ABCA4 miniprotein, nucleic acid encoding ABCA4 miniprotein, and its expression vector for gene therapy

Abstract

FIELD: biotechnology.SUBSTANCE: ABCA4 protein containing the amino acid sequence SEQ ID NO: 6, the nucleic acid encoding it, the vector for expression in eukaryotic cells and the use of said vector for gene therapy of Stargardt disease.EFFECT: invention provides an ABCA4 protein, as well as a vector that ensures stable and high expression of said protein.10 cl, 9 dwg, 4 tbl, 6 ex

Description

[0001] The invention relates to the field of molecular biotechnology, in particular genetic engineering, and specifically to the ABCA4 miniprotein, the nucleic acid encoding the ABCA4 miniprotein, and the expression vector containing it. It can be used in gene therapy for Stargardt disease type 1. Use of a vector containing the nucleic acid encoding the ABCA4 miniprotein results in stable and high expression of the ABCA4 miniprotein.

[0002] Stargardt disease (hereinafter referred to as Stargardt disease, SD) is a progressive macular degeneration of the retina, inherited in an autosomal recessive manner, characterized by damage to the retinal pigment epithelium and photoreceptors. The incidence rate varies in different populations, but is estimated at 1:8,000–10,000 [Blacharski PA Fundus Flavimaculatus. Retinal dystrophies and degenerations. NY: Raven Press 1988; 135–139].

[0003] Interest in studying this disease is primarily due to its social significance, as it leads to disability in childhood and adolescence. The clinical manifestations of BS vary widely in age of onset and phenotype severity. Most often, the disease occurs between the ages of 7 and 15 years, but can debut in older patients, and in rare cases even after 50 years. The clinical picture is characterized by bilateral visual acuity loss, dyschromatopsia, central scotoma formation, and an atrophic lesion in the macular zone with yellow-white spots localized around the lesion or spread throughout the fundus. The degree and rate of progression of morphofunctional impairment are variable: from moderate and slow changes to a significant decline in visual function and pronounced atrophic changes in the chorioretinal complex, leading to the development of severe geographic atrophy.A better prognosis is usually associated with late onset, when visual acuity declines more slowly. However, even in these cases, with long-term observation, visual loss is similar to early-onset BS [Tanna P., Strauss RW, Fujinami K., Michaelides M. Stargardt disease: clinical features, molecular genetics, animal models and therapeutic options / / Br. J. Ophthalmol. 2017. 101. (1). 25–30.]. The different phenotype depends on the genetic variant of the mutation and the degree of residual gene functions [Sheremet N.L., Grushke I.G., Zhorzholadze N.V., Ronzina I.A., Mikaelyan A.A., Kurbatov S.A., Kadyshev V.V., Anoshkin K.I., Strelnikov V.V. Spectrum of molecular genetic disorders and diversity of clinical forms of Stargardt disease. Siberian Scientific Medical Journal. 2018;38(5):65-71].

[0004] It has been established that mutations in the ABCA4 gene are the cause of the development of four clinically polymorphic retinal abiotrophies: Stargardt disease, Franceschetti type retinal abiotrophy, cone-rod and mixed pigment retinal abiotrophy [Shurygina, M. F., Borzenok, S. A., Khlebnikova, O. V., & Solomin, V. A. (2013). Clinical and molecular genetic features of the diagnosis of central retinal abiotrophies in the Russian Federation. Bulletin of the Orenburg State University, (4 (153)), 311-314].

[0005] Currently, there are no medical treatments to prevent, treat, or slow the progression of the disease. There is also no cure for retinitis pigmentosa [Tikhomirova M.A. Usher syndrome / / GENOKAPTA Genetic Encyclopedia, 2019].

[0006] In recent years, gene replacement therapy has been explored as a potential treatment for Stargardt disease. This therapy involves replacing an entire gene with a healthy copy by cloning it into a viral vector, such as the adeno-associated virus (AAV), and delivering this copy to the target cells.

[0007] Currently, there are no registered drugs or treatment methods for SB that have fully undergone clinical trials. However, in recent years, a large number of experimental and clinical studies have been conducted aimed at finding ways to reduce the accumulation of vitamin A dimers, lipofuscin, inhibit complement, as well as RPE regeneration using stem cell transplantation. Research is also being conducted on gene therapy with intravitreal vector administration of the functional ABCA4 gene [Louise JL, Ji L, Ron A. Adelman. Novel therapeutics for Stargardt disease. Graefe's Archive for Clinical and Experimental Ophthalmology. 2017;255(6):1057-1062].

[0008] Lentiviral and nonviral vectors have recently been developed for the delivery of large genes to photoreceptor cells. EIAV-ABCA4 is an equine infectious anemia lentivirus (EIAV) carrying the ABCA4 gene. A safety assessment of subretinal injection of EIAV-ABCA4 in rabbits and macaques revealed transient local inflammatory reactions. At the same time, intraocular pressure and ERG data were not changed, histological evaluation did not reveal any detrimental effects on the retina, and long-term systemic toxicity was not detected [Binley K, Widdowson P, Loader J, Kelleher M, Iqball S, Ferrige G, de Belin J, Carlucci M, Angell-Manning D, Hurst F, Ellis S, Miskin J, Fernandes A, Wong P, Allikmets R, Bergstrom C, Aaberg T, Yan J, Kong J, Gouras P, Prefontaine A, Vezina M, Bussieres M, Naylor S, Mitrophanous KA.Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease. Investigative Ophthalmology and Visual Science. 2013;54:4061–4071.]. Positive results from animal studies prompted the initiation of a clinical trial of SAR422459, formerly known as StarGen™, in patients with SS [ClinicalTrials.gov Identifier: NCT01367444], sponsored by Oxford Biomedica in collaboration with Sanofi. The clinical trial, which began in June 2011, was terminated in September 2019, not for safety reasons, as reported by the sponsors, but due to a review of clinical development plans and priorities (Oxford BioMedica Announces Ocular Clinical Programme Update. 19 / 11 / 2012. Accessed June 23, 2020).

[0009] Despite the ability of lentiviruses to package larger transgenes, their use is associated with potential risks. Lentiviruses can induce unwanted immune responses, have limited transduction capacity, and can integrate the transgene-carrying virus into the patient's DNA, which carries risks of insertional mutagenesis or oncogene activation [Ahmed, H., Shubina-Oleinik, O., Holt, JR 2017. Emerging gene therapies for genetic hearing loss. J Assoc Res Otolaryngol 18, 649-670].

[0010] To improve patient safety, approaches using AAV vectors are known in the art. Given the limited capacity of AAV vectors, several approaches currently exist to address this issue: dual AAV vectors and the minigene approach.

[0011] The use of various dual AAV vectors to evaluate the successful expression of functional myosin VIIa has been described [Dyka, FM, Boye, SL, Chiodo, VA, Hauswirth, WW, Boye, SE 2014. Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene, MYO7A. Hum Gene Ther Methods 25, 166–77]. The studies were successful in an in vitro model system; however, this success did not translate to in vivo experiments. The use of dual vectors has had limited success in animal models when attempting to deliver large genes to the retina [Ferreira, MV; Fernandes, S.; Almeida, AI; Neto, S.; Mendes, JP; Silva, RJS; Peixoto, C.; Coroadinha, AS Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses. Int. J. Mol. Sci. 2023, 24, 10524].

[0012] A dual AAV vector approach for ABCA4 gene delivery was developed. Dual AAV vectors were found to effectively deliver the ABCA4 gene to photoreceptors in mouse and pig models and reverse the retinal phenotype of ABCA4 (–) / (–) mice, demonstrating the potential for this approach to be used in gene therapy for SS [Trapani I. Dual AAV Vectors for Stargardt Disease. Methods in Molecular Biology. 2018;1715:153-175.].

[0013] A minigene approach was used to create a truncated version of the ABCA4 gene. The gene encoding the ABCA4 protein is located on chromosome 1 (chr1: 93,992,834 - 94,121,148) and is approximately 150,000 base pairs in size. The ABCA4 protein has a complex domain organization and consists of 2,273 amino acid residues, weighing 256 kDa (SEQ ID No. 1). Genes (SEQ ID No. 2) of this size cannot be delivered in viral vectors.

[0014] At IVERIC bio (formerly Ophthotech corporation), a biopharmaceutical company specializing in the development of novel gene therapies for orphan hereditary retinal diseases, several ABCA4 minigene constructs were created and evaluated in both in vitro and in vivo experiments. Additional experiments were conducted to optimize the constructs and evaluate their efficacy in a mouse model.

[0015] At present, a phase 2b clinical trial (NCT03364153) is underway, in which 95 patients with BS are being examined for 18 months after IVF of Zimura, which began in 2018. The study assessed the average rate of change in the area of ​​the ellipsoidal zone defect using optical coherence tomography data.

[0016] We analyzed the sequences provided in patent WO2020214809, where the authors propose six minisequences of the ABCA4 gene, ranging in size from 3,500 to 4,400 bp, including variants containing the rhodopsin kinase (GRK) promoter, a constitutive promoter, an inducible promoter, a tissue-specific promoter, and the chicken beta-actin (CBA) promoter. Some of the resulting miniversions resulted in reduced fundus autofluorescence in ABCA4 knockout mice. The analysis was performed using fluorescence microscopy. ZO-1 was used as a cell marker. However, the given sequences of ABCA4 minigenes do not contain key structures (for example, ATP sites and aa interacting with the substrate are cut out), which, according to the literature, are responsible for the functioning of the protein and are necessary for the specific functioning of photoreceptor cells, thus high levels of expression from such structures are impossible.

[0017] Therefore, there is currently no registered effective and safe therapy for Stargardt disease type 1. Despite progress in developing gene therapy for this disease, there remains a need to develop a suitable nucleotide sequence encoding a suitable ABCA4 protein. Such a sequence, protein, and vector are provided in the present invention.

[0018] The resulting ABCA4 miniprotein sequences, which contain a unique combination of domains, were found to accommodate the mutational profile and structural features at both the gene and protein levels. Portions of the ECD1, ECD2, NBD1, NBD2, and TM domains were removed, while retaining the active and ATP-binding sites. The presence of the NBD1 and NBD2 cytoplasmic domains ensures the desired functionality of the ABCA4 protein, while the signal sequence and transmembrane domain in the ABCA4 miniprotein structure ensure its physiological localization. The length of the ABCA4 minigene sequence, corresponding to the protein, allows for its successful delivery into cells within viral vectors.

[0019] The present invention relates to an ABCA4 miniprotein, a nucleic acid encoding the ABCA4 miniprotein, and an expression vector containing the nucleic acid for expression in eukaryotic cells. The vectors are used for gene therapy of Stargardt disease type 1.

[0020] The nucleic acid encoding the ABCA4 miniprotein, according to the present invention, was efficiently packaged into an AAV vector in general, and specifically into AAV vectors of serotypes 2, 9, 5, or 2.7m8. The minimal transgene size allowed for the production of a high-titer AAV vector (>1.0 x 10^12 vg / ml), which will simplify the process of their mass production and purification.

[0021] The present invention is based on the discovery that combining ABCA4 protein domains in a miniprotein reduces the size of the native ABCA4 gene nucleotide sequence by 30%, while still maintaining a nucleic acid sequence length that allows for its successful delivery into cells within viral vectors. The proposed nucleic acid sequence retains the core functional domains of the ABCA4 protein while sequestering the number of domains in varying amounts.

[0022] The nucleic acid also exhibits low immunogenicity. Thus, the ABCA4 miniprotein, nucleic acid, and vector overcome all the shortcomings of prior art technical solutions for gene therapy of Stargardt disease type 1.

[0023] The use of such a miniprotein, nucleic acid, and vector containing it for gene therapy of retinitis pigmentosa in Stargardt disease type 1 has not been reported until recently.

[0024] The invention relates to an ABCA4 miniprotein for gene therapy of Stargardt disease type 1, comprising the amino acid sequence SEQ ID No. 6.

[0025] An expression vector for expression in eukaryotic cells, containing a nucleic acid encoding the ABCA4 miniprotein. Preferably, the vector is a plasmid expression vector containing the following elements:

[0026] – replication start site ori;

[0027] – left inverted terminal repeat ITR;

[0028] – CMV cytomegalovirus promoter enhancer;

[0029] – cytomegalovirus promoter CMV promoter;

[0030] – human b-globin gene intron sequence;

[0031] – optimized ABCA4 gene sequence SEQ ID No. 7;

[0032] – hGH polyadenylation signal sequence poly(A) signal;

[0033] – right inverted terminal repeat ITR;

[0034] – replication origin site for packaging into f1 ori phage particles;

[0035] – promoter of the ampicillin resistance gene AmpR promoter;

[0036] – gene of resistance to the antibiotic ampicillin AmpR.

[0037] In another aspect, the vector is a viral expression vector. In particular, the vector is an adeno-associated virus. In a further aspect, the vector is adeno-associated virus serotype 2, serotype 9, serotype 2.7m8, or serotype 5. In another aspect, the vector is produced in adherent HEK293T cell cultures.

[0038] Use of the vector in the present invention for gene therapy of Stargardt disease type 1. In another aspect, the use of the vector for gene therapy of retinitis pigmentosa Stargardt disease type 1.

[0039] The invention is explained by drawings and diagrams.

[0040] Figure 1 shows the genomic organization of the ABCA4 gene and the variants of the ABCA4 gene transcripts (mRNA). The gene encoding the ABCA4 protein is located on chromosome 1 (chr1: 93,992,834 - 94,121,148) and has a size of 128,315 base pairs.

[0041] Figure 2 shows the domain organization of the ABCA4 miniprotein (4272 base pairs, SEQ ID 7). Portions of the ECD1, ECD2, NBD1, NBD2, and TM domains were deleted, while the active sites and ATP-binding sites were preserved.

[0042] Figure 3 shows the results of bioinformatics analysis of the ABCA4 miniprotein. The amino acid sequence of the ABCA4 miniprotein was used to construct a similarity-based model using the I-TASSER program (https: / / zhanggroup.org / I-TASSER / ) Access date: 01.02.2024. The figure shows the domains and their secondary structure (beta sheets, alpha helices, and non-structural loops). The domain organization of the presented structure completely reproduces the calculated miniprotein sequence in Fig. 2.

