs of Usher syndrome are disorientation in darkness, frequent stumbling, and collisions with obstacles.

[0008] There are currently no medical means to prevent, treat or slow down the progression of the disease. Cochlear implantation, a prosthetic surgical procedure, allows compensating for the loss of hearing in individuals with sensorineural deafness. There is also no treatment available for retinitis pigmentosa (Tikhomirova M.A. Usher syndrome / / GENOKARTA. A Genetic Encyclopedia. 2019).

[0009] Over the last few years, gene replacement therapy has been considered as a potential solution for the problem of treating Usher syndrome. The essence of the replacement therapy lies in replacing an entire gene with a healthy copy thereof by cloning the latter into a viral vector, e.g., an adeno-associated virus (AAV) vector, and delivering this copy to the relevant cells.

[0010] Usher syndrome is a challenging model for the development of gene therapies, as patients with this pathology suffer from consequences of retinal degeneration as well as from hearing loss; with that being the case, many of the researchers focus on one of these problems.

[0011] It is known from the prior art that the first gene therapy clinical trial for Usher syndrome was the phase I / II study No. NTC01505062, sponsored by Sanofi, aimed at evaluating subretinal lentiviral delivery of the USH1B MYO7A gene. The clinical trials were discontinued. Although lentiviruses are capable of packaging larger transgenes, their use is associated with potential risks. Lentiviruses tend to cause adverse immune reactions, have limited transduction ability, and they are also able to integrate a carrier transgene into a patient’s DNA, thus posing a risk of insertional mutagenesis or activation of oncogenes. (Ahmed, H., Shubina-Oleinik, O., Holt, J.R. 2017. Emerging gene therapies for genetic hearing loss. J Assoc Res Otolaryngol 18, 649-670).

[0012] To improve patients’ safety, approaches relying on the use of AAV vectors are known in the art. At present, several ways have been proposed to overcome the limited carrying capacity of AAV vectors, namely dual AAV vectors and a minigene-based approach.

[0013] The use of various dual AAV vectors to assess efficient expression of functional myosin Vila has been described (Dyka, F.M., Boye, S.L., Chiodo, V.A., Hauswirth, W.W., Boye, S.E. 2014. Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene, MY07A. Hum Gene Ther Methods 25, 166-77). The studies were successful in a model system in vitro, however in vivo experiments failed to reproduce this success. The use of dual vectors had limited success in animal models when attempting to deliver large genes to the retina (Ferreira, M.V.; Fernandes, S.; Almeida, A.I.; Neto, S.; Mendes, J.P.; Silva, R.J.S.; Peixoto, C.; Coroadinha, A.S. Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses. Int. J. Mol. Sci. 2023, 24, 10524).

[0014] A minigene-based approach was used to create a shortened version of the USH2A gene. A gene encoding the USH2A protein is located on chromosome 1 (chrl:215,622,891- 216,423,448 (GRCh38 / hg38)) and is 800,558 base pairs in size. The USH2A protein has a complex domain organization and consists of 5202 amino acid residues (SEQ ID NO: 1). Genes (SEQ ID NO:2) of this size cannot be delivered in viral vectors. Several constructs having lengths of 6.8 kb and 4.1 kb were provided; they were cloned into the Tol2 transposon vector pDestTol2CG2 (Vona B, Doll J, Hofrichter M., Haaf T., Varshney G.K., Small fish, big prospects: using zebrafish to unravel the mechanisms of hereditary hearing loss, Hearing Research, Volume 397, 2020; Dona, M. (2018). Towards gene therapy for USH2A -associated retinitis pigmentosa (Doctoral thesis, Radboud Institute for Molecular Life Sciences. Radboud Repository, htps: / / repository.ubn.ru.nl / handle / 2066 / 196854). As a result, authors have successfully achieved partial restoration of retinal function in a mutant line of zebrafish (Danio rerio). However, it must be considered that the Danio rerio model is not optimal for studying retinal pathologies and has a number of physiological distinctive features preventing it from being fully used for studying retinal dystrophies. Furthermore, the resulting USH2A minigene constructs are still too large to be packaged into an AAV vector for carrying out a therapy.

[0015] As can be noted, minigene structures of the USH2A protein proposed by other research teams often contain the fibronectin domain F17-F31 region (CN114402075 A). According to available data in the ClinVar database, the highest number of pathogenic mutations is concentrated in the F17-F31 region of the fibronectin domain of the USH2A protein relative to the first half of the fibronectin domain.

[0016] Analysis of sequences set forth in the patent WO2019166549, wherein the authors provide two main minisequences of the USH2A protein, revealed that in the first case, provided that the major essential regions are preserved, the sequence is 6786 bp (base pairs) in size which does not satisfy further requirements for packaging this sequence into AAV due to capacity limitations. The second minisequence of the USH2A protein is 4125 bp; however, this sequence lacks LamGL, LamNT, and LamG domains. Deletion of exon 13 in the sequence, which is most often referred to as a target region for antisense therapy, results in deletion of a part of the LamEGF protein domain required for the specific photoreceptor cell function.

[0017] The closest analogue of the invention is a technical solution provided in the patent application WO2020214796A1, wherein the authors disclose constructing several USH2A minigenes intended for packaging into AAV, however the application provides no data on testing and efficient expression of the construct. The disclosed sequences of USH2A minigenes do not comprise key structures, such as EGF-like and Lam-G domains, which, according to the published references, are responsible for the protein function, and therefore, such structures will not be expressed at high levels. Additionally, expression of the USH2A minivariants is controlled by the human rhodopsin kinase (hGRK) promoter, which is non-authentic for the USH2A gene, is non-inducible, and has a relatively low expression level. In summary, there is currently no registered effective and safe therapy for Usher syndrome type II. Despite the progress in developing gene therapy for this disease, there remains a need to obtain a suitable nucleotide sequence encoding a suitable USH2A protein. Such a sequence, a protein and a vector are provided in the present invention.

[0018] The obtained USH2A miniprotein sequences comprising a unique combination of domains were found to comply with the mutational profile and structural features at both gene and protein levels. The presence of LamGL, LamNT, EGF Lam, FN3 domains and PDZ-binding motif ensures the required functionality of the USH2A protein, and the signal sequence and transmembrane domain in USH2A miniprotein structure enable its physiological localization. The sequence of the USH2A minigene corresponding to the USH2A protein has a length allowing it to be successfully delivered into cells in viral vectors.

[0019] The present invention relates to a USH2A miniprotein, a nucleic acid encoding the USH2A miniprotein, and an expression vector comprising the nucleic acid for expression in eukaryotic cells. The use of vectors for gene therapy of Usher syndrome type II is disclosed.

[0020] In general, the nucleic acid encoding for the USH2A miniprotein of the present invention has been effectively packaged into an AAV vector, in particular into the AAV vector serotypes 9, 5, or 2.7m8. The minimal size of the transgene allowed high-titer production of AAV vectors (>1.0xl0A12 vg / ml) that will simplify their large scale manufacturing and purification.

[0021] The present invention is based on the discovery that combination of the USH2A protein domains in a miniprotein allows to reduce the size of the native nucleotide sequence of the USH2A gene by 74% compared to the original sequence, and the length of this nucleic acid sequence allows to successfully deliver it into cells in viral vectors. The provided nucleic sequence retains the main functional domains of the USH2A protein while sequestering a number of domains in different versions.

[0022] Unexpectedly, expression of the USH2A minigene in human retinal pigment epithelial cells was found to increase by more than 80-fold precisely after transfection with inventive plasmid vectors and transduction with the inventive vectors, and the presence of the USH2A miniprotein was demonstrated in the retinal pigment epithelial cells after transduction. According to the published data (de Joya E.M., Colbert B.M., Tang P.C., Lam B.L., Yang J., Blanton S.H., Dykxhoom DM, Liu X. Usher Syndrome in the Inner Ear: Etiologies and Advances in Gene Therapy. Int J Mol Sci. 2021 Apr 10;22(8):3910), the presence of the USH2A protein is required for the function of the hair cells in the inner ear. Therefore, delivery of the USH2A miniprotein to these cells may potentially be used for gene therapy of hereditary deafness associated with mutations in the USH2A gene. The use of vectors comprising the nucleic acid encoding the USH2A miniprotein provides stable expression of the USH2A miniprotein. In ocular tissues, this reverses protein deficiency in ocular cells and tissues, and, consequently, restores eye function and stabilizes vision in retinitis pigmentosa. In ear tissues, namely in the hair cells, expression of the USH2A minigene will allow to produce a functional USH2A miniprotein required for the function of the USH2A complex with WHRN, PDZD7, and GPR98, and thereby to restore the missing function.