[0043] Fig. 4 shows the structure of the expression vector pAAV-isoABCA4_1 containing the ABCA4 gene mini-isoform gene. The recombinant plasmid expression vector pAAV-isoABCA4_1 expressing the optimized sequence of the ABCA4 gene mini-isoform with a size of 2940 bp, consisting of the following elements: the replication origin ori; the left inverted terminal repeat ITR; CMV enhancer of the cytomegalovirus promoter; cytomegalovirus promoter CMV promoter; the sequence of the human beta-globin gene intron (intron of the hBG1 gene - the subunit of hemoglobin gamma-1); the ABCA4 gene sequence SEQ ID No.: 4; the sequence of the hGH poly(A) signal polyadenylation signal (hGH poly(A) signal, polyadenylation signal of the human growth hormone gene); right inverted terminal repeat ITR; origin of replication for packaging into phage particles f1 ori; promoter of the ampicillin resistance gene AmpR promoter; gene of resistance to the antibiotic ampicillin AmpR.

[0044] Fig. 5 shows the structure of the expression vector pAAV-isoABCA4_2 containing the ABCA4 gene mini-isoform gene. The recombinant plasmid expression vector pAAV-isoABCA4_2 expressing the optimized sequence of the ABCA4 gene mini-isoform with a size of 3125 bp, consisting of the following elements: the replication origin ori; the left inverted terminal repeat ITR; CMV enhancer of the cytomegalovirus promoter; cytomegalovirus promoter CMV promoter; the sequence of the human beta-globin gene intron (intron of the hBG1 gene - the subunit of hemoglobin gamma-1); the ABCA4 gene sequence SEQ ID No.: 5; the sequence of the hGH poly(A) signal polyadenylation signal (hGH poly(A) signal, polyadenylation signal of the human growth hormone gene); right inverted terminal repeat ITR; origin of replication for packaging into phage particles f1 ori; promoter of the ampicillin resistance gene AmpR promoter; gene of resistance to the antibiotic ampicillin AmpR.

[0045] Figure 6 shows the structure of the expression vector pAAV-cmABCA4_1 containing the gene of the computer-modeled version of the ABCA4 mini-gene. The recombinant plasmid expression vector pAAV-cmABCA4_2 expressing the optimized sequence of the ABCA4 mini-gene of 4272 bp, consisting of the following elements: the origin of replication ori; the left inverted terminal repeat ITR; the CMV enhancer of the cytomegalovirus promoter; the cytomegalovirus promoter CMV promoter; the intron sequence of the human β-globin gene (intron of the hBG1 gene - the subunit of hemoglobin gamma-1); the sequence of the ABCA4 mini-gene SEQ ID No.:7; hGH poly(A) signal (hGH poly(A) signal, human growth hormone gene polyadenylation signal); right inverted terminal repeat ITR; replication origin for packaging into phage particles f1 ori; ampicillin resistance gene promoter AmpR promoter; ampicillin resistance gene AmpR.

[0046] Fig. 7 shows the structure of the expression vector pAAV-cmABCA4_2 containing the gene of the computer-modeled version of the ABCA4 mini-gene. The recombinant plasmid expression vector pAAV-cmABCA4_2 expressing the optimized sequence of the ABCA4 mini-gene of 4269 bp, consisting of the following elements: the origin of replication ori; the left inverted terminal repeat ITR; the CMV enhancer of the cytomegalovirus promoter; the cytomegalovirus promoter CMV promoter; the intron sequence of the human β-globin gene (intron of the hBG1 gene - the subunit of hemoglobin gamma-1); the sequence of the ABCA4 mini-gene SEQ ID No.:8; hGH poly(A) signal (hGH poly(A) signal, human growth hormone gene polyadenylation signal); right inverted terminal repeat ITR; replication origin site for packaging into phage particles f1 ori; ampicillin resistance gene promoter AmpR promoter; ampicillin resistance gene AmpR.

[0047] Figure 8 shows a schematic diagram of the sequence of actions during the production and subsequent analysis of AAV particles for subsequent transduction of cell lines. The result of Western blot analysis of the expression of the abca4 mini-protein in lysates of transfected HEK293T human embryonic kidney cells.

[0048] The HEK293T cell line, into which the pAAV-ABCA4 construct (Seq ID 5) was introduced, produced the abca4 miniprotein protein (Seq ID 6).

[0049] The result of Western blot analysis of the expression of the abca4 mini-protein in lysates of transfected human embryonic kidney cells HEK293T is shown in Fig. 9, where:

[0050] 1, 2 - expression of miniABCA4 in lysates of HEK293T cells transfected with pAAV-ABCA4;

[0051] 3 - no miniABCA4 expression in the lysate of untransfected HEK293T cells.

[0052] Cells were harvested 72 hours after transfection with the PEI reagent. RIPA lysis buffer was used for lysis. Electrophoresis was performed in PAGE, and proteins were transferred to a PVDF membrane. miniABCA4 was detected using a rabbit polyclonal antibody (Affinity Biosciences #DF9852). Horseradish peroxidase-linked anti-rabbit IgG antibody (Wuhan Fine Biotech Co., FNSA-0011) was used as a secondary antibody.

[0053] Unless otherwise specified, 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 this invention pertains. In some cases, definitions of terms with 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 description should not be construed as implying a significant difference in the meaning of the term from that commonly understood in the art.

[0054] Furthermore, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic 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 are widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as commonly practiced in the art, or as described herein.

[0055] All publications, patents and patent applications, and accession numbers cited in this document, whether above or below, are hereby incorporated by reference in their entirety.

[0056] The terms "comprises," "contain," and "comprising" are to be interpreted as inclusive, not exclusive, i.e., as including other unspecified components or process steps. The terms "consists," "consisting," and their variations are to be interpreted as exclusive, not inclusive, i.e., as excluding components or process steps that are not specifically listed.

[0057] As used in the present invention, "Stargardt disease" (SD) is a hereditary disease characterized by congenital progressive pigmentary degeneration of the retina (PDS) (retinitis pigmentosa), leading to gradual loss of central visual acuity and blindness, but the nature of damage to the light perception organs may be different.

[0058] Gene therapy refers to a group of methods aimed at modifying the sequence of genes or controlling their expression, as well as changing the biological properties of cells for their therapeutic or prophylactic use.

[0059] “Mini ABCA4 protein” (“miniABCA4”, “mini ABCA4 protein”) is a truncated version of the natural ABCA4 protein, which is obtained as a result of translation of messenger RNA by ribosomes from the ABCA4 minigene polypeptide product.

[0060] In one embodiment of the present invention, the ABCA4 miniprotein for gene therapy of Stargardt disease type 1 comprises the sequence of SEQ ID No. 6.

[0061] The term "codon-optimized" refers to 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.

[0062] The term "codon optimization" refers to an experimental approach to improving 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, which means 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. Codon optimization can also focus on identifying elements of mRNA instability, mRNA secondary structures, sequence repeats, internal ribosome entry sites, promoter sequences, putative splice 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]. Synonymous codon frequencies vary among different organisms and even between different cells of the same organism. They are decoded by ribosomes at different rates because their corresponding tRNAs are also present in different quantities in different cells, 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 herein by reference.Translation in mitochondria deviates from the universal genetic code, using mechanisms and codon frequencies more similar to their α-proteobacterial ancestors than to the mammalian nuclear genome, so successful cytosolic expression of mitochondrial genes requires codon optimization to recode the mitochondrial gene sequence 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].

[0063] Codon optimization was performed taking into account the codon and tRNA abundance in human cells with a relative adaptivity level of at least 50%. The developed codon-optimized sequence is only 80% identical to the human ABCA4 sequence.

[0064] The term "nucleic acid" refers to a sequence of DNA or RNA. The term encompasses sequences that contain any of the known DNA and RNA base analogs such as, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)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, β-D-mannosylqueuosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid, oxybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of β-uracil-5-hydroxyacetic acid, uracil-5-hydroxyacetic acid, pseudouracil, queuosine, 2-thiocytosine, and 2,6-diaminopurine.

[0065] The term "ABCA4 minigene" refers to a minimal functional copy of the ABCA4 gene, obtained through targeted biodesign to remove exons from the native structure of the natural ABCA4 gene. The nucleotide sequence within the minigene is codon-optimized for expression in human cells. The resulting ABCA4 miniprotein sequences, according to the invention, contain a unique combination of domains, taking into account the mutational profile and structural features at both the gene and protein levels. Portions of the ECD1, ECD2, NBD1, NBD2, and TM domains were removed, while maintaining the active sites and ATP-binding sites. The length of the sequence corresponding to the ABCA4 minigene protein allows for its successful delivery into cells within viral vectors.

[0066] A nucleic acid with the claimed sequence can be obtained 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 cell genome, using conventional cloning technology and PCR and others, as well as chemical synthesis methods.

[0067] In embodiments of the present invention, the nucleic acid comprises a nucleotide sequence selected from SEQ ID No. 4, 5, 7, 8. In preferred embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID No. 7 or SEQ ID No. 8.

[0068] The 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 the nucleotide sequence of the present invention into HEK293T cells by transfection results in expression of the ABCA4 minigene.

[0069] Nucleic acid also has low immunogenicity. Codon-optimized transgene sequences have a lower ability to induce an intracellular immune response due to minimal activation of the expression of interferon-stimulating genes (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 have less immunogenicity compared to adenoviruses (Rabinowitz J, Chan YK, Samulski RJ. Adeno-associated Virus (AAV) versus Immune Response. Viruses, 2019 Jan 25;11(2):102). Additionally, the introduction of the AAV vector under the retina significantly limits its systemic spread and, as a result, the activation of the immune response.The eye is known to be separated by the blood-ocular barrier, lacking an intraocular lymphatic system, and the surface of ocular stromal cells exhibits weak or no expression of MHC class I and II molecules. All of this explains the leading position of gene therapy drug development in ophthalmology. The presented amino acid sequence of the protein is not highly immunogenic, as it is essentially a miniaturized version of the native ABCA4 protein. No new domains or linkers that could be interpreted as foreign by the patient's immune system have been added to the miniABCA4 structure.

[0070] The "coding sequence" or sequence that "encodes" the selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are defined by an initiation codon at the 5' (amino) end and a translation termination codon at the 3' (carboxy) end. A transcription termination sequence may be located 3' relative to the coding sequence.

[0071] The term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication when linked to appropriate control elements and that can transfer gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0072] The term "transfection" is used to refer to the accumulation of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced into the cell membrane. Many 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.

[0073] The term "expression vector" ("expression vector") refers to a vector containing a promoter and other regulatory sequences that ensure efficient transcription of a recombinant gene, followed by translation of mRNA and formation of a recombinant protein. The plasmid and expression vectors used below are used for illustrative purposes and do not limit the scope of the present invention.

[0074] In an embodiment of the invention, a plasmid expression vector is provided, comprising elements in accordance with the physical and genetic map shown in Fig. 1. An expression vector for expression in eukaryotic cells, comprising a nucleic acid, which comprises a nucleotide sequence selected from SEQ ID No. 4, 5, 7, 8. In preferred embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID No. 7 or SEQ ID No. 8.

[0075] The term "promoter" as used herein specifically refers to a DNA nucleotide sequence recognized by RNA polymerase at which transcription initiation occurs of the element to which the promoter is operably linked. A promoter may also be accompanied by an enhancer. Enhancers enhance promoter activity and stimulate transcription. To produce large amounts of protein in eukaryotic cells, and in particular in human cells, it is advisable to use strong promoters active in the target cells. Strong constitutive promoters capable of initiating recombinant gene expression in various cell types are well known in the art. In one embodiment, the cytomegalovirus promoter (CMV promoter) is used as a promoter.

[0077] In an embodiment of the invention, the plasmid expression vector comprises the following elements in the direction from the 5' end to the 3' end:

[0078] – replication start site ori;

[0079] – left inverted terminal repeat ITR;

[0080] – CMV cytomegalovirus promoter enhancer;

[0081] – cytomegalovirus promoter CMV promoter;

[0082] – human b-globin gene intron sequence;

[0083] – optimized sequence of mini ABCA4 gene SEQ ID No.:7;

[0084] – hGH poly(A) signal polyadenylation signal sequence;

[0085] – right inverted terminal repeat ITR;

[0086] – promoter of the ampicillin resistance gene AmpR promoter;

[0087] – gene of resistance to the antibiotic ampicillin AmpR.

[0088] In another embodiment of the invention, the said vector comprises the sequence SEQ ID No. 8 as the sequence encoding the ABCA4 miniprotein (SEQ ID No. 6). Transfection of cells with the plasmid expression vectors of the present invention results in stable and high expression of the ABCA4 miniprotein, suitable for the treatment of Stargardt disease type 1.

[0089] The term "gene expression" refers to the transformation of genetic information encoded in the nucleotide sequence of a gene into a functional product - RNA or protein.

[0090] A "polypeptide" or "peptide," as used herein, refers to two or more independently selected natural or non-natural amino acids joined by a covalent bond (e.g., a peptide bond). A peptide may 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 joined 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, comprising the nucleic acid of the present invention. The nucleic acid of the present invention can be introduced into any vector known in the art, including plasmid, viral, including those based on the SV40 virus, adenoviruses, herpesviruses, retroviruses, lentiviruses, adeno-associated and other viruses. Vectors into which the nucleic acid of the present invention can be introduced are not limited to this list. The choice of expression vector is determined by the tasks for which the vector will be subsequently used and does not limit the scope of the present invention. Methods for producing vectors are known in the art. Vectors containing the nucleic acid of the present invention can be obtained by specialists in the field of genetic engineering.

[0092] Adeno-associated virus (AAV) is a nonpathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome with a nonenveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs), which function as a replication and packaging signal for the viral lineage. The Rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The Cap ORF encodes three structural proteins (VPs 1–3) that assemble to form the 60-mer viral capsid. Finally, an ORF present as an alternative reading frame in the cap gene produces assembly-activating protein (AAP), a viral protein that localizes AAV capsid proteins to the nucleolus and functions in the capsid assembly process.

[0093] There are several natural ("wild-type") serotypes and over 100 known variants of AAV, each differing in amino acid sequence, particularly in the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No link has been established between any AAV and any human disease, making recombinant AAV attractive for clinical applications.

[0094] For purposes of this document, the term "AAV" is an abbreviation for adeno-associated virus, including, but not limited to, the virus itself and its derivatives. Except where otherwise noted, this terminology refers to all subtypes or serotypes and 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 AAVrh10), 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, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, 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 (AAVrh10); the disclosures of which are incorporated herein by reference for instructional purposes of AAV nucleic acid and amino acid sequences. See also, e.g., 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. et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Maurice et al. (2004) Virology 33:375-383; international patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and Pat. US No. 6156303.

[0096] In preferred embodiments of the invention, the vector is an adeno-associated virus of serotype 2, serotype 9, serotype 2.7m8, or serotype 5.

[0097] In one embodiment of the invention, an adeno-associated virus serotype 2 containing a nucleic acid of the present invention, produced in adherent HEK293T cell cultures, is provided as a viral expression vector. Introduction of a plasmid expression vector containing a nucleic acid of the present invention together with a pHelper vector and a pRC2 / 2 packaging plasmid into cells of adherent HEK293T cell cultures results in the production of viral expression vectors representing adeno-associated virus serotype 2 [i1].

[0098] In another embodiment of the invention, an adeno-associated virus of serotype 9 containing a nucleic acid of the present invention, produced in adherent HEK293T cell cultures, is provided as a viral expression vector. Introduction of a plasmid expression vector containing a nucleic acid of the present invention together with a pHelper vector and a pRC2 / 9 packaging plasmid into cells of adherent [4] HEK293T cell cultures results in the production of viral expression vectors representing an adeno-associated virus.

[0099] In another embodiment of the invention, the viral expression vector provided is an adeno-associated virus 2.7m8 serotype containing the nucleic acid of the present invention, produced in adherent HEK293T cell cultures. Introduction of the plasmid expression vector containing the nucleic acid of the present invention together with the pHelper vector and the pRC2.7m8 packaging plasmid into cells of adherent [i2] [MS3] HEK293T cell cultures results in the production of viral expression vectors representing the adeno-associated virus 2.7m8 serotype.

[0100] In a further embodiment of the invention, an adeno-associated virus of serotype 5 containing the nucleic acid of the present invention, produced in adherent HEK293T cell cultures, is provided as a viral expression vector. Introduction of a plasmid expression vector containing the nucleic acid of the present invention together with a pHelper vector and a pRC2 / 5 packaging plasmid into cells of adherent [4] HEK293T cell cultures results in the production of viral expression vectors representing an adeno-associated virus.

[0101] The expression vectors of the present invention demonstrate stable and high expression of the ABCA4 miniprotein in cells. This suggests that administration of any of the claimed expression vectors of the present invention to ocular tissue can lead to stable expression of the ABCA4 miniprotein and compensate for the deficiency of the Abca4 protein in ocular cells and tissues, thereby restoring its function and stabilizing vision in Stargardt disease. Administration of the plasmid and viral vectors of the present invention to cells with a natural mutation of the ABCA4 gene leads to accumulation of the ABCA4 miniprotein.

[0102] 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; sporting mammals (e.g., horses); farmed mammals (e.g., sheep, goats, etc.); mammalian animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some embodiments, the "individual" is a human.

[0103] As used herein, a cell is said to be "stably" altered, transduced, genetically modified, or transformed by a genetic or nucleotide sequence if this sequence is available to perform its function during prolonged cell culture in vitro and / or over an extended period of time in vivo. Typically, such a cell is "heritably" altered (genetically modified) in that the introduced genetic change is also inherited by the progeny of the altered cell.

[0104] The terms "therapy," "treatment," "treatment," and the like as used herein refer to achieving a desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or a side effect associated with the disease.

[0105] "Treatment" as used herein encompasses any treatment of a disease in a mammal, especially a human, and includes: (a) preventing the onset of a disease (and / or symptoms caused by the disease) in a subject that may be predisposed to the disease or at risk of contracting the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., stopping its development; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., causing regression of the disease (and / or symptoms caused by the disease), i.e., reducing the intensity of the disease and / or one or more symptoms of the disease. For example, the proposed ABCA4 miniprotein, a nucleic acid encoding the ABCA4 miniprotein, and an expression vector containing the same can be directed to the treatment of Stargardt disease type 1.

[0106] As used herein, the term "effective amount" is an amount sufficient to achieve beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For the purposes of this description, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount sufficient to temporarily alleviate, improve, stabilize, reverse, prevent, slow, or halt the progression of (and / or symptoms associated with) a particular disease state (e.g., retinitis pigmentosa, hearing loss).

[0107] The term "retinal cell" refers herein to any of the cell types that are contained in the retina, such as, but not limited to, retinal ganglion cells (RG), amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, Müller glial cells, microglial cells, and retinal pigment epithelium.

[0108] The term "administering," as used in the methods, refers to delivering the composition to a selected target cell characteristic of an eye and / or ear disease. In one embodiment, the composition is delivered by subretinal injection into photoreceptor cells or other ocular cells. In another embodiment, intravitreal injection into ocular cells is used. In yet another embodiment, injection via an eyelid vein may be used to deliver the ocular cells. Other administration methods may be selected by those skilled in the art, taking into account this description.

[0109] "Administration" or "route of administration" means the delivery of a composition described herein to a subject, with or without a pharmaceutical carrier or excipient. Routes of administration may be combined, if desired. In some embodiments, administration is repeated periodically.

[0110] The pharmaceutical compositions described herein are designed for delivery to subjects in need thereof via any suitable route or a combination of different routes. Direct delivery to the eye (optionally via intraocular delivery, intraretinal injection, intravitreal, topical route) or delivery via systemic routes, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal and other parenteral administration routes. The nucleic acid molecules and / or vectors described herein can be delivered in a single composition or a plurality of compositions. Optionally, two or more different AAVs or a plurality of viruses can be delivered (see, for example, WO 202011 / 126808 and WO 2013 / 049493). In another embodiment, the plurality of viruses may comprise different replication-defective viruses (e.g., AAV and adenovirus) alone or in combination with proteins.

[0111] In one embodiment, the viral constructs may be delivered in concentrations of at least 1×10 6 up to about at least 1×10 11 Viral genomes (vg) are injected in volumes ranging from about 1 µl to about 3 µl for small animals such as mice. For larger animals with eyes nearly the same size as humans, larger human doses and volumes are used, as indicated above, for example, from 1 x 10 6 up to about 1×10 15 vg per dose. For a discussion of good practice for administering substances to various animals, see, for example, Diehl et al, J. Applied Toxicology, 21:15–23 (2001). That document is incorporated herein by reference.

[0112] It is preferable that the lowest effective concentration of virus or other delivery vehicle be used to reduce the risk of adverse effects such as toxicity, dysplasia and retinal detachment.

[0113] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by prior invention.

[0114] It should be understood that all numerical designations are preceded by the term "approximately," although this is not always explicitly stated. It should also be understood that the reagents described in this document are merely exemplary and that their equivalents are known in the art, although this is not always explicitly stated.

[0115] Before describing the present invention in detail, it should be understood that this invention is not limited to specific compositions or process parameters, which, of course, are subject to change. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention only and is not intended to be limiting.

[0116] 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 can be used in the practice of the present invention, and the invention should be construed as including any and all applications contemplated herein and all variations of equivalents within the skill of an ordinary skilled artisan.

[0117] The central feature of the present invention is the discovery that the ABCA4 miniprotein and the nucleic acid encoding it, packaged in an AAV vector, exhibit stable and high expression of the ABCA4 minigene and the production of the ABCA4 miniprotein necessary for the therapy of Stargardt disease type 1.

[0118] The invention is confirmed by clinical examples.

[0119] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, but they are not intended to limit the scope of what the inventors consider to be their invention.

[0120] Example 1. Creation of plasmid expression vectors

[0121] Example 1.1. Optimization of codons of the nucleotide sequence encoding the ABCA4 minigene

[0122] Annotation of the ABCA4 gene coding sequence was performed using the NCBI / CCD service. For alignment of nucleotide sequences, the UniPro UGENE tool was used (Okonechnikov K, Golosova O, Fursov M, the UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics, 2012, 28:1166-1167). Translation of the nucleotide sequence into amino acid sequence was performed using the tool at the link: https: / / web.expasy.org / translate. Since there are differences in the representation of transfer RNAs in cells of different tissues, optimization of the nucleotide sequence was performed to exclude stop codons and non-synonymous codons. To study the stability of the transcript, the RNA fold program was used (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi). Nucleic acid with an optimized ABCA4 gene sequence (SEQ ID No. 7), encoding the ABCA4 miniprotein sequence (SEQ ID No.6) were synthesized using a service from TopGenetech (Canada). Additionally, unstructured protein regions were analyzed to select optimal regions for optimization using IUPred2 (https: / / iupred2a.elte.hu).

[0123] Example 1.2. Preparation of an expression vector for ABCA4 expression

[0124] pAAV-MCS (cat#VPK-410, Cell biolabs) was used as the plasmid expression vector.

[0125] The insertion of various ABCA4 sequence variants was performed according to the method recommended by the vector manufacturer, using restriction endonucleases SalI and XhoI (New England Biolabs Inc., USA) and T4 DNA ligase according to standard protocols.

[0126] As a result, the recombinant plasmid expression vector pAAV-ABCA4 was obtained (Fig. 6).

[0127] Cell transformation was performed according to the standard protocol (Sambrook, J. and Russell, DW (2001) Molecular Cloning: A Laboratory Manual. 3rd Edition, Vol. 1, Cold Spring Harbor Laboratory Press, New York). To carry out bacterial transformation, 100 μl of chemically competent Escherichia coli DH5α cells stored at -80 °C were used and placed on ice for slow thawing. Then, 10 μl of the ligation mixture were added to the cells and the cells were incubated on ice for 30 min. Plasmid DNA was delivered to the bacterial cells using heat shock for 30 sec at 42 °C in a water bath, then the tube with the cells was transferred to ice and incubated for 2 min. Next, 1 ml of LB nutrient medium preheated to 37 °C was added to the cells and they were incubated in a thermostat for 1 hour at 37 °C and stirred at 180-200 rpm.The transformed cell suspension was plated onto Petri dishes containing LB agar and ampicillin, pre-dried in an incubator at 37°C (100 µl per dish). The cells were incubated in an incubator at 37°C for 16-18 hours.

[0128] To analyze transformed cell colonies, PCR screening was performed using the vector-specific primers pAAV_For_seq2 and pAAV_Rev_seq2 (Table 1) and the ScreenMix ready-to-use PCR mixture (Eurogen, Russia). The reaction mixture was prepared according to the manufacturer's recommendations. Thermolysates of bacterial monoclones were used as a template. For this, each of the 8 monoclones was first transferred by tip injection to a Petri dish with the prepared template, and then the tip was dipped into a tube containing 20 μl of mQ water. The Petri dish was incubated in a thermostat at 37°C for 24 hours, and the tubes (strips) containing water and bacteria were heated at 95°C for 5 minutes. 2 μl of the suspension were used for the PCR. PCR was performed with the following parameters: preliminary melting of DNA 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 confirmed to contain the correct-sized insert by PCR were submitted for sequencing. Clones with the correct sequence were selected based on the sequencing results.

[0129] Table 1

[0130] PCR conditions

[0131] PCR reaction mixture Total volume 20 µl № Components Concentration Volume, µl Final concentration 1 Purified water - 13.5 - 2 10 mM dNTP (50X) 5 mM 0,5 0.2 mM 3 5x Fusion Polymerase Reaction Buffer (Alpha Enzyme) 5X 4,0 1X 4 Forward primer 10 μM 10 μM 0,5 0.2 μM 5 Reverse primer 10 μM 10 μM 0,5 0.2 μM 6 DNA polymerase Fusion (Alpha enzyme) 2 U / µl 0,5 1 U / µl 7 ABCA4 10 ng 1,5 10 ng

[0132] As a result of culturing the selected clones, recombinant plasmid expression vectors were obtained, each of which contained the ABCA4 sequence.

[0133] The recombinant plasmid expression vector pAAV-ABCA4 expressing the codon-optimized sequence of the ABCA gene (ABCA, SEQ ID No. 7) contains the following elements (Fig. 6):

[0134] – replication start site ori;

[0135] – left inverted terminal repeat ITR;

[0136] – CMV cytomegalovirus promoter enhancer;

[0137] – cytomegalovirus promoter CMV promoter;

[0138] – sequence of the intron of the human b-globin gene (intron of the hBG1 gene – a subunit of hemoglobin gamma-1);

[0139] – ABCA4 sequence SEQ ID No. 7;

[0140] – hGH poly(A) signal polyadenylation signal sequence (hGH poly(A) signal, human growth hormone gene polyadenylation signal);

[0141] – right inverted terminal repeat ITR;

[0142] – promoter of the ampicillin resistance gene AmpR promoter;

[0143] – gene of resistance to the antibiotic ampicillin AmpR.

[0144] Example 1.3. In silico modeling

[0145] In silico selection of designed variants yielded the nucleotide sequence ABCA4 (SEQ ID No. 7). Modeling the secondary structure of a protein molecule can be performed de novo, in the absence of homologous structural models, or by comparison with protein secondary structures available in databases, such as the Protein Data Bank (https: / / www.rcsb.org / ). The accuracy and adequacy of the model are assessed by analyzing known protein domains, or by the conservation of catalytic centers or conserved regions of the molecule necessary for the protein to perform physiological functions.DNA with the claimed sequence can be obtained 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 cell genome, using conventional cloning technology and PCR and others, as well as chemical synthesis methods.

[0146] The gene with the codon-optimized nucleotide sequence of the present invention can be introduced into a plasmid expression vector, such as a plasmid or pAAV-based one. Introduction of the plasmid expression vector containing the gene with the nucleotide sequence of the present invention into eukaryotic cells, such as HEK293T cells, by transfection results in the expression of the heterologous ABCA4 gene.

[0147] Introduction of a plasmid expression vector containing a gene with the nucleotide sequence of the present invention, together with the pHelper vector and the pRC2 / 9, pRC2 / 5, or pRC2.7m8 vector, into HEK293T cells results in the production of a viral expression vector. Introduction of the resulting viral expression vector into HEK293T cells by transduction results in the expression of the heterologous ABCA4 gene.

[0148] Example 2. Evaluation of the expression level of the miniABCA4 gene after transfection of HEK293T

[0149] Example 2.1. Evaluation of the miniABCA4 gene mRNA level after HEK293T transfection

[0150] For transfection, cells were seeded into a 6-well plate at a density of 0.5 million cells / well. Transfection was performed 30 hours after cell seeding (when cell confluency reached 80%). Transfection was performed with a DNA-PEI complex at a ratio of 1:5, with a DNA load of 1 μg / million cells.

[0151] After transfection, incubate the cells for 2 days. Lyse the cells by two freeze-thaw cycles. After thawing, isolate RNA using the RIBO-PRep and AmpliPrime kits. The isolated RNA was used as a template to obtain cDNA.

[0152] The obtained cDNA was used for PCR using 2 primers: for the target gene and for the housekeeping genes (PPIA and GAPDH).