[0023] In addition, nucleic acids have low immunogenicity. Thus, the USH2A miniprotein, the nucleic acid, and the vector of the invention successfully overcome disadvantages of the technical solutions suggested in the prior art for the gene therapy of Usher syndrome type II.

[0024] To date, the use of such a miniprotein, a nucleic acid and a vector comprising the same for the gene therapy of retinitis pigmentosa and hearing loss in Usher syndrome type II has not been reported.

[0025] SUMMARY OF THE INVENTION

[0026] The invention relates to the USH2A miniprotein for the gene therapy of Usher Syndrome type II comprising an amino acid sequence selected from SEQ ID Nos 4, 6, 8, 10, and 12. Preferably, the invention relates to the USH2A miniprotein for the gene therapy of Usher Syndrome type II comprising the amino acid sequence of SEQ ID NO 4 or 6.

[0027] In another aspect, the invention relates to a nucleic acid encoding the USH2A miniprotein. Preferably, the invention relates to the nucleic acid encoding the USH2A miniprotein comprising a nucleotide sequence selected from SEQ ID Nos 3, 7, 9, 11, and 13. In particular, the invention relates to the nucleic acid encoding the USH2A miniprotein comprising the nucleotide sequence of SEQ ID NO 3 or 7.

[0028] An expression vector for expression in eukaryotic cells comprises the nucleic acid encoding the USH2A miniprotein. Preferably, the vector is a plasmid expression vector comprising the following elements:

[0029] - the origin of replication (ori);

[0030] - a left inverted terminal repeat (ITR);

[0031] - a cytomegalovirus (CMV) promoter enhancer;

[0032] - a cytomegalovirus (CMV) promoter;

[0033] - the intron sequence of human b-globin gene;

[0034] - the optimized fUSH2A gene sequence of SEQ ID NO 3;

[0035] - a hGH polyadenylation (poly(A)) signal sequence;

[0036] - a right inverted terminal repeat (ITR);

[0037] - the origin of replication (ori) for packaging into phage fl particles;

[0038] - the ampicillin resistance gene promoter (AmpR); - the ampicillin antibiotic resistance gene (AmpR).

[0039] In another aspect, the vector is a viral expression vector. In particular, the vector is the adeno-associated virus. In the next aspect, the vector is the adeno-associated virus serotype 9, serotype 2.7m8, or serotype 5. In another aspect, the vector is produced in adherent HEK293T cell cultures.

[0040] The present invention provides the use of the vector for the gene therapy of Usher syndrome type II. In another aspect, the present invention provides the use of the vector for the gene therapy of retinitis pigmentosa and deafness associated with Usher syndrome type II.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Fig. 1 shows a schematic structure of the expression vector pAAV-fUSH2A. The recombinant plasmid expression vector pAAV_fUSH2A expressing the optimized 4011 bp fUSH2A sequence consists of the following elements: the origin of replication (ori); the left inverted terminal repeat (ITR); the enhancer of cytomegalovirus (CMV) promoter; the cytomegalovirus (CMV) promoter; the intron sequence of human b-globin gene (the hBGl gene intron - hemoglobin gamma-1 subunits); the fUSH2A sequence of SEQ ID NO 1; the hGH polyadenylation signal sequence (hGH poly(A) signal, the human growth hormone gene polyadenylation signal); the right inverted terminal repeat (ITR); origin of replication (ori) for packaging into phage fl particles; the ampicillin resistance gene promoter (AmpR promoter); the ampicillin antibiotic resistance gene (AmpR).

[0043] Fig. 2 shows the domain organization of the human USH2A protein and the fUSH2A minigene.

[0044] Fig. 3 shows amplification plot for cell lysates using primers for the fUSH2A gene and housekeeping PPIA gene. The X-axis shows amplification cycle numbers, and the Y-axis shows relative fluorescent signal intensities. Control and eGFP are control samples where cell lysates without transfection or transfected with eGFP-plasmid were used; pUSH2A, fUSH2A denote cells transfected with pUSH2A or fUSH2A plasmids; PPIA denotes all samples with primers for PPIA.

[0045] Fig. 4 shows the results of PAGE analysis in non-reducing conditions. Tested samples are as follows: 11 - molecular marker, 1 - original cell lysate, 2 - concentrated cell lysate, 3 - breakthrough fraction of the original lysate, 4 - breakthrough fraction of the concentrated lysate, 5 - eluate fraction 1 (hereinafter, for concentrated lysate sample), 6 - eluate fraction 2, 7 - eluate fraction 3, 8 - eluate fraction 4, 9 - eluate fraction 5, 10 - eluate fraction 3 (for non-concentrated lysate sample). Distribution of molecular markers by weight is shown on the left of the plot; capsid VP1, VP2 and VP3 proteins’ positions are shown on the right of the plot. Fig. 5 shows a standard curve for assessing the level of miniprotein production using enzyme-linked immunosorbent assay (ELISA) plotted using the optical density measurements in USH2A miniprotein standard samples with concentrations 1000 pg / ml to 15.1 pg / ml. The predictive trend line is indicated in the plot; the correlation coefficient was 0.97. The X-axis shows the USH2A protein concentration (pg / ml), the Y-axis shows optical density (450 nm).

[0046] Fig. 6 shows the results of the fUSH2A and uUSH2A miniprotein synthesis measurements 72 hours after HEK293T cells transfection using pAAV-fUSH2A and pAAV-uUSH2A vectors, and the results for the non-transfected HEK293T cells lysate.

[0047] Fig. 7 shows a plot of AAV genome amplification by digital droplet PCR using a fluorescent probe (A). The range of the primer annealing temperatures is 52.8 to 62.7°C. The fluorescent signal was detected using the FAM channel.

[0048] Fig. 8 shows a plot of AAV genome amplification by digital droplet PCR using an intercalating dye (EvaGreen) (B). The range of the primer annealing temperatures is 52.8 to 62.7°C. The fluorescent signal was detected using the FAM channel.

[0049] Fig. 9 shows the distribution of droplets by signal intensities in the FAM (Green) channel. A high density of positive droplets (indicated in blue) indicates the presence of AAV genomes. Each positive droplet in the plot indicates the presence of one AAV genome copy therein.

[0050] Fig. 10 shows a signal correlation plot in the FAM channel, where the signal is obtained by qPCR and ddPCR methods. The linear relationship in the plot reflects reproducibility provided by the qPCR and ddPCR methods used to produce AAV vectors. The correlation coefficient I was 0.99. The test results indicate that the qPCR and ddPCR methods can be used interchangeably for detecting AAV genomes in the selected numerical range of vg / ml.

[0051] Fig. 11 shows the results of the fUSH2A miniprotein bioinformatic analysis. The USH2A miniprotein amino acid sequence was used to build a similarity-based model using the I- TASSER program (https : / / zhanggroup .org / l-TAS S ER / ) Query date 10 / 28 / 2023. The figure shows domains and their secondary structure (beta-sheets, alpha-helices, and non-structural loops). Domain organization of the presented structure exactly reproduces the estimated sequence of the miniprotein in Fig. 2. The color scheme was selected arbitrarily.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] Unless defined otherwise, all terms, designations and other scientific terms used in this application are intended to have the same meanings as commonly understood by those skilled in the art to which this invention belongs. In certain cases, definitions of terms that have generally accepted meanings are provided in this application for clarity and / or for quick reference and understanding, and the inclusion of such definitions in this specification should not be construed as representative of a substantial difference compared to the meaning of the term generally understood in the art.