[0153] To assess miniABCA4 gene expression, a miniABCA4 construct was transfected using PEI as the transfection agent. A miniABCA4-PEI complex was obtained and then added to the cells. Two days after transfection, RNA was isolated from the cell mass and treated with DNase. The resulting RNA was used as a template for cDNA production.

[0154] The resulting cDNA samples from the transfected and non-transfected cells were analyzed by quantitative PCR using three types of primers: one for the target miniABCA4 gene and the PPIA and GAPDH enzyme genes as reference housekeeping genes. Transfected cells were used as a control.

[0155] Samples:

[0156] 1 – cell line transfected with 1 μg of plasmid per 1 million cells.

[0157] 2 – control, where all transfection reagents were added to the cell line except the plasmid itself.

[0158] RNA was isolated from the samples and treated with DNase I (containing no RNase activity) from NEB for 10 min at 37°C. After treatment, DNase was removed from the RNA samples by treatment with TRI reagent, followed by RNA reprecipitation with isopropanol and washing with 75% ethanol. RNA loss after reprecipitation was approximately 25% of the initial RNA amount. A reverse transcription reaction was then performed (samples are designated as 1RT, 2RT) using a random primer from Eurogen. Control samples for the reverse transcription reaction, in which all components, including RNA except reverse transcriptase, were added (samples are designated as 1C), were also mixed and incubated in parallel.

[0159] Next, a real-time PCR reaction was performed with primers 147-148 and 1 µl of the mixture, followed by a reverse transcription reaction using 25 µl of the PCR mixture. The resulting St values ​​for the experiment are presented in Table 2.

[0160] Data from a typical ABCA4 minigene expression experiment following transfection with a plasmid encoding the ABCA4 minigene under the control of the CMV promoter. Table of average St values ​​for triplicate PCR reactions. cDNA obtained by reverse transcription of RNA samples pretreated with DNase was used as the template.

[0161] Table 2

[0162] Data from a typical ABCA4 minigene expression experiment after transfection with a plasmid encoding the ABCA4 minigene under the control of the CMV promoter

[0163] Detectable mRNA 1RTD 1 CD 2RTD ABCA4 minigene (Seq ID 7) 21.284 - - GAPDH 18.569 - 18.787 PPIA 17.328 - 17.275

[0164] Description of Table 2 samples:

[0165] 1RTD - HEK293 cells were transfected using a plasmid carrying a mini-version of the ABCA4 gene under the control of a constitutive promoter, RNA was isolated from the cells, treated with DNase, and a reverse transcription reaction was performed.

[0166] 1CD - transfection of HEK293 cells using a plasmid carrying a mini-version of the ABCA4 gene under the control of a constitutive promoter, RNA was isolated from the cells and treated with DNase (the reverse transcription reaction was not performed).

[0167] 2RTD - RNA was isolated from HEK293 cells, treated with DNase, and subjected to a reverse transcription reaction.

[0168] Table 2 shows that no significant signals were detected for any primer pairs used in the 1CD control sample, whereas in the 1RTD experiment, expression of the ABCA4 miniversion, as well as expression of the housekeeping genes GAPDH and PPIA, was detected. Without transfection with the ABCA4 miniversion plasmid, its expression could not be detected (sample 2RTD).

[0169] The results of normalizing the expression of the mini-version of the ABCA4 gene to the expression of the corresponding housekeeping genes are presented in Table 3. Normalization was carried out by calculating how many times the expression of the mini-version of the ABCA4 gene differs from that of the housekeeping gene using the formula “2^(Ct[housekeeping gene]-Ct[miniABCA4])”.

[0170] Table 3

[0171] Results of normalization of the expression of the mini-version of the ABCA4 gene to the expression of the corresponding housekeeping genes

[0172] sample number 1RTD normalization by GAPDH 0.1523917 PPIA normalization 0.06445685

[0173] Thus, using a plasmid carrying the mini-version of the ABCA4 gene to transfect the HEK293 cell line results in the production of the mini-version of the ABCA4 gene transcript.

[0174] Example 2.2 Detection of the mini-version of ABCA4 protein was assessed by Western blot analysis after transfection of HEK293T

[0175] For transfection, HEK293T cells were seeded into a 6-well plate at a density of 0.5 million cells / well. Transfection was performed 30 hours after cell seeding (when the cells reached 80% confluency). Transfection was performed with a DNA-PEI complex at a ratio of 1:5, with a DNA load of 1 μg / million cells.

[0176] After 72 hours of incubation, cells were harvested in RIPA lysis buffer (Tris-HCl, NaCl, EDTA, sodium deoxycholate) for further processing. Protein concentrations were determined using the RC DC Protein Assay kit.

[0177] Protein lysates were loaded onto a 7.5% PAA gel, electrophoresed, and transferred to a PVDF membrane. The membrane was blocked in 5% milk in TBS buffer and then incubated for 1 h at room temperature with anti-ABCA4 primary antibodies in 1% milk in TBS buffer. After washing, the membrane was incubated with anti-rabbit secondary antibodies. The signal was detected using a chemiluminescent (ECL) substrate (Fig. 9).

[0178] Result

[0179] It was found that cells transfected with a plasmid containing a mini-version of the ABCA4 gene produce ABCA4 protein, while no protein expression was observed in untransfected cells. This confirms that the delivered construct is synthesized in HEK293T cells.

[0180] Example 4. Production of AAV2.7m8-ABCA4 viral vector

[0181] HEK293T cell culture was used to produce the AAV2.7m8-ABCA4 viral vector by triple transfection. Cells were cultured in DMEM and 5% FBS in the presence of penicillin / streptomycin antibiotics. One hour before transfection, the culture medium was changed to DMEM supplemented with 2% FBS. Transfection was performed by adding 1.5 μg of total DNA / million cells (the pGOI:pHelper:pRepCap plasmid ratio was 1:2:5). pHelper pRepCap plasmid. The DNA:PEI ratio was 1:5. Twenty-four hours after transfection, the culture medium was changed to DMEM supplemented with 5% FBS. Cells were lysed 72 hours after transfection by two freeze / thaw cycles and the addition of Triton X-100 to a final concentration of 0.1%.

[0182] The cell lysate was concentrated 10-fold using centrifuge cassettes with a cutoff of 100 kDa. The resulting solution was dialyzed against PBS with a 10-fold excess of the initial concentrated solution. The lysate, filtered through a 0.22 μm filter, and the dialyzed concentrated sample were used as the initial solution for purification.

[0183] The resulting filtrate was used for isolation on the ThermoFisher POROS AAVX chromatographic sorbent.

[0184] After storage, the sorbent was pre-washed with purified water, regenerated with a solution of 100 mM glycine, pH 3.0, and 10 mM NaOH, pH 11. Then, it was washed with water and equilibrated with a PBS solution. After applying the cell lysate, the column was washed with an equilibration buffer. An additional wash was performed in a 0.1 M citrate buffer solution, pH 6.2. Elution was carried out with a solution of 100 mM glycine, pH 3.0. Strip washing was carried out with a solution of 100 mM glycine, pH 2.2. The sorbent was regenerated with a solution of 10 mM NaOH, pH 11. A description of the stages of the purification process is presented in Table 4.

[0185] Table 4

[0186] Sequence of steps for purification of viral particles

[0187] Item No. Stage Composition of the buffer solution Volume, ml Stage time, min 1 Flushing H2O 10 5 2 Regeneration 0.1 M Gly, pH 2.2 10 5 3 Balancing PBS, pH 7.4 10 5 4 Application Cell lysate 28 30 5 Balancing PBS, pH 7.4 10 10 6 Flushing 0.1 M Na3Cit, pH 6.2 10 10 7 Elution 0.1 M Gly, pH 3.0 5 10 8 Strip washing 0.1 M Gly, pH 2.2 5 10 9 Flushing H2O 10 10 10 Regeneration 10 mM NaOH 10 10 9 Flushing PBS, pH 7.4 10 10 10 Flushing H2O 10 10 11 Canning 20% EtOH 10 10

[0188] The resulting eluate fractions were analyzed by quantitative PCR and PAGE. PAGE detected the presence of three capsid proteins characteristic of the adeno-associated virus: VP1 (84 kDa), VP2 (69 kDa), and VP3 (62 kDa). Electrophoresis also demonstrated the absence of other proteins in all eluate fractions.

[0189] Example 5. Evaluation of the expression level of the ABCA4 mini-version sequence after transduction in AAV-based viral vectors.

[0190] Plasmid expression vectors pAAV-ABCA4, as well as commercially available helper plasmid (pHelper) and packaging plasmid pRC2.7m8 were used to obtain viral AAV vectors.

[0191] HEK293T adherent cell line cells were thawed and cultured according to standard operating procedures and techniques. Upon receipt, the estimated cell culture density was 106 cells / ml, the cell culture volume was 30 ml. Transfection was performed in 500 ml Erlenmeyer flasks (working volume 175 ml). The seeding dose was 5⋅105 cells / ml. Transfection with plasmids was performed 12 hours after seeding. Transfection conditions: cell concentration during transfection 106 cells / ml, viability 85-90%, DNA mass 1.5 μg / 1 million cell biomass. DNA:PEI ratio 1:5, the volume of the transfection mixture 5% of the cell culture volume. After transfection, cells were incubated at 37°C, 5%CO2, 75% humidity, 100 rpm for 120 h.

[0192] To lyse the cells, Tween-20 was added to a concentration of 0.05% and the flask was incubated for 1 hour. Benzonase was then added to a concentration of 20 IU / ml and MgCl2 to 1-2 mM, and the flask was incubated for 1 hour. The lysates were centrifuged for 10 min at 3000 g and filtered through filters with a pore size of 0.22 μm. The filtrates were concentrated by tangential filtration with a cut-off of molecules larger than 100 kDa, from 525 ml to 50 ml, at a transmembrane pressure of 1.5-2 bar. Affinity chromatography was performed on AAVX sorbent (Thermo Fisher Scientific, USA) according to the manufacturer's recommendations.

[0193] The viral titer of the obtained AAV samples was assessed by RT-PCR, and the physical titer and the presence of impurities (low molecular weight or high molecular weight aggregates) were assessed by PAGE and DLS.

[0194] All samples contained particles predominantly 20–25 nm in size and a minor amount of high-molecular impurities (aggregates).

[0195] Example 6. In silico modeling

[0196] To ​​confirm the correctness and authenticity of the secondary structure of the ABCA4 miniprotein variants obtained by the rational design method, in silico modeling was performed to predict the secondary structure of the proteins. For this purpose, the ABCA4 minigene sequences were translated into amino acid sequences using the EMBOSS TransSeq online utility (https: / / www.ebi.ac.uk / Tools / st / emboss_transeq / ), access date 27.10.2023. Then, the obtained amino acid sequences were used to build models using the AlphaFold2 (https: / / github.com / google-deepmind / alphafold), Robetta (https: / / robetta.bakerlab.org / ) and I-TASSER (https: / / zhanggroup.org / I-TASSER / ) services. Data on the domain structure of the natural human ABCA4 protein was used as a comparison model. Miniproteins SEQ ID No: 3, 6 showed the predicted secondary structure according to the domain organization and the generated nucleotide sequence (Fig. 2 and 3).The model parameters for the miniprotein SEQ ID No. 6 were assessed as acceptable.

[0197] Thus, the creation of variants of the ABCA4 minigene encoding the ABCA4 miniprotein is described. The results of codon optimization for this gene and the development of AAV vectors for packaging the ABCA4 minigene are also described. Delivery and expression of the ABCA4 minigene within the AAV2.7m8 viral vector in HEK293T cells are demonstrated. Production of the ABCA4 miniprotein is confirmed by interaction with antibodies in immunoblotting.

[0198] Although embodiments of the present invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined herein.

[0199] --->

[0200] <?xml version="1.0" encoding="UTF-8"?>

[0201] <!DOCTYPE ST26SequenceListing PUBLIC "- / / WIPO / / DTD Sequence Listing

[0202] 1.3 / / EN" "ST26SequenceListing_V1_3.dtd">

[0203] <st26sequencelisting dtdversion="V1_3" filename="2024137394.xml"

[0204] softwarename="WIPO Sequence" softwareversion="2.3.0"

[0205] productiondate="2025-11-13">

[0206] <applicationidentification>

[0207] <ipofficecode> RU< / ipofficecode>

[0208] <applicationnumbertext> 2024137394< / applicationnumbertext>

[0209] <filingdate> 2024-12-12< / filingdate>

[0210] < / applicationidentification>

[0211] <applicantfilereference> 2024137394< / applicantfilereference>

[0212] <applicantname languagecode="ru">FEDERAL STATE

[0213] AUTONOMOUS EDUCATIONAL INSTITUTION OF HIGHER EDUCATION THE FIRST

[0214] MOSCOW STATE MEDICAL UNIVERSITY NAMED AFTER I.M.

[0215] SECHENOV MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION

[0216] (SECHENOV UNIVERSITY< / applicantname>

[0217] <applicantnamelatin>FEDERAL STATE AUTONOMOUS

[0218] EDUCATIONAL INSTITUTION OF HIGHER EDUCATION FIRST MOSCOW

[0219] GOSUDARSTVENNYI MEDITSINSKII UNIVERSITET IMENI IM SECHENOVA

[0220] MINISTERSTVA ZDRAVOOKHRANENIIA ROSSIISKOI FEDERATSLL (SECHENOVSKII

[0221] UNIVERSITET)< / applicantnamelatin>

[0222] <inventiontitle languagecode="ru">ABCA4 MINIPROTEIN, NUCLEIN

[0223] ACID ENCODING MINIPROTEIN ABCA4 AND ITS EXPRESSION LINK

[0224] VECTOR FOR GENE THERAPY< / inventiontitle>

[0225] <sequencetotalquantity> 8< / sequencetotalquantity>

[0226] <sequencedata sequenceidnumber="1">

[0227] <insdseq>

[0228] <INSDSeq_length> 2273< / INSDSeq_length>

[0229] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0230] <INSDSeq_division> PAT< / INSDSeq_division>

[0231] <INSDSeq_feature-table>

[0232] <insdfeature>

[0233] <INSDFeature_key>source< / INSDFeature_key>

[0234] <INSDFeature_location>1..2273< / INSDFeature_location>

[0235] <INSDFeature_quals>

[0236] <insdqualifier>

[0237] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0238] <INSDQualifier_value>protein< / INSDQualifier_value>

[0239] < / insdqualifier>

[0240] <insdqualifier id="q11">

[0241] <INSDQualifier_name>organism< / INSDQualifier_name>

[0242] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0243] < / insdqualifier>

[0244] < / INSDFeature_quals>

[0245] < / insdfeature>

[0246] < / INSDSeq_feature-table>

[0247] <INSDSeq_sequence> MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLIWLRNANPLY

[0248] SHHECHFPNKAMPSAGMLPWLQGIFCNVNNPCFQSPTPGESPGIVSNYNNSILARVYRDFQELLMNAPES

[0249] QHLGRIWTELHILSQFMDTLRTHPERIAGRGIRIRDILKDEETLTLFLIKNIGLSDSVVYLLINSQVRPE

[0250] QFAHGVPDLALKDIACSEALLERFIIFSQRRGAKTVRYALCSLSQGTLQWIEDTLYANVDFFKLFRVLPT

[0251] LLDSRSQGINLRSWGGILSDMSPRIQEFIHRPSMQDLLWVTRPLMQNGGPETFTKLMGILSDLLCGYPEG

[0252] GGSRVLSFNWYEDNNYKAFLGIDSTRKDPIYSYDRRTTSFCNALIQSLESNPLTKIAWRAAKPLLMGKIL

[0253] YTPDSPAARRILKNANSTFEELEHVRKLVKAWEEVGPQIWYFFDNSTQMNMIRDTLGNPTVKDFLNRQLG

[0254] EEGITAEAILNFLYKGPRESQADDMANFDWRDIFNITDRTLRLVNQYLECLVLDKFESYNDETQLTQRAL

[0255] SLLEENMFWAGVVFPDMYPWTSSLPPHVKYKIRMDIDVVEKTNKIKDRYWDSGPRADPVEDFRYIWGGFA

[0256] YLQDMVEQGITRSQVQAEAPVGIYLQQMPYPCFVDDSFMIILNRCFPIFMVLAWIYSVSMTVKSIVLEKE

[0257] LRLKETLKNQGVSNAVIWCTWFLDSFSIMSMSIFLLTIFIMHGRILHYSDPFILFLFLLAFSTATIMLCF

[0258] LLSTFFSKASLAAACSGVIYFTLYLPHILCFAWQDRMTAELKKAVSLLSPVAFGFGTEYLVRFEEQGLGL

[0259] QWSNIGNSPTEGDEFSFLLSMQMMLLDAAVYGLLAWYLDQVFPGDYGTPLPWYFLLQESYWLGGEGCSTR

[0260] EERALEKTEPLTEETEDPEHPEGIHDSFFEREHPGWVPGVCVKNLVKIFEPCGRPAVDRLNITFYENQIT

[0261] AFLGHNGAGKTTTLSILTGLLPPTSGTVLVGGRDIETSLDAVRQSLGMCPQHNILFHHLTVAEHMLFYAQ

[0262] LKGKSQEEAQLEMEAMLEDTGLHHKRNEEAQDLSGGMQRKLSVAIAFVGDAKVVILDEPTSGVDPYSRRS

[0263] IWDLLLKYRSGRTIIMSTHHMDEADLLGDRIAIIAQGRLYCSGTPLFLKNCFGTGLYLTLVRKMKNIQSQ

[0264] RKGSEGTCSCSSKGFSTTCPAHVDDLTPEQVLDGDVNELMDVVLHHVPEAKLVECIGQELIFLLPNKNFK

[0265] HRAYASLFRELEETLADLGLSSFGISDTPLEEIFLKVTEDSDSGPLFAGGAQQKRENVNPRHPCLGPREK

[0266] AGQTPQDSNVCSPGAPAAHPEGQPPPEPECPGPQLNTGTQLVLQHVQALLVKRFQHTIRSHKDFLAQIVL

[0267] PATFVFLALMLSIVIPPFGEYPALTLHPWIYGQQYTFFSMDEPGSEQFTVLADVLLNKPGFGNRCLKEGW

[0268] LPEYPCGNSTPWKTPSVSPNITQLFQKQKWTQVNPSPSCRCSTREKLTMLPECPEGAGGLPPPQRTQRST

[0269] EILQDLTDRNISDFLVKTYPALIRSSLKSKFWVNEQRYGGISIGGKLPVVPITGEALVGFLSDLGRIMNV

[0270] SGGPITREASKEIPDFLKHLETEDNIKVWFNNKGWHALVSFLNVAHNAILRASLPKDRSPEEYGITVISQ

[0271] PLNLTKEQLSEITVLTTSVDAVVAICVIFSMSFVPASFVLYLIQERVNKSKHLQFISGVSPTTYWVTNFL

[0272] WDIMNYSVSAGLVVGIFIGFQKKAYTSPENLPALVALLLLYGWAVIPMMYPASFLFDVPSTAYVALSCAN

[0273] LFIGINSSAITFILELFENNRTLLRFNAVLRKLLIVFPHFCLGRGLIDLALSQAVTDVYARFGEEHSANP

[0274] FHWDLIGKNLFAMVVEGVVYFLLTLLVQRHFFLSQWIAEPTKEPIVDEDDDVAEERQRIITGGNKTDILR

[0275] LHELTKIYPGTSSPAVDRLCVGVRPGECFGLLGVNGAGKTTTFKMLTGDTTVTSGDATVAGKSILTNISE

[0276] VHQNMGYCPQFDAIDELLTGREHLYLYARLRGVPAEEIEKVANWSIKSLGLTVYADCLAGTYSGGNKRKL

[0277] STAIALIGCPPLVLLDEPTTGMDPQARRMLWNVIVSIIREGRAVVLTSHSMEECEALCTRLAIMVKGAFR

[0278] CMGTIQHLKSKFGDGYIVTMKIKSPKDDLLPDLNPVEQFFQGNFPGSVQRERHYNMLQFQVSSSSLARIF

[0279] QLLLSHKDSLLIEEYSVTQTTLDQVFVNFAKQQTESHDLPLHPRAAGASRQAQD< / INSDSeq_sequen

[0280] ce>

[0281] < / insdseq>

[0282] < / sequencedata>

[0283] <sequencedata sequenceidnumber="2">

[0284] <insdseq>

[0285] <INSDSeq_length>7328< / INSDSeq_length>

[0286] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0287] <INSDSeq_division>PAT< / INSDSeq_division>

[0288] <INSDSeq_feature-table>

[0289] <insdfeature>

[0290] <INSDFeature_key>source< / INSDFeature_key>

[0291] <INSDFeature_location>1..7328< / INSDFeature_location>

[0292] <INSDFeature_quals>

[0293] <insdqualifier>

[0294] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0295] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0296] < / insdqualifier>

[0297] <insdqualifier id="q4">

[0298] <INSDQualifier_name>organism< / INSDQualifier_name>

[0299] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0300] < / insdqualifier>

[0301] < / INSDFeature_quals>

[0302] < / insdfeature>

[0303] < / INSDSeq_feature-table>

[0304] <INSDSeq_sequence>ggacacagcgtccggagccagaggcgctcttaacggcgtttatgtcctt

[0305] tgctgtctgaggggcctcagctctgaccaatctggtcttcgtgtggtcattagcatgggcttcgtgagac

[0306] agatacagcttttgctctggaagaactggaccctgcggaaaaggcaaaagattcgctttgtggtggaact

[0307] cgtgtggcctttatctttatttctggtcttgatctggttaaggaatgccaacccactctacagccatcat

[0308] gaatgccatttccccaacaaggcgatgccctcagcaggaatgctgccgtggctccaggggatcttctgca

[0309] atgtgaacaatccctgttttcaaagccccaccccaggagaatctcctggaattgtgtcaaactataacaa

[0310] ctccatcttggcaagggtatatcgagattttcaagaactcctcatgaatgcaccagagagccagcacctt

[0311] ggccgtatttggacagagctacacatcttgtcccaattcatggacaccctccggactcacccggagagaa

[0312] ttgcaggaagaggaatacgaataagggatatcttgaaagatgaagaaacactgacactatttctcattaa

[0313] aaacatcggcctgtctgactcagtggtctaccttctgatcaactctcaagtccgtccagagcagttcgct

[0314] catggagtcccggacctggcgctgaaggacatcgcctgcagcgaggccctcctggagcgcttcatcatct

[0315] tcagccagagacgcggggcaaagacggtgcgctatgccctgtgctccctctcccagggcaccctacagtg

[0316] gatagaagacactctgtatgccaacgtggacttcttcaagctcttccgtgtgcttcccacactcctagac

[0317] agccgttctcaaggtatcaatctgagatcttggggaggaatattatctgatatgtcaccaagaattcaag

[0318] agtttatccatcggccgagtatgcaggacttgctgtgggtgaccaggcccctcatgcagaatggtggtcc

[0319] agagacctttacaaagctgatgggcatcctgtctgacctcctgtgtggctaccccgagggaggtggctct

[0320] cgggtgctctccttcaactggtatgaagacaataactataaggcctttctggggattgactccacaagga

[0321] aggatcctatctattcttatgacagaagaacaacatccttttgtaatgcattgatccagagcctggagtc

[0322] aaatcctttaaccaaaatcgcttggagggcggcaaagcctttgctgatgggaaaaatcctgtacactcct

[0323] gattcacctgcagcacgaaggatactgaagaatgccaactcaacttttgaagaactggaacacgttagga

[0324] agttggtcaaagcctgggaagaagtagggccccagatctggtacttctttgacaacagcacacagatgaa

[0325] catgatcagagataccctggggaacccaacagtaaaagactttttgaataggcagcttggtgaagaaggt

[0326] attactgctgaagccatcctaaacttcctctacaagggccctcgggaaagccaggctgacgacatggcca

[0327] acttcgactggagggacatatttaacatcactgatcgcaccctccgcctggtcaatcaatacctggagtg

[0328] cttggtcctggataagtttgaaagctacaatgatgaaactcagctcacccaacgtgccctctctctactg

[0329] gaggaaaacatgttctgggccggagtggtattccctgacatgtatccctggaccagctctctaccacccc

[0330] acgtgaagtataagatccgaatggacatagacgtggtggagaaaaccaataagattaaagacaggtattg

[0331] ggattctggtcccagagctgatcccgtggaagatttccggtacatctggggcgggtttgcctatctgcag

[0332] gacatggttgaacaggggatcacaaggagccaggtgcaggcggaggctccagttggaatctacctccagc

[0333] agatgccctacccctgcttcgtggacgattctttcatgatcatcctgaaccgctgtttccctatcttcat

[0334] ggtgctggcatggatctactctgtctccatgactgtgaagaagcatcgtcttggagaaggagttgcgactg

[0335] aaggagaccttgaaaaatcagggtgtctccaatgcagtgatttggtgtacctggttcctggacagcttct

[0336] ccatcatgtcgatgagcatcttctctcctgacgatatcatcatgcatggaagaatcctacattacagcga

[0337] cccattcatcctcttcctgttcttgttggctttctccactgccaccatcatgctgtgctttctgctcagc

[0338] accttcttctccaaggccagtctggcagcagcctgtagtggtgtcatctatttcaccctctacctgccac

[0339] acatcctgtgcttcgcctggcaggaccgcatgaccgctgagctgaagaaggctgtgagcttactgtctcc

[0340] ggtggcatttggatttggcactgagtacctggttcgctttgaagaagcaaggcctggggctgcagtggagc

[0341] aacatcgggaacagtcccacggaaggggacgaattcagcttcctgctgtccatgcagatgatgctccttg

[0342] atgctgctgtctatggcttactcgcttggtaccttgatcaggtgtttccaggagactatggaaccccact

[0343]