[0054] Furthermore, unless the context requires otherwise, terms in the singular form include plural, and vice versa. Generally, classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and chemistry of proteins and nucleic acids as used herein are well known to those skilled in the art and are widely used in these fields. Enzymatic reactions and purification methods are carried out in accordance with manufacturers’ instructions, as commonly practiced in the art or described herein.

[0055] All publications, patents and patent applications, and accession numbers cited throughout the present application, are hereby incorporated by reference in their entirety.

[0056] The terms “comprises”, “comprise” and “comprising”, “contains”, “contain” and “containing” should be interpreted as inclusive rather then exclusive, i.e., these terms include other unspecified components or process steps. The terms “consists”, “consisting” and variations thereof shell be interpreted as exclusive rather than inclusive, i.e., these terms exclude components or steps not specifically indicated.

[0057] As used herein, “Usher syndrome” is a hereditary disease characterized by congenital hearing impairments of varying severity (hearing loss), vestibular dysfunction, and progressive retinal pigmentary degeneration (RPD) (retinitis pigmentosa) resulting in gradual narrowing of visual fields and blindness, though the impairments of the light and sound perception organs may have different nature.

[0058] “Gene therapy” as used herein relate to multiple methods aimed at modifying gene sequences or controlling their expression, and altering cellular biological properties for therapeutic or prophylactic applications thereof.

[0059] “USH2A miniprotein” (“mini -usherin”, “usherin miniprotein”) is a shortened version of the native USH2A protein produced by ribosomal translation of USH2A minigene mRNA to a polypeptide product.

[0060] In one embodiment of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 12. In yet another embodiment of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 10. In yet another embodiment of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 8. In yet another embodiment of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 6. In yet another embodiment of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 4.

[0061] In particular embodiments of the present invention, the USH2A miniprotein for the gene therapy of Usher syndrome type II comprises the sequence of SEQ ID NO 4 or SEQ ID NO 6.

[0062] The term “codon-optimized” means a nucleotide sequence where one or more codons have been replaced with synonymous codons without changing a protein sequence that is synthesized from the template of this sequence.

[0063] The term “codon optimization” refers to an experimental approach aimed at improving codon usage of a recombinant gene without changing the resulting amino acid sequence, based on various criteria. Codon optimization is possible due to genetic code degeneracy which means the majority of amino acids are encoded by more than one codon. Most approaches to codon optimization are based on avoidance of rare codons. Codon optimization can also be directed to identification of mRNA instability elements, mRNA secondary structures, sequence repeats, internal ribosome entry sites, promoter sequences, putative splicing sites and the like. Exemplary technologies for such an optimization are described in the literature (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, el7596). Synonymous codons frequencies vary among different organisms and even in different cells of the same organism. They are decoded by ribosomes at different rates as the corresponding tRNAs have different abundances in different cells, as set out 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) which is incorporated herein by reference in its entirety. Mitochondrial translation deviates from the universal genetic code via the use of mechanisms and codon frequencies more similar to their a-proteobacterial ancestors than to the mammalian nuclear genome, and thus a successful expression of mitochondrial genes in cytosol requires codon optimization to recode mitochondrial gene sequences into the universal code (Lewis CJ, Dixit B, Batiuk E, et al. Codon optimization is an essential parameter for the efficient allotopic expression of mtDNA genes. Redox Biol. 2020 Feb;30: 101429. Doi: 10.1016 / j.redox.2020.101429). Codon optimization was performed in view of codon and tRNA abundances in human cells with relative adaptiveness of at least 50%. The designed codon-optimized sequence is only 73% identical to the human USH2A sequence.

[0064] The term “nucleic acid” refers to a DNA or RNA sequence. The term encompasses sequences comprising any known nucleobase analogues for DNA and RNA, including, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5 -fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl -2 -thiouracil, 5 -carboxymethyl aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxyaminomethyl -2 -thiouracil, P-D-mannosylqueuosine, 5 ’-methoxy carbonylmethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5 -oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methyl ester, uracil- 5-oxyacetic acid, pseudouracil, queuosine, 2-thiocytosine and 2,6-diaminopurine.

[0065] The term “USH2A minigene” refers to a minimal functional copy of USH2a gene produced using targeted biodesign to remove exons from the native structure of the natural USH2a gene. The nucleotide sequence composing the minigene is codon-optimized for expression in human cells. The resulting inventive USH2A miniprotein sequences comprising a unique combination of domains, comply with the mutational profile and structural features at both gene and protein levels. The presence of LamGL, LamNT, EGF Lam, FN3 domains and PDZ-binding motif ensures the required functionality of the USH2A protein, and the signal sequence and transmembrane domain in USH2A miniprotein structure enable its physiological localization. The sequence length of the USH2A minigene corresponding to the USH2A protein permits successful delivery into cells in viral vectors.

[0066] A nucleic acid with the claimed sequence can be produced by any method known in the art including, but not limited to, recombinant methods such as cloning nucleic acid sequences from a recombinant library or cellular genome using conventional cloning and PCR technologies etc., and methods of chemical synthesis.

[0067] In embodiments of the present invention, the nucleic acid comprises a nucleotide sequence selected from SEQ ID Nos 3, 7, 9, 11, and 13. In preferred embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO 3 or SEQ ID NO 7.

[0068] The nucleotide sequence of the present invention can be inserted into a plasmid expression vector, for example, a pAAV-based vector. Insertion of the plasmid expression vector comprising the nucleotide sequence of the present invention into HEK293T cells by transfection results in the USH2A minigene expression.

[0069] In addition, nucleic acids have low immunogenicity. Codon-optimized transgene sequences are less able to induce intracellular immune responses due to the minimal activation of interferon-stimulating genes expression (Galieva A., Egorov A., Malogolovkin A., Brovin A., Karabelsky A. RNA-Seq Analysis of Trans-Differentiated ARPE-19 Cells Transduced by AAV9-AIPL1 Vectors. International Journal of Molecular Sciences. 2024; 25(1): 197). Moreover, AAV vectors themselves are less immunogenic compared to adenoviruses (Rabinowitz J., Chan Y.K., Samulski R.J. Adeno-associated Virus (AAV) versus Immune Response. Viruses. 2019 Jan 25; 11(2): 102). In addition, administering an AAV vector under the retina significantly limits its systemic distribution and, consequently, activation of immune response. The eye is known to be separated by the blood-ocular barrier, and it lacks an intraocular lymphatic system; ocular stromal cells have low or no expression of MHC class I and II molecules on their surface. All of this defines predominance of gene therapies development in the field of ophthalmology. The amino acid protein sequence of the invention is not highly immunogenic due to being essentially a miniaturized version of the native Usherin protein; there are no new domains or linkers added to the mini-Usherin structure which could be recognized as foreign by a patient’s immune system.

[0070] A “coding sequence” or a sequence “encoding for” a selected polypeptide is a nucleic acid molecule that is transcribed (for DNA) and translated (for mRNA) into a polypeptide when placed under control of suitable regulatory sequences. The coding sequence boundaries are defined by the start codon at the 5 ’ (amino) terminal end, and by translation termination codon at the 3’ (carboxy) terminal end. The transcription termination sequence may be located downstream of the coding sequence.

[0071] A “vector” means any genetic element, such as a plasmid, a phage, a transposon, a cosmid, a chromosome, a virus, a virion, etc., which is capable of replication when linked with the proper control elements, and can transfer gene sequences into cells. Therefore, the term includes carriers for cloning and expression, as well as viral vectors.

[0072] The term “transfection” is used to refer to accumulation of foreign DNA by a cell, and a cell is said to be “transfected” when exogenous DNA is introduced across the cell membrane. A variety of transfection methods are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13: 197. Such methods can be used to introduce one or more exogenous molecules into suitable host cells. The term “expression vector” means a vector Comprising promoter sequence and other regulatory sequences which enable efficient transcription of a recombinant gene followed by the mRNA translation and production of a recombinant protein. The utilized plasmid and expression vectors disclosed below are exemplary only and do not limit the scope of the present invention.

[0073] In one embodiment, the present invention provides a plasmid expression vector comprising elements according to the physical and genetic map shown in Fig. 1. The expression vector for eukaryotic expression, comprising a nucleic acid that comprises a nucleotide sequence selected from SEQ ID Nos 3, 7, 9, 11, and 13, is disclosed. In preferred embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NO 3 or SEQ ID NO 7.

[0074] The term “promoter” as used herein specifically refers to a DNA nucleotide sequence recognized by a RNA polymerase whereon the transcription of an element operatively linked to a promoter is initiated. A promoter can also be accompanied by an enhancer.

[0075] Enhancers increase promoter activity and stimulate the transcription process. To produce large amounts of a protein in eukaryotic cells and, in particular, in human cells, it is beneficial to use strong promoters that are active in target cells. Strong constitutive promoters capable of driving recombinant gene expression in different cell types are well known in the art. In one embodiment of the invention, the cytomegalovirus promoter (CMV promoter) is used as a promoter.

[0076] In one embodiment of the invention, a plasmid expression vector comprises the following elements from the 5’-terminus to the 3’-terminus:

[0077] - the origin of replication (ori);

[0078] - a left inverted terminal repeat (ITR);

[0079] - a cytomegalovirus (CMV) promoter enhancer;

[0080] - the cytomegalovirus (CMV) promoter;

[0081] - the intron sequence of human b-globin gene;

[0082] - the optimized fUSH2A gene sequence of SEQ ID NO 3;

[0083] - a hGH polyadenylation (poly(A)) signal sequence;

[0084] - a right inverted terminal repeat (ITR);

[0085] - the ampicillin resistance gene promoter (AmpR);

[0086] - the ampicillin antibiotic resistance gene (AmpR).

[0087] In yet another embodiment of the invention, said vector comprises the sequence of SEQ ID NO 7 for the coding sequence for the USH2A miniprotein (uUSH2A, SEQ ID NO 6). In yet another embodiment of the invention, said vector comprises the sequence of SEQ ID NO 9 for the coding sequence for the USH2A miniprotein (cUSH2A, SEQ ID NO 8). In yet another embodiment of the invention, said vector comprises the sequence of SEQ ID NO 11 for the coding sequence for the USH2A miniprotein (kUSH2A, SEQ ID NO 10). In yet another embodiment of the invention, said vector comprises the sequence of SEQ ID NO 13 for the coding sequence for the USH2A miniprotein (yUSH2A, SEQ ID NO 12).

[0088] Transfection of cells with the plasmid expression vectors of the present invention results in the stable and high expression of the USH2A miniprotein suitable for therapy of Usher syndrome type II.

[0089] The term “gene expression” refers to translating hereditary information encoded in the nucleotide sequence of a gene into a functional product such as RNA or protein.

[0090] A “polypeptide” or “peptide”, as used in this specification, means two or more independently selected natural or non-natural amino acids linked by a covalent bond (e.g., peptide bond). A peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more natural or non-natural amino acids linked by peptide bonds. Polypeptides as described herein include full-length proteins (e.g., fully processed proteins) as well as shorter amino acid sequences (e.g., fragments of naturally occurring proteins, or synthetic polypeptide fragments).

[0091] In one embodiment, the invention provides an expression vector for expression in eukaryotic cells, said vector comprising the nucleic acid of the present invention. The nucleic acid of the present invention can be inserted into any vector known in the prior art, including plasmid and viral vectors, such as based on SV40 virus, adenoviruses, herpes viruses, retroviruses, lentiviruses, adeno-associated viruses and the like. Vectors useful for the insertion of the disclosed nucleic acid are not limited to the above list. The choice of expression vector depends on the intended applications and does not limit the scope of the present invention; the methods for producing vectors are known in the art; vectors comprising the nucleic acid of the present invention can be produced by those skilled in the field of gene engineering.

[0092] Adeno-associated virus is a non-pathogenic parvovirus comprised of 4.7 kb single-stranded DNA genome, and has a non-enveloped, icosahedral capsid. The viral genome comprises three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) which act as viral replication and packaging signals. Rep ORF encodes four nonstructural proteins that contribute to viral replication, transcription regulation, site-specific integration, and virion assembly. Cap ORF encodes three structural proteins (VP 1-3) that assemble to form a 60-mer viral capsid. Finally, ORF is present as an alternative reading frame in the cap gene and produces the assembly-activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions during capsid assembly.

[0093] There are several natural (“wild type”) serotypes and over 100 known AAV variants, all of them having different amino acid sequences, particularly in hypervariable regions of capsid proteins, and thus differing in their gene delivery properties. No association has been demonstrated between any AAV and human disease of any kind, making a recombinant AAV an attractive candidate for clinical applications.

[0094] For descriptive purposes herein, the term “AAV” is an abbreviation for adeno-associated virus including, without limitation, the virus itself and its derivatives. Except where otherwise noted, this terminology refers to all subtypes or serotypes, and to both replication competent and recombinant forms. The term “AAV” includes, but is not limited to AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrhlO), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infects primates, “non-primate AAV” refers to AAV that infects non-primate mammals, “bovine AAV” refers to AAV that infects bovine mammals, etc.

[0095] The genomic sequences of various AAV serotypes are known in the art, as well as sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits. Such sequences can be found in the literature or in the open databases such as GenBank. See, e.g., GenBank accession numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10), and AAO88208 (AAVrhlO) which descriptions are incorporated herein by reference for instructional purposes of AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et. Al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and US patent No. 6,156,303.

[0096] In preferred embodiments of the invention, the vector is the adeno-associated virus serotype 9, serotype 2.7m8, or serotype 5.

[0097] In one embodiment of the invention, the adeno-associated virus serotype 9, comprising the nucleic acid of the present invention and produced in the adherent HEK293T cell cultures, is provided as a viral expression vector. Co-administration of the plasmid expression vector comprising the nucleic acid of the present invention along with the pHelper vector and the packaging plasmid pRC2 into adherent HEK293T cell cultures leads to production of viral expression vectors representing the adeno-associated virus serotype.

[0098] In another embodiment of the invention, the adeno-associated virus serotype 2.7m8, comprising the nucleic acid of the present invention and produced in the adherent HEK293T cell cultures, is provided as a viral expression vector. Co-administration of the plasmid expression vector comprising the nucleic acid of the present invention along with the pHelper vector and the packaging plasmid pRC2.7m8 into adherent HEK293T cell cultures leads to production of viral expression vectors representing the adeno-associated virus serotype 2.7m8.

[0099] In the further embodiment of the invention, the adeno-associated virus serotype 5 comprising the nucleic acid of the present invention and produced in the adherent HEK293T cell cultures, is provided as a viral expression vector. Co-administration of the plasmid expression vector comprising the nucleic acid of the present invention along with the pHelper vector and the packaging plasmid pRC2 / 5 into adherent HEK293T cell cultures leads to the production of viral expression vectors representing the adeno-associated virus serotype 5.

[0100] The expression vectors of the present invention demonstrate stable and high cellular expression of the USH2A miniprotein. This suggests that administering any of the claimed expression vectors of the present invention into ocular tissue can result in stable expression of the USH2A miniprotein and elimination of the USH2A protein deficit in ocular cells and tissues, and, consequently, its functional recovery and vision stabilization in retinitis pigmentosa associated with Usher syndrome type II. Delivering plasmid and viral vectors of the present invention into cells harboring a natural mutation of the USH2A gene leads to accumulation of the USH2A miniprotein.

[0101] The terms “individual”, “host”, “subject” and “patient” are used interchangeably herein and refer to a mammal, including, but not limited to humans; non-human primates, including monkeys and apes; mammals used in sport (for example, horses); farm mammals (e.g., sheep, goats, etc.); mammals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some embodiments of the invention, the “individual” is a human.