[0344] gccctggaaaagaccgagcccctaacagagaaacggaggattccagagcaccccagaaggaatacacgact

[0345] ccttctttgaacgtgagcatccagggtgggttcctggggtatgcgtgaagaatctggtaaagatttttga

[0346] gccctgtggccggccagctgtggaccgtctgaacatcaccttctacgagaaccagatcaccgcattcctg

[0347] ggccacaatggagctgggaaaaccaccaccttgtccatcctgacgggtctgttgccaccaacctctggga

[0348] ctgtgctcgttgggggaagggacattgaaaccagcctggatgcagtccggcagagccttggcatgtgtcc

[0349] acagcacaacatcctgttccaccacctcacggtggctgagcacatgctgttctatgcccagctgaaagga

[0350] aagtcccaggaggaggcccagctggagatggaagccatgttggaggacacaggcctccaccacaagcgga

[0351] atgaagaggctcaggacctatcaggtggcatgcagagaaagctgtcggttgccattgcctttgtgggaga

[0352] tgccaaggtggtgattctggacgaacccacctctggggtggacccttactcgagacgctcaatctgggat

[0353] ctgctcctgaagtatcgctcaggcagaaccatcatcatgtccactcaccacatggacgaggccgacctcc

[0354] ttggggaccgcattgccatcattgcccagggaaggctctactgctcaggcaccccactcttcctgaagaa

[0355] ctgctttggcacaggcttgtacttaaccttggtgcgcaagatgaaaaacatccagagccaaaggaaaggc

[0356] agtgaggggacctgcagctgctcgtctaagggtttctccaccacgtgtccagcccacgtcgatgacctaa

[0357] ctccagaacaagtcctggatggggatgtaaatgagctgatggatgtagttctccaccatgttccagaggc

[0358] aaagctggtggagtgcattggtcaagaacttatcttccttcttccaaataagaacttcaagcacagagca

[0359] tatgccagccttttcagagagctggaggagacgctggctgaccttggtctcagcagttttggaatttctg

[0360] acactcccctggaagagatttttctgaaggtcacggaggattctgattcaggacctctgtttgcgggtgg

[0361] cgctcagcagaaaagagaaaacgtcaacccccgacacccctgcttgggtcccagagagaaggctggacag

[0362] acaccccaggactccaatgtctgctccccaggggcgccggctgctcacccagagggccagcctcccccag

[0363] agccagagtgcccaggcccgcagctcaacacggggacacagctggtcctccagcatgtgcaggcgctgct

[0364] ggtcaagagattccaacacaccatccgcagccacaaggacttcctggcgcagatcgtgctcccggctacc

[0365] tttgtgtttttggctctgatgctttctattgttatccctccttttggcgaataccccgctttgacccttc

[0366] acccctggatatatgggcagcagtacaccttcttcagcatggatgaaccaggcagtgagcagttcacggt

[0367] acttgcagacgtcctcctgaataagccaggctttggcaaccgctgcctgaaggaagggtggcttccggag

[0368] tacccctgtggcaactcaacaccctggaagactccttctgtgtccccaaacatcacccagctgttccaga

[0369] agcagaaatggacacaggtcaacccttcaccatcctgcaggtgcagcaccagggagaagctcaccatgct

[0370] gccagagtgccccgagggtgccgggggcctcccgcccccccagagaacacagcgcagcacggaaattcta

[0371] caagacctgacggacaggaacatctccgacttcttggtaaaaacgtatcctgctcttataagaagcagct

[0372] taaagagcaaattctgggtcaatgaacagaggtatggaggaatttccattggaggaaagctcccagtcgt

[0373] ccccatcacgggggaagcacttgttgggtttttaagcgaccttggccggatcatgaatgtgagcgggggc

[0374] cctatcactagagaggcctctaaagaaatacctgatttccttaaacatctagaaactgaagacaacatta

[0375] aggtgtggtttaataacaaaggctggcatgccctggtcagctttctcaatgtggcccacaacgccatctt

[0376] acgggccagcctgcctaaggacaggagccccgaggagtatggaatcaccgtcattagccaacccctgaac

[0377] ctgaccaaggagcagctctcagagattacagtgctgaccacttcagtggatgctgtggttgccatctgcg

[0378] tgattttctccatgtccttcgtcccagccagctttgtcctttatttgatccaggagcgggtgaacaaatc

[0379] caagcacctccagtttatcagtggagtgagccccaccacctactgggtgaccaacttcctctgggacatc

[0380] atgaattattccgtgagtgctgggctggtggtgggcatcttcatcgggtttcagaagaaagcctacactt

[0381] ctccagaaaaccttcctgcccttgtggcactgctcctgctgtatggatgggcggtcattcccatgatgta

[0382] cccagcatccttcctgtttgatgtccccagcacagcctatgtggctttatcttgtgctaatctgttcatc

[0383] ggcatcaacagcagtgctattaccttcatcttggaattatttgagaataaccggacgctgctcaggttca

[0384] acgccgtgctgaggaagctgctcattgtcttcccccacttctgcctgggccggggcctcattgaccttgc

[0385] actgagccaggctgtgacagatgtctatgcccggtttggtgaggagcactctgcaaatccgttccactgg

[0386] gacctgattgggaagaacctgtttgccatggtggtggaaggggtggtgtacttcctcctgaccctgctgg

[0387] tccagcgccacttcttcctctcccaatggattgccgagcccactaaggagcccattgttgatgaagatga

[0388] tgatgtggctgaagaaagacaaagaattattactggtggaaataaaactgacatcttaaggctacatgaa

[0389] ctaaccaagatttatccaggcacctccagcccagcagtggacaggctgtgtgtcggagttcgccctggag

[0390] agtgctttggcctcctgggagtgaatggtgccggcaaaacaaccacattcaagatgctcactggggacac

[0391] cacagtgacctcaggggatgccaccgtagcaggcaagagtattttaaccaatatttctgaagtccatcaa

[0392] aatatgggctactgtcctcagtttgatgcaattgatgagctgctcacaggacgagaacatctttaccttt

[0393] atgcccggcttcgaggtgtaccagcagaagaaatcgaaaaggttgcaaactggagtattaagagcctggg

[0394] cctgactgtctacgccgactgcctggctggcacgtacagtgggggcaacaagcggaaactctccacagcc

[0395] atcgcactcattggctgcccaccgctggtgctgctggatgagcccaccacagggatggacccccaggcac

[0396] gccgcatgctgtggaacgtcatcgtgagcatcatcagagaagggagggctgtggtcctcacatcccacag

[0397] catggaagaatgtgaggcactgtgtacccggctggccatcatggtaaagggcgcctttcgatgtatgggc

[0398] accattcagcatctcaagtccaaatttggagatggctatatcgtcacaatgaagatcaaatccccgaagg

[0399] acgacctgcttcctgacctgaaccctgtggagcagttcttccaggggaacttcccaggcagtgtgcagag

[0400] ggagaggcactacaacatgctccagttccaggtctcctcctcctccctggcgaggatcttccagctcctc

[0401] ctctcccacaaggacagcctgctcatcgaggagtactcagtcacacagaccacactggaccaggtgtttg

[0402] taaattttgctaaacagcagactgaaagtcatgacctccctctgcaccctcgagctgctggagccagtcg

[0403] acaagcccaggactgatctttcacaccgctcgttcctgcagccagaaaggaactctgggcagctggaggc

[0404] gcaggagcctgtgcccatatggtcatccaaatggactggccagcgtaaatgaccccactgcagcagaaaa

[0405] caaacacacgaggagcatgcagcgaattcagaaagaggtctttcagaaggaaaccgaaactgacttgctc

[0406] acctggaacacctgatggtgaaaccaaacaaatacaaaatccttctccagaccccagaactagaaacccc

[0407] gggccatcccactagcagctttggcctccatattgctctcatttcaagcagatctgcttttctgcatgtt

[0408] tgtctgtgtgtctgcgttgtgtgtgattttcatggaaaaataaaatgcaaatgcactcatcacaaacta<

[0409] / INSDSeq_sequence>

[0410] < / insdseq>

[0411] < / sequencedata>

[0412] <sequencedata sequenceidnumber="3">

[0413] <insdseq>

[0414] <INSDSeq_length> 979< / INSDSeq_length>

[0415] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0416] <INSDSeq_division> PAT< / INSDSeq_division>

[0417] <INSDSeq_feature-table>

[0418] <insdfeature>

[0419] <INSDFeature_key>source< / INSDFeature_key>

[0420] <INSDFeature_location>1..979< / INSDFeature_location>

[0421] <INSDFeature_quals>

[0422] <insdqualifier>

[0423] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0424] <INSDQualifier_value>protein< / INSDQualifier_value>

[0425] < / insdqualifier>

[0426] <insdqualifier id="q6">

[0427] <INSDQualifier_name>organism< / INSDQualifier_name>

[0428] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0429] < / insdqualifier>

[0430] < / INSDFeature_quals>

[0431] < / insdfeature>

[0432] < / INSDSeq_feature-table>

[0433] <INSDSeq_sequence> MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLIWLRNANPLY

[0434] SHHECHFPNKAMPSAGMLPWLQGIFCNVNNPCFQSPTPGESPGIVSNYNNSILARVYRDFQELLMNAPES

[0435] QHLGRIWTELHILSQFMDTLRTHPERIAGRGIRIRDILKDEETLTLFLIKNIGLSDSVVYLLINSQVRPE

[0436] QFAHGVPDLALKDIACSEALLERFIIFSQRRGAKTVRYALCSLSQGTLQWIEDTLYANVDFFKLFRVLPT

[0437] LLDSRSQGINLRSWGGILSDMSPRIQEFIHRPSMQDLLWVTRPLMQNGGPETFTKLMGILSDLLCGYPEG

[0438] GGSRVLSFNWYEDNNYKAFLGIDSTRKDPIYSYDRRTTSFCNALIQSLESNPLTKIAWRAAKPLLMGKIL

[0439] YTPDSPAARRILKNANSTFEELEHVRKLVKAWEEVGPQIWYFFDNSTQMNMIRDTLGNPTVKDFLNRQLG

[0440] EEGITAEAILNFLYKGPRESQADDMANFDWRDIFNITDRTLRLVNQYLECLVLDKFESYNDETQLTQRAL

[0441] SLLEENMFWAGVVFPDMYPWTSSLPPHVKYKIRMDIDVVEKTNKIKDRYWDSGPRADPVEDFRYIWGGFA

[0442] YLQDMVEQGITRSQVQAEAPVGIYLQQMPYPCFVDDSFMIILNRCFPIFMVLAWIYSVSMTVKSIVLEKE

[0443] LRLKETLKNQGVSNAVIWCTWFLDSFSIMSMSIFLLTIFIMSLLSPVAFGFGTEYLVRFEEQGLGLQWSN

[0444] IGNSPTEGDEFSFLLSMQMMLLDAAVYGLLAWYLDQVFPGDYGTPLPWYFLLQESYWLGGEGCSTREERA

[0445] LEKTEPLTEETEDPEHPEGIHDSFFEREHPGWVPGVCVKNLVKIFEPCGRPAVDRLNITFYENQITAFLG

[0446] HNGAGKTTTFSAGAPLSSKGPERHLLRGEHGPPWFPKEQQTRTSQGAANPRDTVFKSFGEPQEGTTACTP

[0447] CDGMFSRAVQCEWNVGPVPF< / INSDSeq_sequence>

[0448] < / insdseq>

[0449] < / sequencedata>

[0450] <sequencedata sequenceidnumber="4">

[0451] <insdseq>

[0452] <INSDSeq_length>3105< / INSDSeq_length>

[0453] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0454] <INSDSeq_division>PAT< / INSDSeq_division>

[0455] <INSDSeq_feature-table>

[0456] <insdfeature>

[0457] <INSDFeature_key>source< / INSDFeature_key>

[0458] <INSDFeature_location>1..3105< / INSDFeature_location>

[0459] <INSDFeature_quals>

[0460] <insdqualifier>

[0461] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0462] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0463] < / insdqualifier>

[0464] <insdqualifier id="q12">

[0465] <INSDQualifier_name>organism< / INSDQualifier_name>

[0466] <INSDQualifier_value>unidentified< / INSDQualifier_value>

[0467] < / insdqualifier>

[0468] < / INSDFeature_quals>

[0469] < / insdfeature>

[0470] < / INSDSeq_feature-table>

[0471] <INSDSeq_sequence>gcgctcttaacggcgtttatgtcctttgctgtctgaggggcctcagctc

[0472] tgaccaatctggtcttcgtgtggtcattagcatgggcttcgtgagacagatacagcttttgctctggaag

[0473] aactggaccctgcggaaaaggcaaaagattcgctttgtggtggaactcgtgtggcctttatctttatttc

[0474] tggtcttgatctggttaaggaatgccaacccgctctacagccatcatgaatgccatttccccaacaaggc

[0475] gatgccctcagcaggaatgctgccgtggctccaggggatcttctgcaatgtgaacaatccctgttttcaa

[0476] agccccaccccaggagaatctcctggaattgtgtcaaactataacaactccatcttggcaagggtatatc

[0477] gagattttcaagaactcctcatgaatgcaccagagagccagcaccttggccgtatttggacagagctaca

[0478] catcttgtcccaattcatggacaccctccggactcacccggagagaattgcaggaagaggaatacgaata

[0479] agggatatcttgaaagatgaagaaacactgacactatttctcattaaaaacatcggcctgtctgactcag

[0480] tggtctaccttctgatcaactctcaagtccgtccagagcagttcgctcatggagtcccggacctggcgct

[0481] gaaggacatcgcctgcagcgaggccctcctggagcgcttcatcatcttcagccagagacgcggggcaaag

[0482] acggtgcgctatgccctgtgctccctctcccagggcaccctacagtggatagaagacactctgtatgcca

[0483] acgtggacttcttcaagctcttccgtgtgcttcccacactcctagacagccgttctcaaggtatcaatct

[0484] gagatcttggggaggaatattatctgatatgtcaccaagaattcaagagtttatccatcggccgagtatg

[0485] caggacttgctgtgggtgaccaggcccctcatgcagaatggtggtccagagacctttacaaagctgatgg

[0486] gcatcctgtctgacctcctgtgtggctaccccgagggaggtggctctcgggtgctctccttcaactggta

[0487] tgaagacaataactataaggcctttctggggattgactccacaaggaaggatcctatctattcttatgac

[0488] agaagaacaacatccttttgtaatgcattgatccagagcctggagtcaaatcctttaaccaaaatcgctt

[0489] ggagggcggcaaagcctttgctgatgggaaaaatcctgtacactcctgattcacctgcagcacgaaggat 0490

[0491] gtagggccccagatctggtacttctttgaacaagcacacagaatgaaacatgatcagagataccctgggga 0492 0493

[0494] aacatcactgatcgcaccctccgcctggtcaatcaatacctggagtgcttggtcctggataagtttgaaa

[0495] gctacaatgatgaaactcagctcacccaacgtgccctctctctactggaggaaaacatgttctgggccgg

[0496] agtggtattccctgacatgtatccctggaccagctctctaccaccccacgtgaagtaagatccgaatg

[0497] gacatagacgtggtggagaaaaaccaataagattaaagacaggtattgggattctggtcccagagctgatc

[0498] ccgtggagaatttccggtacatctggggcgggtttgcctatctgcaggacatggttgaacaggggatcac

[0499] aaggagccaggtgcaggcggaggctccagttggaatctacctccagcagatgccctaccctgcttcgtg

[0500] gacgattctttcatgatcatcctgaaccgctgtttccctatcttcatggtgctggcatggatctactctg

[0501] tctccatgactgtgaagagcatcgtcttggagaaggagttgcgactgaaggagaccttgaaaaatcaggg

[0502] tgtctccaatgcagtgatttggtgtacctggttcctggacagcttctccatcatgtcgatgagcatcttc

[0503] ctcctgacgatattcatcatgagcttactgtctccggtggcatttggatttggcactgagtacctggttc

[0504] gctttgaagagcaaggcctggggctgcagtggagcaacatcgggaacagtcccacggaaggggacgaatt

[0505] cagcttcctgctgtccatgcagatgatgctccttgatgctgctgtctatggcttactcgcttggtacctt

[0506] gatcaggtgtttccaggagactatggaaccccacttccttggtactttcttctacaagagtcgtattggc

[0507] ttggcggtgaagggtgttcaaccagagaagaaagagccctggaaaagaccgagcccctaacagaggaaac

[0508] ggaggatccagagcacccagaaggaatacacgactccttctttgaacgtgagcatccagggtgggttcct

[0509] ggggtatgcgtgaagaatctggtaaagatttttgagccctgtggccggccagctgtggaccgtctgatca

[0510] tcaccttctacgagaaccagatcaccgcattcctgggccacaatggagctgggaaaaccaccaccttctc

[0511] cgcaggcgccccactctcctccaagggacccgagcgccatctgctgagaggagaacacggcccgccatgg

[0512] tttcccaaggagcagcagacacggacctcgcagggggcagcgaacccacgtgacacagtcttcaagtcct

[0513] ttggagagccccaggaaggaacaacagcgtgtacaccctgtgatggaatgttctctagggcggttcagtg

[0514] tgaatggaatgtggggccggtgccattctaattggttctgtttccctctagtggttgatcgcggagattt

[0515] cggcttctccatcaggacaagttcagatagcctgagatggtatcag< / INSDSeq_sequence>

[0516] < / insdseq>

[0517] < / sequencedata>

[0518] <sequencedata sequenceidnumber="5">

[0519] <insdseq>

[0520] <INSDSeq_length>2937< / INSDSeq_length>

[0521] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0522] <INSDSeq_division>PAT< / INSDSeq_division>