[0102] As used herein, a cell is referred to as “stably” altered, transduced, genetically modified or transformed by a genetic nucleotide sequence if that sequence is able to perform its function during long-term culture of this cell in vitro and / or in vivo. Typically, this cell is “inheritably” altered (genetically modified) in that the introduced genetic alteration is also inherited by progeny of the altered cell. As used herein, the terms “therapy”, “treating”, “treatment” and the like refer to achieving a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or symptom thereof, and / or can be therapeutic in terms of partial or complete cure of a disease and / or side effect associated with the disease.

[0103] “Treatment” as used herein encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) prevention of the disease (and / or symptoms caused by the disease) in a subject who can be predisposed to the disease or at risk of contracting the disease, but has not yet been diagnosed as having the disease; (b) inhibition of the disease (and / or symptoms caused by the disease), i.e. cessation of the disease progression; and (c) alleviation of the disease (and / or symptoms caused by the disease), i.e. induction of the disease regression (and / or regression of symptoms caused by the disease), i.e. reduction in disease intensity and / or one or more symptoms thereof. For example, the provided USH2A miniprotein, the nucleic acid encoding the USH2A miniprotein, the expression vector comprising the same can be used for the treatment of Usher syndrome type II.

[0104] As used herein, the term “effective amount” is an amount sufficient to produce beneficial or desired clinical outcomes. An effective amount can be administered in one or more administrations. For the purposes of this specification, an effective amount of a compound (e.g., infectious rAAV virion) is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, prevent, slow down, or arrest the progression of (and / or symptoms associated with) a particular disease state (e.g. retinitis pigmentosa, hearing loss).

[0105] The term “retinal cell” refers herein to any of the cell types composing the retina, such as, but not limited to, retinal ganglion (RG) cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, Muller glial cells, microglia, and retinal pigment epithelium.

[0106] The term “administration” as used in the provided methods means delivery of a composition to a selected target cell that is characteristic for a disease of the eye and / or ear. In one embodiment, the composition is delivered by subretinal injection into photoreceptor cells or other ocular cells. In another embodiment, an intravitreal injection into ocular cells is used. In yet another embodiment, an injection into the eyelid vein may be used for delivery to ocular cells. Other routes of administration may be chosen by one skilled in the art taking into account this specification.

[0107] “Administration” or a “route of administration” means delivery of the composition described herein, with or without a pharmaceutical carrier or excipient, to a subject. Routes of administration can be combined if desired. In some embodiments, administration is repeated periodically. Pharmaceutical compositions described herein are designed for delivery to subjects in need thereof using any suitable route or a combination of different routes. Direct delivery to the eye (optionally by intraocular delivery, intraretinal injection, intravitreal, and local route), or systemic delivery via intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral routes are contemplated. Nucleic acid molecules and / or vectors described herein can be delivered in a single composition or multiple compositions. Optionally, two or more different AAVs or multiple viruses can be delivered (see, for example, WO 202011 / 126808 and WO 2013 / 049493). In another embodiment, multiple viruses can include various replication defective viruses (e.g., AAV and adenovirus), alone or in combination with proteins.

[0108] In one embodiment, viral constructs can be delivered in concentrations from at least I xlO6to at least IxlO11viral genomes (vg) in volumes from about 1 pl to about 3 pl for small animals, such as mice. For larger animals having eyes nearly the same size as human eyes, the higher human doses and volumes indicated above are used, for example, 1 xlO6to about IxlO15vg per dose. Good practice for administering substances to various animals is discussed in, for example, Diehl et al., J. Applied Toxicology, 21: 15-23 (2001). This document is incorporated herein by reference.

[0109] It is preferable to use the lowest effective concentration of a virus or other delivery means to reduce the risk of adverse effects such as toxicity, dysplasia, and retinal detachment.

[0110] Until otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, exemplary methods, devices and materials are described below. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0111] It should be understood that all numerical designations are preceded by the term “approximately” even though this is not necessarily explicitly stated. It should also be understood that reagents described herein are exemplary only, and their equivalents are known in the art, although this is not necessarily explicitly stated.

[0112] Before describing the present invention in detail, it should be noted that this invention is not limited to specific compositions or process parameters, which, of course, may actually vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. It should be understood that the invention should not be construed as limited to the examples described herein. Methods and materials similar or equivalent to those described herein may be used in practicing the present invention, and the invention should be construed as encompassing any and all uses contemplated herein, and all variations of equivalents within the skill of one of ordinary skill in the art.

[0113] The present invention primarily resides in the discovery that the USH2A miniprotein and nucleic acid encoding the same and packaged into the AAV vector exhibit stable and high expression of the USH2A minigene and production of the USH2A miniprotein required for the therapy of Usher syndrome type II.

[0114] EXAMPLES

[0115] The following examples are set forth so as to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0116] Example 1. Generation of plasmid expression vectors

[0117] Example 1.1. Codon optimization of the nucleotide sequence encoding fUSH2A, uUSH2A, cUSH2A, kUSH2A, and yUSH2A genes

[0118] The coding sequence for USH2A gene was annotated using the NCBI / CDD service. The UniPro UGENE tool was employed to align nucleotide sequences (Okonechnikov K., Golosova O., Fursov M., the UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics, 2012, 28: 1166-1167). The nucleotide sequence was translated to the amino acid sequence using a tool which can be found at https : / / web . expasy . org / translate . To account for the differences in the transfer RNAs abundances between cells of different tissues, the nucleotide sequence was optimized to exclude stop codons and non-synonymous codons. Transcript stability was investigated using the RNAfold program (http: / / ma.tbi.univie.ac.at / cgi- bin / RNAWebSuite / RNAfold.cgi). The nucleic acid having the optimized USH2A gene sequence (fUSH2A, SEQ ID NO 3) encoding the USH2A miniprotein sequence (fUSH2A, SEQ ID NO 4) was synthesized using TopGenetech (Canada) service. In addition, unstructured regions of the protein were analyzed using IUPred2 (https: / / iupred2a.elte.hu) to select optimal regions for optimization.

[0119] Example 1.2. Generation of the expression vector for fUSH2A expression pAAV-MCS (Cell biolabs, cat# VPK-410) was used as a plasmid expression vector. fUSH2A and uUSH2A sequences were inserted according to a technique recommended by the vector manufacturer, using restriction endonucleases BamHI and Hindlll (New England Biolabs Inc., USA) and T4 DNA ligase according to standard protocols. As a result, the recombinant plasmid expression vector pAAV-fUSH2A was obtained (Fig.

[0120] 1).

[0121] Cells were transformed according to a standard protocol (Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. 3rdEdition, Vol. 1, Cold Spring Harbor Laboratory Press, New York). For bacterial transformation, 100 pl of chemically competent Escherichia coli DH5a cells stored at -80°C were used and placed on ice for slow thawing. Next, 10 pl of ligase mixture was added to the cells and incubated on ice for 30 min. Plasmid DNA was delivered to bacterial cells using the heat shock method for 30 sec at 42°C in a water bath, then the tube with cells was transferred on ice and incubated for 2 min. Then, 1 ml of the LB nutrient medium preheated to 37°C was added to the cells and incubated in a thermostat for 1 hour at 37°C, followed by stirring at 180-200 rpm. 100 pl per plate of the transformed cell suspension was plated on Petri dishes with LB agar and ampicillin, pre-dried in a thermostat at 37°C. The cells were cultured in a thermostat at 37°C for 16-18 hours.

[0122] Table 1. Conditions for PCR reaction

[0123] To analyze colonies of transformed cells, PCR screening was performed using vectorspecific primers pAAV_For_seq2 and pAAV_Rev_seq2 (Table 1) and a ready-to-use mixture ScreenMix for PCR (Evrogen, Russia). The reaction mixture was prepared according to the manufacturer’s recommendations. Thermal lysates of single bacterial clones were used as templates. To this end, each of 8 single clones was first picked up with a tip and transferred to a Petri dish containing prepared template, and then the tip was immersed into a test tube containing 20 pl of mQ water. The Petri dish was incubated in a thermostat at 37°C for a day, and the test tubes (strips) containing water and bacteria were heated at 95°C for 5 minutes. 2 pl of suspension was taken for PCR reactions. PCR reaction was carried out using the following parameters: DNA pre-melting at 95°C for 3 minutes, 25 amplification cycles, including melting for 20 seconds at 95°C, annealing for 20 seconds at 55°C and elongation at 72°C for 2 minutes, final elongation at 72°C for 5 minutes. Clones carrying a correct-sized insert as confirmed by PCR were submitted for sequencing. Clones with the correct sequence were selected based on the sequencing results.

[0124] Culturing the selected clones yielded recombinant plasmid expression vectors, each of them comprising the USH2A sequence.

[0125] The recombinant plasmid expression vector pAAV-f7757724 expressing codon-optimized USH2A gene sequence (IUSH2A, SEQ ID NO 3) comprises the following elements (see Fig. 1):

[0126] - the origin of replication (ori);

[0127] - the left inverted terminal repeat (ITR);

[0128] - the cytomegalovirus (CMV) promoter enhancer;

[0129] - the cytomegalovirus (CMV) promoter;

[0130] - the human b-globin gene intron sequence (the hBGl gene intron - subunits of hemoglobin gamma- 1);

[0131] - the £USH2A sequence of SEQ ID NO 3;

[0132] - the hGH polyadenylation (poly(A)) signal sequence (the human growth hormone gene polyadenylation signal);

[0133] - the right inverted terminal repeat (ITR);

[0134] - the ampicillin resistance gene promoter (AmpR);

[0135] - the ampicillin antibiotic resistance gene (AmpR).

[0136] Example 1.3. In silico modeling

[0137] The IUSH2A nucleotide sequence (SEQ ID NO: 3) was obtained by in silico selection from designed variants. Modeling the secondary structure of a protein molecule can be carried out de novo, if there are no homologous structural models, or by comparison with proteins secondary structures available in databases, for example, in the Protein Data Bank (https: / / www.rcsb.org / ). Correctness and adequacy of the model are evaluated by analyzing known domains of a protein, or by preserving catalytic centers or conserved regions of a molecule which are required for the protein physiological functions. DNA having the claimed sequence can be produced by any method known in the art, including, but not limited to, recombinant methods such as cloning nucleic acid sequences from a recombinant library or a cell genome, using conventional cloning and PCR technologies and the like, as well as methods of chemical synthesis. The gene having the codon-optimized nucleotide sequence of the present invention can be inserted into a plasmid expression vector, for example, a plasmid or pAAV-based vectors. Introduction of the plasmid expression vector carrying the gene having the nucleotide sequence according to the present invention into eukaryotic cells, for example, HEK293T, by transfection results in expression of the heterologous USH2A gene.

[0138] Introduction of the plasmid expression vector carrying the gene with the nucleotide sequence of the present invention together with the pHelper vector and pRC2 / 9 or pRC2 / 5 or pRC2.7m8 vector into HEK293T cells results in the production of viral expression vector. Introduction of the resulting viral expression vector into HEK293T cells by transduction leads to the expression of heterologous £USH2A gene.

[0139] Example 2. Evaluation of the fUSH2A gene expression levels after HEK293T transfection

[0140] In order to perform transfection, cells were seeded into a 6-well plate at a density of 0.5 million cells / well. Transfection was carried out 30 hours after cell seeding (after cell confluency reaches 80%). Transfection was carried out using DNA-PEI complex at a ratio of 1:5, DNA load was 1 pg / 1 million cells.

[0141] After transfection, the cells were incubated for 2 days. The cells were lysed using two freeze / thaw cycles. After cell thawing RNAs were extracted using the RIBO-Prep kit, AmpliPrime®. The extracted RNA was used as a template for producing cDNA.

[0142] The resulting cDNA was used for running PCR reaction with 2 primers: for the target gene and for the housekeeping gene (PPIA).

[0143] Expression was assessed using the AACt method.

[0144] To evaluate the expression of the fUSH2A gene, the fUSH2A construct was transfected using PEI as a transfection agent. The IUSH2A-PEI complex was prepared and then added to the cells. Two days after transfection, RNA was extracted from the cell pellet. The resulting RNA was used as a template for producing cDNA.

[0145] The resulting isolated DNA samples from transfected and non-transfected cells were analyzed by quantitative PCR using 2 types of primers: for the fUSH2A target gene and the PPIA enzyme gene as a housekeeping gene. Non-transfected cells and cells transfected with the eGFP gene were used as controls. The amplification results are shown in Fig. 3.

[0146] Table 2. Results of calculation using the AACt method

[0147] AAV9-fUSH2A viral vector was produced by triple transfection of HEK293T cell culture.

[0148] HEK293T cells were cultured in DMEM supplemented with 5% FBS in the presence of penicillin / streptomycin antibiotics. 1 hour before transfection, the culture medium was replaced with DMEM supplemented with 2% FBS. Transfection was performed by adding 1.5 pg of total DNA / million cells (at a 1:2:5 ratio for pGOI:pHelper:pRepCap plasmids). For pHelper and pRepCap plasmids, a ratio DNA:PEI was 1:5. 24 hours after transfection, the culture medium was replaced with DMEM supplemented with 5% FBS. The cells were lysed 72 hours after transfection by two cycles of freezing / thawing, and by adding Triton X-100 to a final concentration of 0.1%.

[0149] Cell lysate was concentrated 10-fold using centrifuge cassettes with a 100 kDa cutoff. The resulting solution was dialyzed against PBS solution with 10-fold excess of initial concentrated solution. Lysate filtered through a 0.22 pm filter and the dialyzed concentrated sample were used as the initial solution for purification.

[0150] The resulting filtrate was used for isolation on POROS AAVX chromatography sorbent (ThermoFisher).

[0151] After storage, the sorbent was pre-washed with purified water and regenerated using 100 mM glycine solution, pH 3.0, and 10 mM NaOH, pH 11. Next, it was washed with water and equilibrated with PBS solution. After applying the cell lysate, the column was washed with equilibration buffer. Additional washing was carried out in 0.1 M citrate buffer solution, pH 6.2. Elution was conducted using 100 mM glycine solution, pH 3.0. A strip washing was conducted using 100 mM glycine solution, pH 2.2. The sorbent was regenerated using 10 mM NaOH solution, pH 11. Stages of the purification process are described in Table 3.

[0152] Table 3. The order of stages for viral particle purification.

[0153] The resulting eluate fractions were analyzed by quantitative PCR and PAGE. The results of PAGE analysis are shown in Fig. 4. This figure shows the presence of three capsid proteins specific for an adeno-associated virus with VP1, VP2, and VP3 MWs equal to 84 kDa, 69 kDa, and 62 kDa, respectively. Electrophoresis also shows the absence of irrelevant protein impurities in all eluate fractions. In this case, a ratio of VP1:VP2:VP3 proteins in the viral capsid is about 1: 1: 10, which is confirmed by the presented electrophoregram.

[0154] For cells transfected with the pUSH2A plasmid, RNA expression levels were 50,082 times higher compared to the non-transfected cell sample control. For cells transfected with the fUSH2A plasmid, this value was 72,405 (see Table 2). This confirms the RNA synthesis from the delivered construct in HEK293T cells. Furthermore, due to the lack of USH2A protein synthesis in the evaluated cell culture, a significant difference in protein expression levels is observed compared to non-transfected cells (tens of thousands).

[0155] Example 3. Evaluation of the fUSH2A sequence expression levels after transduction in AAV-hased viral vectors

[0156] Viral AAV vectors were produced using the plasmid expression vector pAAV-fUSH2A and a commercially available helper plasmid (pHelper), and the packaging plasmid pRC9 for AAV9 (Cell Biolabs Inc., USA).

[0157] Adherent HEK293T cell line was thawed and cultured according to standard operating procedures and techniques. At the time of production, the estimated density of the cell culture was 106cells / ml, the volume of the cell culture was 30 ml. Transfection was carried out in 500 ml Erlenmeyer flasks (working volume 175 ml). A seeding dose was 5x105cells / ml. Plasmid transfection was carried out 12 hours after seeding. Transfection conditions: cell density during transfection was 106cells / ml, 85-90% viability, amount of DNA was 1.5 pg / 1 million cells. A DNA:PEI ratio was 1:5, the transfection mixture volume was 5% of the cell culture volume. After transfection, the cells were incubated at 37°C, 5% CO2, 75% humidity, and 100 rpm for 120 h.