[0523] <INSDSeq_feature-table>

[0524] <insdfeature>

[0525] <INSDFeature_key>source< / INSDFeature_key>

[0526] <INSDFeature_location>1..2937< / INSDFeature_location>

[0527] <INSDFeature_quals>

[0528] <insdqualifier>

[0529] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0530] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0531] < / insdqualifier>

[0532] <insdqualifier id="q10">

[0533] <INSDQualifier_name>organism< / INSDQualifier_name>

[0534] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0535] < / insdqualifier>

[0536] < / INSDFeature_quals>

[0537] < / insdfeature>

[0538] < / INSDSeq_feature-table>

[0539] <INSDSeq_sequence>atgggcttcgtgcgccagatccaattgctcctgtggaaaaattggacac

[0540] tgcgaaaacgacagaagattcggttcgttgtcgagctcgtctggcctttgtctctgtttctggtgcttat

[0541] ctggttgagaaatgccaacccactgtactcacaccatgaatgtcactttccaaataaagccatgccgtct

[0542] gccggcatgcttccctggttgcagggcatcttttgtaatgtgaataacccctgcttccaatcacccacac

[0543] ctggggagtctcccggaattgttagcaactacaataattccattctcgctcgcgtctaccgagacttcca

[0544] agagcttctcatgaacgcaccggaaagtcaacacctcggtaggatttggacagagttgcacattctcagc

[0545] cagtttatggataccctcagaactcaccctgaacgaattgcgggccggggaattagaatcagagatatac

[0546] tcaaggacgaggaaacacttaccctgttcttgatcaaaaatataggtttgtcagactccgtggtctacct

[0547] gctgatcaatagccaagtcaggcccgagcaattcgcgcacggcgttcccgacctggcacttaaagatatt

[0548] gcctgttcagaagctctgttggaacgatttataatcttctcacaaaggcggggtgcaaagacggtccgat

[0549] atgctctgtgtagtcttagtcagggaacacttcaatggatcgaagataccctctatgccaatgtggattt

[0550] cttcaaactttttcgagtgctcccaactcttcttgacagcagaagtcagggcattaaccttcggagttgg

[0551] ggcggtatactctcagacatgagtccacgcatacaagagttcatacataggccctccatgcaggacttgc

[0552] tttgggttacgagacctttgatgcaaaacggcggtcccgaaacgtttactaagcttatgggaatcctctc

[0553] tgacctgctttgcggttacccggaaggtggcggctctagagtcctgtctttcaattggtacgaggacaat

[0554] aattacaaggcatttttgggcatcgattctacccgcaaagatcctatctattcatacgaccgacgaacaa

[0555] cgtccttctgcaacgcactgattcaatcactcgagagtaatccactgacaaaaatcgcctggcgagctgc

[0556] caaaccattgttgatggggaagatattgtacacacctgactccccggcagcgagacggatcttgaagaat

[0557] gcaaattctacgttcgaagaactcgaacatgtgaggaaactggtcaaagcgtgggaagaagtaggccccc

[0558] aaatctggtacttctttgataactcaacgcagatgaatatgatacgagatacgttggggaatcctacggt

[0559] caaagacttcttgaaccgacaacttggagaagaaggtataactgccgaagctatcctcaatttcctttat

[0560] aaaggcccacgcgaaagccaagctgatgacatggcgaacttcgattggcgcgacatttttaatattacgg

[0561] acaggacgcttaggctcgtaaaccagtatttggaatgtttggtgcttgataaattcgagtcatacaatga

[0562] cgagacacagcttacacagagggcgctctccttgctcgaagagaacatgttttgggccggagttgttttc

[0563] cctgatatgtatccgtggacatcctcattgccacctcatgtgaagtataaaatccgcatggacatcgatg

[0564] tggtggagaagacaaataagatcaaagatagatattgggactccggacctagagccgatccggtggaaga

[0565] tttccgctacatctgggggggattcgcctatctgcaagatatggtagaacagggaatcactcgcagccaa

[0566] gttcaagcagaggcccctgtcggtatatacttgcagcaaatgccgtacccgtgcttcgtagacgattcat

[0567] ttatgatcattctcaaccgatgcttccccatatttatggtcctcgcctggatatattccgtgagcatgac

[0568] cgtgaagtccattgtcttggagaaggaactgcgattgaaggaaacgttgaaaaaccaaggcgtatcaaac

[0569] gcagttatctggtgcacatggttccttgatagcttcagtatcatgtcaatgtcaattttcttgttgacta

[0570] tctttataatgtctctgctctcccccgttgcctttggtttcggaaccgaatatttggtacgatttgaaga

[0571] gcagggactcggcctccaatggagtaacataggcaactcccccaccgagggagacgagtttagttttctt

[0572] ttgtccatgcagatgatgttgcttgacgctgcagtgtacgggctccttgcatggtacttggatcaggtat

[0573] ttccgggggactatgggacgcccctcccatggtattttttgctgcaggaatcctactggcttgggggtga

[0574] ggggtgctctacacgcgaagagcgagctctggaaaagacagaacccctgacagaggagacagaagatcca

[0575] gagcaccctgagggcatacacgatagcttttttgagcgggagcaccctgggtgggttccaggggtttgtg

[0576] taaagaaccttgtcaaaatctttgagccgtgtggcagacccgctgtagatcgcctcaatattacgttcta

[0577] tgagaatcagataacggccttccttggtcataatggcgcgggcaagacaactacattctctgcgggcgct

[0578] cccctgtcatcaaaaggaccagagagacatctcttgcggggggaacatggcccaccatggttcccaaagg

[0579] aacaacaaacgagaacctctcaaggtgccgcaaatccaagagatacagttttcaagtctttcggggagcc

[0580] acaagaggggaccacggcttgtaccccttgtgatggtatgttctctatagctgtacaatgtgaatggaac

[0581] gttggacctgtgcccttt< / INSDSeq_sequence>

[0582] < / insdseq>

[0583] < / sequencedata>

[0584] <sequencedata sequenceidnumber="6">

[0585] <insdseq>

[0586] <INSDSeq_length>1422< / INSDSeq_length>

[0587] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0588] <INSDSeq_division>PAT< / INSDSeq_division>

[0589] <INSDSeq_feature-table>

[0590] <insdfeature>

[0591] <INSDFeature_key>source< / INSDFeature_key>

[0592] <INSDFeature_location>1..1422< / INSDFeature_location>

[0593] <INSDFeature_quals>

[0594] <insdqualifier>

[0595] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0596] <INSDQualifier_value>protein< / INSDQualifier_value>

[0597] < / insdqualifier>

[0598] <insdqualifier id="q14">

[0599] <INSDQualifier_name>organism< / INSDQualifier_name>

[0600] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0601] < / insdqualifier>

[0602] < / INSDFeature_quals>

[0603] < / insdfeature>

[0604] < / INSDSeq_feature-table>

[0605] <INSDSeq_sequence>MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLIWLRNANPLY

[0606] SHHEFCNALIQSLESNPLTKIAWRAAKPLLMGKILYTPDSPAARRILKNANSTFEELEHVRKLVKAWEEV

[0607] GPQIWYFFDNSTQMNMIRDTLGNPTVKDFLNRQLGEEGITAEAILNFLYKGPRESQADDMANFDWRDIFN

[0608] ITDRTLRLVNQYLECLVLDKFESYNDETQLTQRALSLLEENMFWAGVVFPDMYPWTSSLPPHVKYKIRMD

[0609] IDVVEKTNKIKDRYWDSGPRADPVEDFRYIWGGFAYLQDMVEQGITRSQVQAEAPVGIYLQQMPYPCFVD

[0610] DSFMIILNRCFPIFMVLAWIYSVSMTVKSIVLEKELRLKETLKNQGVSNAVFSKASLAAACSGVIYFTLY

[0611] LPHILCFAWQDRMTAELKKAVSLLSPVAFGFGTEYLVRFEEQGLGLQWSNIGNSPTEGDEFSFLLSMQMM

[0612] LLDAAVYGLLAWYLDQVFPGDYGTPLPWYFLLQESYWLGGEGCSTREERALEKTEPLTEETEDPEHPEGI

[0613] HKGKSQEEAQLEMEAMLEDTGLHHKRNEEAQDLSGGMQRKLSVAIAFVGDAKVVILDEPTSGVDPYSRRS

[0614] IWDLLLKYRSGRTIIMSTHHMDEADLLGDRIAIIAQGRLYCSGTPLFLKNCFGTGLYLTLVRKMKNIQSQ

[0615] RKGSEGTCSCSSKGFSTTCPAHVDDLTPEQVLDGDVNELMDVVLHHVPEAKLVECIGQELIFLLPNKNFK

[0616] HRAYASLFRELEETLADLGLSSFGISDTPLEEIFLKVTEDSDSGPLFAGGAQQKRENVNPRHPCLGPREK

[0617] AGQTPQDSNVCSPGAPAAHPEGQPPPEPECPGPQLNTGTQLVLQHVQALLVKRFQHTIRSHKDFLAQIVL

[0618] PATFVFLALMLSIVIPPFGEYPALTLHPWIYGQQYTFFSMDEPGSEQFTVLADVLLNKPGFGNRCLKEGW

[0619] LPEYPCGNSTPWKTPSVSPNITQLFQKQKWTQVNPSPSCRCSVDAVVAICVIFSMSFVPASFVLYLIQER

[0620] VNKSKHLQFISGVSPTTYWVTNFLFDVPSTAYVALSCANLFIGINSSAITFILELFENNRTLLRFNAVLR

[0621] KLLIVFPHFCLGRGLIDLALSQAVTDVYARFGEEHSANPFHWDLIGKNLFAMVVEGVVYFLLTLLVQRHF

[0622] FLSQWIAEPTKEPIVDEDDDVAEERQRIITGGNKTDILRLLTVYADCLAGTYSGGNKRKLSTAIALIGCP

[0623] PLVLLDEPTTGMDPQARRMLWNVIVSIIREGRAVVLTSHSMEECEALCTRLAIMVKGAFRCMGTIQHLKS

[0624] KFGDGYIVTMKIKSPKDDLLPDLNPVEQFFQGNFPGSVQRERHYNMLQFQVSSSSLARIFQLLLSHKDSL

[0625] LIEEYSVTQTTLDQVFVNFAKQQTESHDLPLHPRAAGASRQAQ< / INSDSeq_sequence>

[0626] < / insdseq>

[0627] < / sequencedata>

[0628] <sequencedata sequenceidnumber="7">

[0629] <insdseq>

[0630] <INSDSeq_length>4271< / INSDSeq_length>

[0631] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0632] <INSDSeq_division>PAT< / INSDSeq_division>

[0633] <INSDSeq_feature-table>

[0634] <insdfeature>

[0635] <INSDFeature_key>source< / INSDFeature_key>

[0636] <INSDFeature_location>1..4271< / INSDFeature_location>

[0637] <INSDFeature_quals>

[0638] <insdqualifier>

[0639] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0640] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0641] < / insdqualifier>

[0642] <insdqualifier id="q16">

[0643] <INSDQualifier_name>organism< / INSDQualifier_name>

[0644] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0645] < / insdqualifier>