[0158] For cell lysis, Tween-20 was added to the flask to a concentration of 0.05% and incubated for 1 h. Next, benzonase was added to 20 lU / ml, and MgCU to 1-2 mM, and the cells were incubated for 1 h. Lysates were centrifuged for 10 min at 3000 g and filtered through 0.22 pm filters. Filtrates were concentrated from 525 ml to 50 ml by tangential filtration with molecular weight cutoff of 100 kDa at a transmembrane pressure of 1.5-2 bars. Affinity chromatography was carried out on AAVX sorbent (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s recommendations.

[0159] Viral titers in resulting AAV samples were measured using RT-PCR, and physical titers and impurities (low molecular weight or high molecular weight aggregates) were assessed using PAGE (polyacrylamide gel electrophoresis) and DLS.

[0160] All samples were found to predominantly contain particles in the 20-25 nm size range, and a minor amount of high molecular weight impurities (aggregates).

[0161] Example 4. Evaluation of the USH2A miniprotein amounts in cell lysates by enzyme-linked immunosorbent assay (ELISA)

[0162] To assess USH2A protein levels, the Human Usherin USH2A EEISA Kit (Abclonal, Cat. No. RK12051) was used. The protein was produced in HEK293T cell culture by transfection with pAAV-fUSH2A and pAAV-uUSH2A plasmids in AAV2.7m8 comprising fUSH2A miniprotein (SEQ ID NO 4), and the uUSH2A miniprotein (SEQ ID NO 6) sequences.

[0163] HEK293T cells cultivation. One day before transfection, the cells were transferred to a plate; 1 hour before transfection, the cells were transferred to serum-free medium and incubated in the serum-free medium prior to transfection; 5 hours after transfection, the culture medium was replaced with DMEM supplemented with 5% FBS. The cells were seeded into a six-well plate at 1 million cells per well. Transfection was performed using PEI max (1 mg / ml polyethylene amine from Polyscience (Cat. No. 24675-1) and 1 pg of plasmid DNA per well). A PEI:DNA ratio was 5: 1. The cells were incubated for 372 h after transfection. Non-transfected HEK293T cells were used as a control. The following lysis buffer was used to lyse the cell pellet: 50 mM Tris, 150 mM NaCl, 1% NP-40, pH 8.0 (a hydrochloric acid solution was used for adjusting pH levels). The limit of quantification is 3.9 pg / ml. For the protein quantification, a series of standards included in the kit (1000 - 15.6 pg / ml) were pre-prepared. The reaction was designed in accordance with the manufacturer’s recommendations. Optical density measurements were conducted on Allsheng Feyond-A300 plate reader (China) at a wavelength of 450 nm. The amount of the USH2A miniprotein was calculated using Excel software.

[0164] The results of the fUSH2A miniprotein (70.22 pg / ml) and uUSH2A miniprotein (93.58 pg / ml) synthesis evaluations 72 hours after pAAV-fUSH2A and pAAV-uUSH2A vector transfection in HEK293T cells, and in the non-transfected HEK293T lysate (30,15).

[0165] As can be seen in Figs 5 and 6, the results show a 2.5-3-fold increase in the miniprotein amount in transduced HEK293T cells compared to non-transfected cells. The miniprotein detected in cell lysates indicates the presence of the miniprotein in its authentic conformation. The interaction of antibodies with the miniprotein suggests that the obtained proteins present the antibody-binding sites, also confirming the correct secondary structure thereof.

[0166] Taken together, the results of the enzyme immunoassays confirm the presence of the miniprotein protein in eukaryotic cell resulting from delivery in an expression construct.

[0167] Example 5. Evaluation of the AAV titer by digital droplet PCR (ddPCR)

[0168] AAV samples for analysis were obtained in an adherent HEK293T cell line using a three- plasmid system. A detailed protocol for obtaining AAV is set forth in the Example of AAV production. Briefly, the cell suspension was lysed by adding of buffer solution containing 10 mM Tris, 20 mM MgCU, 1% Triton X-100, pH 7.5, to 10% of the total volume. 90 U of benzonuclease per ml of cell suspension was also added to the solution. The resulting solution was incubated for 1 h at 37°C with stirring. After incubation, the solution was centrifuged at 3000xg for 20 minutes. A supernatant was collected and filtered through a 0.22 pm PES filter. The resulting filtrate was used for isolation on POROS AAVX chromatography sorbent (ThermoFisher Scientific, USA). The volume of the chromatography sorbent was 0.8 ml. Hanbon Bio-lab 30 chromatograph and Diba Omnifit 6.6x150 mm chromatography column were employed. After storage, the sorbent was pre-washed with purified water and regenerated with 100 mM glycine solution, pH 3.0, and 10 mM NaOH, pH 2.2. Next, it was washed with water and equilibrated with PBS solution. After applying the cell lysate, the column was washed with equilibration buffer. Elution was carried out using 100 mM glycine solution, pH 3.0.

[0169] In order to measure the number of AAV genomes, 2 types of primers were employed depending on the gene of interest used (see Table 4). A system with a fluorescent probe was used for the primers directed to AAV-ITR (inverted terminal repeats), and gene-specific primers and Eva Green system with an intercalating dye were used for the transgene detection. RainSure Supermix ddPCR Reaction mixture for probes (186-3026). EvaGreen Reaction mixture for QX200ddPCR (186-4033). Amplifier RainSure QX200 for digital droplet PCR (186-4001).

[0170] Table 4. The ddPCR mixture composition

[0171] The following criteria were used to validate the digital droplet PCR method: 1. Primer annealing temperature . Linearity

[0172] 3. Intermediate precision

[0173] 4. Limit of quantification

[0174] Primer annealing temperature

[0175] To determine the optimal primer annealing temperature, the temperature range with a center point of 570°C and a temperature difference of ±50°C was used. In the gradient mode of the cycler software 8 points for screening were selected. HEK293T cell lysate comprising the AAV2.7m8-GFP construct was used as a sample for experimental setup. As the two dye-systems were used for the digital PCR setup, z.e., Eva Green and AAV-P probe, annealing temperatures for the primers were evaluated in the indicated temperature range for both variants. The results obtained are presented in Table 5 and Figs. 7 and 8.

[0176] Table 5. Optimization parameters for primer annealing temperatures and AAV amounts (vg / ml) primers was 55 °C.

[0177] Linearity

[0178] Three-fold dilutions over the range of 100 to 220,000 were chosen for plotting a linear function for the tested eluate sample.

[0179] The samples were prepared according to the following scheme. 90 pl of purified water and 10 pl of eluate sample containing AAV2.7m8 encoding fUSH2A and uUSH2A miniprotein variants, were added to a 1.5 ml polypropylene tube. The resulting solution was stirred (lOx predilution - solution 1). 90 pl purified water and 10 pl of solution 1 were added to a 1.5 ml polypropylene tube. The resulting solution was stirred (lOOx dilution - solution 2). 20 pl purified water and 10 pl of solution 2 were added to a 1.5 ml polypropylene tube. The resulting solution was stirred (300x dilution - solution 3). 20 pl purified water and 10 pl of solution 3 were added to a 1.5 ml polypropylene tube. The resulting solution was stirred (900x dilution - solution 4). 20 pl purified water and 10 pl of solution 4 were added to a test tube. The resulting solution was stirred (dilution 2,700x - solution 5). 20 pl purified water and 10 pl of solution 5 were added to a test tube. The resulting solution was stirred (dilution 8100x - solution 6). 20 pl purified water and 10 pl of solution 6 were added to a test tube. The resulting solution was stirred (dilution 24,000x - solution 7). 20 pl purified water and 10 pl of solution 7 were added to a test tube. The resulting solution was stirred (dilution 73,000 - solution 8). 20 pl purified water and 10 pl of solution 8 were added to a test tube. The resulting solution was stirred (dilution 220,000 - solution 9). The following parameters were used for the amplification process: polymerase activation at 95°C for 10 minutes; DNA melting at 95°C for 30 seconds; primers annealing at 55 °C for 30 seconds. Number of cycles: 40.

[0180] After the process was completed, chips with the samples were loaded to an imager and scanned. The assay results are presented in Figs. 9 and 10, and in Table 6.

[0181] Table 6. Results of detection limit determination for AAV genomes using digital droplet

[0182] PCR

[0183] The results suggest that the linear relationship is maintained if the number of positive droplets is 90% to 1%. Testing AAV amounts above and below this specified range will yield concentrations outside the linear range. The results suggest that converging results could be obtained over the range from 90 to 1% of positive droplets. For AAV2.7m8-fUSH samples in a dilution range of 900 to 220,000, the relative standard deviation (RSD) is 9.06%, the average value is 1.44+E10 vg / ml.

[0184] Intermediate precision

[0185] AAV-fUSH sample was used to establish precision.

[0186] First, a series of dilutions from lOx to 80,000x was prepared. The mixture for setting PCR was prepared according to the following scheme. The following reagents were added to a 1.5 ml polypropylene tube: 44 pl of master mix for fluorescent probe, 8 pl of forward primer, 8 pl of reverse primer, 4.4 of AAV-P fluorescent probe, 2 pl of purified water. The resulting solution was stirred and stored on ice.

[0187] Samples of solutions Nos. 12-15 were used for loading (Table 5). To this end, 5.5 pl of each solution was transferred into a separate tube. Next, 16.5 pl of the mixture for PCR setup was added to each test tube and thoroughly mixed. The chip for the digital PCR setup was prepared by adding 75 pl of the oil for probes to the rectangular well, and 20 pl of sample to the sample well, sealed with caps and placed to the amplifier.

[0188] The assay results for three independent experiments are presented in Table 7.

[0189] For each of the three experiments, the relative standard deviation was 14.5, 11.5, and 26.8% for comparison between different concentrations.

[0190] The RSD for comparison of average values between each of the measurements is 12.9%.

[0191] Table 7. The results of intermediate precision measurements and calculation of standard deviation

[0192] Limit of quantification for AAV genomes by digital droplet PCR

[0193] AAV-fUSH sample was used to determine the limit of quantification. The mixture for the PCR setup was prepared according to the following scheme. The following reagents were added to a 1.5 ml polypropylene tube: 44 pl of master mix for fluorescent probe, 8 pl of forward primer, 8 pl of reverse primer, 4.4 of AAV-P fluorescent probe, 2 pl of purified water. The resulting solution was stirred and stored on ice. Samples of solutions Nos. 20-27 were used for loading (Table 8). To this end, 5.5 pl of each solution was transferred into a separate tube. 16.5 pl of the mixture for the PCR setup was added to each test tube and thoroughly mixed. The chip for the digital PCR setup was prepared by adding 75 pl of the oil for probes to the rectangular well and 20 pl of sample to the sample well, sealed with caps and placed to the amplifier.

[0194] The assay results for three independent experiments are presented in Table 8.

[0195] Table 8. Test results for three independent experiments Thus, the limit of quantification for the digital droplet PCR utilizing fluorescent probe system is 12.2 AAV vg / pl, corresponding to 3.0+E05 and 2.4+E02 vg / well dilutions, with 0.8% of positive spots. The provided method allows to determine the number of genomes for viral AAV-based vectors with high precision which is extremely important for dosing a drug for the gene therapy of Usher syndrome type IL Failure to comply with dosing regimens, in particular using doses exceeding an optimal dose can result in toxic effects in patients and cause adverse side effects associated with body responses to administration of large amounts of a viral vector. At the same time, dosages of gene therapeutics lower then recommended ones may often result in the lack of therapeutic effect. Digital droplet PCR, in contrast to quantitative PCR, provides accurate absolute quantitative values for AAV titers while not requiring comparative approximations to standard samples.

[0196] Example 6. In silica modeling

[0197] To confirm the correctness and authenticity of the secondary structure for the USH2A miniprotein variants generated by rational design, in silico modeling was performed to predict the secondary structure of the proteins. For this purpose, the fUSH2A, uUSH2A, cUSH2A, kUSH2A, and yUSH2A minigenes’ sequences were translated into amino acid sequences using the EMBOSS TransSeq online tool (https: / / www.ebi.ac.uk / Tools / st / emboss transeq / ), query date: 10 / 27 / 2023. Next, the obtained amino acid sequences were used to build models using the AlphaFold2 (https: / / githiib.com / googlc-dcepmind / alphafold). Roberta

[0198] (https : / / robetta.bakerlab . org / ) and I-TASSER services (https: / / zhanggroup.org / I-TASSER / ). Data on the domain structure of the natural human USH2a protein (LamGL, LamNT, EGF Lam, FN3, LamG, TM, PDZ) were used as a comparison model. Miniproteins of SEQ ID Nos 4, 6, 8, 10, and 12 showed the claimed secondary structure conforming to the domain organization and generated nucleotide sequence (Fig. 10). The model parameters for miniproteins of SEQ ID Nos 4 and 6 were considered acceptable.

[0199] In summary, generation of fUSH2A, uUSH2A, cUSH2A, kUSH2A and yUSH2A minigene variants, encoding fUSH2A, uUSH2A, cUSH2A, kUSH2A and yUSH2A miniproteins, is disclosed. Also disclosed are the results of the codon optimization of said genes, and the results of production of AAV vectors for packaging the fUSH2A and uUSH2A minigenes. The fUSH2A minigene delivery and expression as part of viral AAV2.7m8 vector in human retinal pigment epithelial cells were shown. The fUSH2A and uUSH2A miniproteins production was confirmed by interaction with anti-USH2A protein antibodies in the enzyme-linked immunosorbent assay. While embodiments of the subject invention have been described in some detail, it will be understood that obvious variations may be made without departing from the spirit and scope of the invention as defined herein.

Claims

CLAIMS1. A USH2A miniprotein for the gene therapy of Usher syndrome type II, comprising an amino acid sequence selected from SEQ ID Nos 4, 6, 8, 10, and 12.

2. The USH2A miniprotein of claim 1, comprising the amino acid sequence of any one of SEQ ID NO 4, SEQ ID NO 6.

3. A nucleic acid encoding the USH2A miniprotein of claim 1.

4. The nucleic acid of claim 3, comprising a nucleotide sequence selected from SEQ ID Nos 3, 7, 9, 11, 13.

5. The nucleic acid of claim 4, comprising the nucleotide sequence of any one of SEQ ID NO 3, SEQ ID NO 7.

6. An expression vector for expression in eukaryotic cells, said vector comprising the nucleic acid of claims 3-5.

7. The vector of claim 6, wherein the vector is a plasmid expression vector comprising the following elements:- the origin of replication (ori);- a left inverted terminal repeat (ITR);- a cytomegalovirus (CMV) promoter enhancer;- a cytomegalovirus (CMV) promoter;- the human b-globin gene intron sequence;- the optimized sequence of fUSH2A gene of SEQ ID NO 3;- a hGH polyadenylation (poly(A)) signal sequence;- a right inverted terminal repeat (ITR);- the ampicillin resistance gene promoter (AmpR);- the ampicillin antibiotic resistance gene (AmpR).

8. The vector of claim 7, wherein the vector is a viral expression vector.

9. The vector of claim 8, wherein the vector is an adeno-associated virus.

10. The vector of claim 9, wherein the vector is the adeno-associated virus serotype 9, serotype 2.7m8 or serotype 5.

11. The vector of claim 10, produced in adherent HEK293T cell cultures.

12. The use of the vector according to claims 6-11 for the gene therapy of Usher syndrome type II.

13. The use of the vector of claim 12 for the gene therapy of retinitis pigmentosa and hearing loss associated with Usher syndrome type II.

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