[0646] < / INSDFeature_quals>

[0647] < / insdfeature>

[0648] < / INSDSeq_feature-table>

[0649] <INSDSeq_sequence>tgggcttcgtgagacagatccagctgctgctgtggaagaactggaccct

[0650] gagaaagagacagaagatcagattcgttgtggaactggtgtggcctctgagcctgttcctggtgctgatc

[0651] tggctgagaaacgccaaccccctgtacagccaccacgagttctgcaacgccctgatccagtctctggaaa

[0652] gcaatcccctgaccaagatcgcctggagagccgccaagcccctgctgatgggcaagatcctgtacacacc

[0653] tgacagccctgccgccagaagaatcctgaagaacgccaacagcaccttcgaggagctggagcacgtgaga

[0654] aagctggtgaaggcctgggaggaggttggccctcagatttggtacttcttcgacaattccacccagatga

[0655] acatgatcagagacaccctgggcaaccccaccgtgaaggacttcctgaacagacagctgggcgaggaggg

[0656] catcaccgccgaggccatcctgaacttcctgtacaagggacccagagagagccaggccgacgacatggcc

[0657] aacttcgactggagagacatcttcaacatcaccgacagaaccctgagactggtgaaccagtacctggagt

[0658] gcctggttctggacaagttcgagagctacaacgacgagacacagctgacacagagagccctgagcctgct

[0659] ggaggagaacatgttctgggccggcgtggtgttccccgacatgtacccctggaccagcagcctgcctccc

[0660] cacgtgaagtacaagatcagaatggacatcgacgtggtggagaagaccaacaagatcaaggacagatact

[0661] gggacagcggccccagagccgaccccgtggaggacttcagatacatctggggcggcttcgcctacctgca

[0662] ggacatggtggaacagggaatcaccagaagccaggtgcaggccgaggcccccgtgggcatctacctgcag

[0663] cagatgccctacccctgcttcgtggacgacagcttcatgatcatcctgaacagatgcttccccatcttca

[0664] tggttctggcttggatctacagcgtgagcatgaccgtgaagagcatcgtgctggagaaggagctgagact

[0665] gaaggagaccctgaagaaccagggcgtgagcaacgccgtgttcagcaaggccagcctggctgccgcctgc

[0666] agcggcgtgatctacttcaccctgtacctgccccacatcctgtgcttcgcctggcaggacagaatgaccg

[0667] ccgagctgaagaaggccgtgagcctgctgagccccgtggccttcggcttcggcaccgagtacctggtgag

[0668] attcgaggagcagggcctgggcctgcagtggagcaacatcggcaacagccccaccgagggcgacgagttc

[0669] tcttttctgctgtctatgcagatgatgctgctggacgctgccgtgtacggcctgctggcctggtatctgg

[0670] accaggtgttccccggcgactacggcacccccctgccctggtacttcctgctgcaggagagctactggct

[0671] gggcggcgagggctgcagcaccagagaggagagagccctggagaagaccgagcccctgaccgaggagaca

[0672] gaggaccccgagcaccccgagggcatccacaagggcaagagccaggaggaagctcagctggagatggagg

[0673] ccatgctggaggacaccggcctgcaccacaagagaaacgaggaggcccaggacctgagcggcggcatgca

[0674] gagaaagctgagcgtggccatcgccttcgtgggcgacgccaaggtggtgatcctggacgagcccaccagc

[0675] ggcgtggacccctactctaggagaagcatctgggacctgctgctgaagtacagaagcggcagaaccatca

[0676] tcatgagcacccaccacatggacgaggctgatctgctgggcgacagaatcgccatcatcgcccagggcag

[0677] actgtactgcagcggcacccccctgttcctgaagaactgcttcggcaccggcctgtacctgaccctggtg

[0678] agaaagatgaagaacatccagagccagagaaagggcagcgagggcacctgcagctgcagcagcaagggct

[0679] tcagcaccacctgccccgcccacgtggacgacctgacccccgagcaggtgctggacggcgacgtgaacga

[0680] gctgatggacgtggtgctgcaccacgtgcccgaggccaagctggtggagtgcatcggccaggagctgatc

[0681] ttcctgctgcccaacaagaacttcaagcacagagcctacgccagcctgttcagagagctggaggagaccc

[0682] tggccgacctgggcctgagcagcttcggcatcagcgacacacccctggaggaaatcttcctgaaggtgac

[0683] agaggacagcgacagcggccctctgttcgccggaggcgctcagcagaagagagagaacgtgaatcccaga

[0684] cacccctgtctgggccctagagagaaggccggccagacaccccaggacagcaacgtgtgcagccctggcg

[0685] ctcctgccgcccaccctgagggccagcctcctcccgagcccgagtgccctggacctcagctgaacaccgg

[0686] cacacagctggtgctgcagcacgtgcaggccctgctggtgaagagattccagcacaccatcagaagccac

[0687] aaggacttcctggcccagatcgtgctgcccgccaccttcgtgtttctggctctgatgctgagcatcgtga

[0688] tccctcccttcggcgaatatcctgccctgaccctgcacccctggatctacggccagcagtacaccttctt

[0689] ctctatggacgagcccggcagcgagcagttcaccgtgctggccgacgtgctgctgaacaagcccggcttc

[0690] ggcaacagatgcctgaaggagggctggctgcccgagtacccctgcggcaacagcacaccctggaagaccc

[0691] cctccgtgtctcccaacatcacccagctgttccagaagcagaagtggacccaggtgaaccccagccccag

[0692] ctgcagatgcagcgtggacgccgtggtggccatctgcgtgatcttcagcatgtcctttgtgcccgccagc

[0693] ttcgtgctgtacctgatccaggagagagtgaacaagagcaagcacctgcagttcatcagcggcgtgagcc

[0694] ccaccacctactgggtgaccaacttcctgttcgacgtgcccagcaccgcctacgtggccctgagctgcgc

[0695] caacctgttcatcggcatcaacagcagcgccatcaccttcatcctggagctgttcgagaacaacagaacc

[0696] ctgctgagattcaacgccgtgctgagaaagctgctcattgtgttcccccacttctgcctgggcagaggcc

[0697] tgatcgacctggctctgtctcaggccgtgaccgacgtgtacgccagattcggcgaggagcacagcgccaa

[0698] ccccttccactgggacctgatcggcaagaacctgttcgctatggtggtggagggcgtggtgtacttcctg

[0699] ctgaccctgctggtgcagagacacttcttcctgtctcagtggatcgccgagcccaccaaggagcccatcg

[0700] tggacgaggacgacgacgtggccgaggagagacagagaatcatcacaggcggaaacaagaccgacatcct

[0701] gagactgctgacagtgtatgccgactgcctggccggcacctacagcggcggcaacaaaaggaagctgagc

[0702] accgccatcgctctgattggctgcccccctctggtgctgctggacgaacctacaaccggcatggaccccc

[0703] aggccagaagaatgctgtggaacgtgatcgtgagcatcatcagagagggcagagccgtggtgctgaccag

[0704] ccacagcatggaggagtgcgaggccctgtgcaccagactggccatcatggtgaagggcgccttcagatgc

[0705] atgggcaccatccagcacctgaagagcaagttcggcgacggctacatcgtgaccatgaagatcaagagcc

[0706] ccaaggacgacctgctgcccgacctgaaccccgtggagcagttcttccagggcaacttccccggcagcgt

[0707] gcagagagagagacactacaacatgctgcagttccaggtgagcagcagcagcctggccagaatcttccag

[0708] ctgctgctgagccacaaggacagcctgctgatcgaggagtacagcgtgacccagacaaccctggaccagg

[0709] tgttcgtgaacttcgccaagcagcagaccgagagccacgacctgcccctgcaccccagagctgctggagc

[0710] cagcagacaggcccaggactag< / INSDSeq_sequence>

[0711] < / insdseq>

[0712] < / sequencedata>

[0713] <sequencedata sequenceidnumber="8">

[0714] <insdseq>

[0715] <INSDSeq_length> 4269< / INSDSeq_length>

[0716] <INSDSeq_moltype> DNA< / INSDSeq_moltype>

[0717] <INSDSeq_division> PAT< / INSDSeq_division>

[0718] <INSDSeq_feature-table>

[0719] <insdfeature>

[0720] <INSDFeature_key>source< / INSDFeature_key>

[0721] <INSDFeature_location>1..4269< / INSDFeature_location>

[0722] <INSDFeature_quals>

[0723] <insdqualifier>

[0724] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0725] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0726] < / insdqualifier>

[0727] <insdqualifier id="q18">

[0728] <INSDQualifier_name>organism< / INSDQualifier_name>

[0729] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0730] < / insdqualifier>

[0731] < / INSDFeature_quals>

[0732] < / insdfeature>

[0733] < / INSDSeq_feature-table>

[0734] <INSDSeq_sequence> atggggttcgtacgccagattcaattgctgttgtggaaaaaattggacct

[0735] tgcggaagaggcaaaaataaggttcgttgtcgaacttgtttggcctctgttcttccttgttcttat

[0736] atggctccgcaacgcgaatcctctctatagccatcacgagttctgcaatgcgctgattcagtcattggaa

[0737] agtaatccccttacaaagatcgcctggcgcgctgcaaagccgttgctcatgggcaaaatactttacaccc

[0738] ccgacagccccgctgcgagacgcatactgaaaaatgccaattcaacgttcgaagaattggagcatgtcag

[0739] gaaactggttaaagcgtgggaagaggtaggtccccaaatttggtatttctttgacaactctctcagatg

[0740] aatatgatcagggacacgttggggaatccaacggtcaaggacttccttaataggcaattgggcgaagaag

[0741] ggattacggcagaagctattcttaatttcttgtataaggggccacgcgagtcccaggcagacgatatggc

[0742] aaactttgactggagggacattttcaacataactgatagaacacttaggctggttaaccagtatctcgag

[0743] tgcctggtcctcgacaaattcgaaagctacaacgacgagacccaattgactcaaagagctctgagtcttt

[0744] tggagaaaaacatgttctgggctggtgtagttttccccgatatgtacccatggacctcatcattgcccc

[0745] acacgtgaagtaataagattcgaatggacatcgatgttgtagaaaaaaacaaataaaattaaggacagatat

[0746] tgggattcagggcccagagccgacccggttgaagatttccggtatatctggggcggttttgcgtaccttc

[0747] aggatatggtagagcaggcataacaagatctcaggttcaggccgaagcgcccgtaggtatctatcttca

[0748] acaaatgccttatccctgtttcgtagatgactcctttatgatcatcctgaatcggtgctttccaatcttc

[0749] atggtactcgcgtggatatactcagtcagtatgacggtcaaatctatagtattggaaaggaactgcggc

[0750] tcaaagagacgctgaaaaaccaaggtgtttctaatgcagtatttagtaaagcatcccttgcggctgcttg

[0751] ttccggcgttatatacttcactttgtatttgccccacatcctgtgtttcgcctggcaggaccgaatgacc

[0752] gccgagttgaaaaaagctgtgtctcttctgtctcctgtagcgtttgggtttggcaccggaatatcttgtgc

[0753] gctttgaagagcaaggtctgggcttgcagtggtcaaatatagggaattccccaaccgagggcgatgaatt

[0754] ctccttcctgctcagcatgcaaatgatgcttcttgatgccgctgtctatgggctgcttgcttggtacctg

[0755] gatcaggtgttccccggtgattacggtaccccacttccatggtattttttgttgcaggagtcatattggt

[0756] tgggcggcgagggatgcagtacaagggaggagagggcccttgaaaagaccgaaccgcttactgaagaaac

[0757] ggaagatcccgagcatccagaagggatccacaaaggaaaatctcaggaggaggcacagttggagatggaa

[0758] gcgatgctcgaagatacgggtctgcatcataagcggaatgaagaagcacaagacttgtccggtggaatgc

[0759] agcgcaagttgtcagtagctatagctttcgtgggcgatgctaaagtcgttatcctggatgaaccaacgtc

[0760] aggcgttgatccatattcaaggcgctctatctgggacctcctgctgaagtaccgcagcggcaggactatc

[0761] attatgagtacgcaccatatggatgaagcagatttgctgggcgatagaatagccattattgcccaggggc

[0762] gattgtattgtagcggtactccgctcttcttgaaaaattgttttggcaccggtttgtacttgactctcgt

[0763] gaggaaaatgaagaatatacagagtcagagaaaaggaagcgaagggacgtgcagttgcagctcaaagggt

[0764] ttcagcaccacatgtccagcccacgtcgacgatttgactcctgagcaagtcctcgatggggatgtaaacg

[0765] agctcatggatgtggttctgcaccaccgtaccagaggccaaattggtcgagtgtataggacaggagctgat

[0766] cttccttctgccgaataaaaaatttcaagcatcgagcgtatgcgagtctcttccgagaacttgaagagacc

[0767] cttgcggacctcgggctgtcttcctttggaatctccgacactcccttggaagaaatttttttgaaagtta

[0768] ctgaggactccgactccgggcccctgttcgcgggcggcgcgcagcagaagcgagagaacgttaatccacg

[0769] ccatccctgccttggtcctaggggaaaaagccggacagactccacaggagacagcaacgtatgttcccctggc

[0770] gcgcccgcagcacaccccgaaggccagccacccccagagccggaatgcccgggtccccagctgaacaccg

[0771] gtacacagctggtactccaacatgtccaggccctccttgtgaaaaggttccagcatacaataaggagcca

[0772] caaggactttctggcccagatcgtactcccggcgacatttgtgtttcttgctctttatgctgagcattgta

[0773] atccccccatttggagagtatcctgcactgaccctgcatccatggattttacggccagcaatatacattct

[0774] tttccatggatgaaccgggaagtgagcaatttactgtactcgctgatgttttgctcaacaagccaggctt

[0775] cggaaacaggtgtctgaaagaaggttggcttcccgagtacccttgcgggaactccacgccttggaaaaca

[0776] ccttcagtctctcctaatatcacacagctctttcaaaaacagaaatggacacaagtaaatccctccccgt

[0777] catgccggtgttccgttgatgcagtcgttgcaatctgtgtaatcttctccatgtcttttgtgcctgcgag

[0778] cttcgtcttgtacctcatccaagagcgagttaataagagtaaacatttgcagtttattagtggagtcagt

[0779] cccaccacgtattgggttacaaatttcctcttcgacgtaccctccaccgcttatgtggccctgagctgtg

[0780] ctaaccttttcattgggataaacagtagtgctattacatttattttggaactctttgagaacaatcggac

[0781] gctccttcgcttcaacgccgtccttcgaaagctcctgatcgtttttcctcatttctgcttggggcgcggc

[0782] ctcatagacctcgcactgagtcaagcggtaaccgatgtttatgcacgatttggagaggaacactccgcta

[0783] acccttttcattgggatctgataggcaaaaatctcttcgcgatggttgttgagggagttgtgtactttct

[0784] cctcacgctcttggtgcagaggcatttctttcttagtcagtggatcgcggaacccacgaaggaaccaatt

[0785] gtggatgaagatgacgatgttgccgaggaaaggcagcgaattattacgggcggaaataagactgatatat

[0786] tgagacttctcacggtgtacgcagattgtctcgctgggacatacagtggcggaaacaaacggaaactcag

[0787] cacagctatcgcgcttataggatgtccccccctggttctcctcgatgaaccaacgacggggatggacccg

[0788] caggcacggcgaatgctctggaacgtgatagtaagtattatacgggaaggtagggctgtcgttctcactt

[0789] cccattccatggaggagtgtgaagctctctgtacgaggttggcgattatggtgaaaggcgcgtttcggtg

[0790] tatgggcacaatacagcaccttaagagcaaattcggcgatggttatatagtgaccatgaagataaagtcc

[0791] cccaaggatgatctgcttcctgatttgaacccggtcgagcagtttttccaggggaactttcctgggtcag

[0792] tgcaacgggaacgacattacaacatgcttcaattccaagtttcctcttccagtctggctcgaattttcca

[0793] acttctgttgagccataaggacagtttgctcatagaggaatactccgtgacacagaccacactggaccag

[0794] gtatttgtgaactttgctaagcaacagacagaaagtcatgatctccccctgcatccccgggcagcgggcg

[0795] cgtcccgccaagcgcaagac< / INSDSeq_sequence>

[0796] < / insdseq>

[0797] < / sequencedata>

[0798] < / st26sequencelisting>

[0799] <---

Claims

1. ABCA4 miniprotein for gene therapy of Stargardt disease, containing the amino acid sequence SEQ ID NO:

6.

2. A nucleic acid encoding the ABCA4 miniprotein according to claim 1, containing a nucleotide sequence selected from SEQ ID NO: 5, 7, 8.

3. The nucleic acid according to claim 2, containing a nucleotide sequence selected from SEQ ID NO: 7, 8.

4. An expression vector for expression in eukaryotic cells, containing a nucleic acid of any of paragraphs 2, 3.

5. The expression vector according to claim 4, which is a plasmid expression vector containing the following elements: – the site of the origin of replication ori; – left inverted terminal repeat ITR; – CMV enhancer of cytomegalovirus promoter; – cytomegalovirus promoter CMV promoter; – the sequence of the intron of the human b-globin gene; – optimized ABCA4 gene sequence SEQ ID NO: 7; – hGH polyadenylation signal sequence poly(A) signal; – right inverted terminal repeat ITR; – promoter of the ampicillin resistance gene AmpR promoter; – the gene of resistance to the antibiotic ampicillin AmpR.

6. The expression vector according to claim 5, which is a viral expression vector.

7. The expression vector according to claim 6, which is an adeno-associated virus.

8. The expression vector according to claim 7, which is an adeno-associated virus of serotype 9.

9. The expression vector according to item 8, produced in adherent HEK293T cell cultures.

10. Use of an expression vector according to any one of claims 4-9 for gene therapy of Stargardt disease.

Images