RU2809065C1 - Nucleic acid for allotopic expression of mt-nd4 gene - Google Patents

Abstract

FIELD: biotechnology; molecular biology; genetic engineering.

SUBSTANCE: nucleic acid is proposed for allotopic expression of MT-ND4 gene, as well as an expression vector containing the said nucleic acid, and its use for the treatment of Leber hereditary optic neuropathy. The nucleic acid contains the nucleotide sequence SEQ ID No. 1 and the sequence encoding the mitochondrial localization peptide of cytochrome c oxidase subunit 8. The inventions make it possible to reduce the level of reactive oxygen species in the cell by at least 25% and the level of mitochondrial calcium by at least 29%.

EFFECT: inventions can be used for gene replacement therapy for Leber hereditary optic neuropathy.

11 cl, 11 dwg, 2 tbl, 8 ex

Description

Field of technology

The invention relates to the field of biotechnology and molecular biology, in particular genetic engineering, namely to a nucleic acid molecule for allotopic expression of a nucleotide sequence encoding the MT-ND4 protein.

State of the art

Elucidation of the molecular mechanisms of development of hereditary retinal diseases (HRD) determines progress in the development of new strategies for the treatment of this disease [Scholl H.P.N, et al. Emerging therapies for inherited retinal degeneration // Sci. Transl. Med. American Association for the Advancement of Science, 2016. Vol.8, No. 368]. There is a specific type of cells of neural origin, the dysfunction of which also causes the manifestation of NDS - retinal ganglion cells.

The first clinical description of a patient suffering from a hereditary disease manifesting itself in the form of optic neuropathy (Leber's Hereditary Optic Neuropathy, LHON) was made more than 150 years ago by Albrecht von Graefe. Later in 1871, the disease was identified as a separate nosological unit by the German ophthalmologist Theodor Leber [Leber T. Ueber hereditare und congenital-angelegte Sehnervenleiden // Albr. von Graefe's Arch. für Ophthalmol. Springer, 1871. Vol.17, No.2. P. 249-291]. Because there is significant gender disproportion, and hereditary optic neuropathy is more common in young men, it has been shown that the mode of inheritance was maternal and the disease was caused by mutations in the mitochondrial genome, or mtDNA [Erickson R.P. Leber's optic atrophy, a possible example of maternal inheritance. //Am. J.Hum. Genet. Elsevier, 1972. Vol.24, No.3. P. 348]. Douglas Wallace and colleagues in 1988 discovered the first mitochondrial DNA (mtDNA) mutation associated with LHON in the MT-ND4 gene, encoding the MT-ND4 protein, one of the subunits of the NADH dehydrogenase complex of the mitochondrial electron transport chain [Brown M.D. et al. Functional Analysis of Lymphoblast and Cybrid Mitochondria Containing the 3460, 11778, or 14484 Leber's Hereditary Optic Neuropathy Mitochondrial DNA Mutation * // J. Biol. Chem. Elsevier, 2000. Vol.275, No.51. P. 39831-39836]. Many other mutations have been described in genes encoding components of the NADH dehydrogenase complex that are involved in the pathogenesis of LHON, but mutations in the MT-ND4 gene (G11778A) [Wallace D.C. et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy // Science, 1988. Vol. 242, No. 4884. P. 1427-1430], MT-ND1 (G3460A) and MT-ND6 (T14484C) [Huoponen K. et al. A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy. //Am. J.Hum. Genet. Elsevier, 1991. Vol.48, No.6. P. 1147] are collectively responsible for 90-95% of all cases of the disease. Moreover, about 70% of all cases are associated with a mutation in the MT-ND4 gene [Yu-Wai-Man P., Griffiths P.G., Chinnery P.F. Mitochondrial optic neuropathies-disease mechanisms and therapeutic strategies //Progress in retinal and eye research. 2011. T. 30. No. 2. P. 81-114].

These replacements cause acute or subacute death of retinal ganglion cells (RGCs) and their axons that make up the optic nerve, leading to loss of central vision and blindness. The manifestation of the pathology is bilateral: most patients experience vision loss first in one eye, and then, usually after 6-8 weeks, in the other. Only in 25% of cases there is a simultaneous manifestation of disorders in both eyes [Yu-Wai-Man P. et al. Treatment strategies for inherited optic neuropathies: past, present and future // Eye 2014 285. Nature Publishing Group, 2014. Vol.28, No. 5. P. 521-537].

Mutations leading to LHON disrupt the functioning of complex I of the respiratory chain, namely, they significantly reduce the NADH-ubiquinone oxidoreductase activity of the NADH dehydrogenase complex [Brown M.D. et al. Functional Analysis of Lymphoblast and Cybrid Mitochondria Containing the 3460, 11778, or 14484 Leber's Hereditary Optic Neuropathy Mitochondrial DNA Mutation * // J. Biol. Chem. Elsevier, 2000. Vol.275, No.51. P. 39831-39836; Majander A. et al. Electron transfer properties of NADH: Ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON) // FEBS Lett. No longer published by Elsevier, 1991. Vol.292, No.12. P. 289 292; Howell N. et al. Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees. //Am. J.Hum. Genet. Elsevier, 1991. Vol.49, No.5. P. 939; Smith P.R. et al. Platelet mitochondrial function in Leber's hereditary optic neuropathy // J. Neurol. Sci. Elsevier, 1994. Vol.122, No.1. P. 80-83; Carelli V. et al. Leber's hereditary optic neuropathy // Neurology. Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology, 1997. Vol.48, No.6. P. 1623-1632], which leads to the death of neurons due to depletion of energy in the form of ATP. The most common mutations are in the MT-ND1, MT-ND6, MT-ND4 genes, with mutations in the MT-ND4 gene accounting for the majority of LHON cases [Majander A. et al. Electron transfer properties of NADH: Ubiquinone reductase in the ND1/3460 and the ND4/11778 mutations of the Leber hereditary optic neuroretinopathy (LHON) // FEBS Lett. No longer published by Elsevier, 1991. Vol.292, No. 1-2. P. 289-292; Carelli V. et al. Biochemical Features of mtDNA 14484 (ND6/M64V) Point Mutation Associated with Leber's Hereditary Optic Neuropathy. 1999; Hofhaus G. et al. Respiration and Growth Defects in Transmitochondrial Cell Lines Carrying the 11778 Mutation Associated with Leber's Hereditary Optic Neuropathy * // J. Biol. Chem. Elsevier, 1996. Vol.271, No.22. P. 13155-13161].

Studies of cell cultures of cytoplasmic hybrids (cybrids) have revealed a decrease in the growth rate of mutant cells along with lower oxygen consumption. Valerio Carelli's group showed that energy deficiency determines cell fate in the LHON cytoplasmic hybrid model [Vergani L. et al. MtDNA Mutations Associated with Leber's Hereditary Optic Neuropathy: Studies on Cytoplasmic Hybrid (Cybrid) Cells // Biochem. Biophys. Res. Commun. Academic Press, 1995. Vol. 210, No. 3. P. 880-888]. Complex I mutations can also cause oxidative stress, the potential pathogenic role of which has received less attention.

Mitochondrial respiration is the basis of metabolic processes in the cells of all multicellular eukaryotes. Disruption of mitochondrial processes, whether fragmentation or other morphological changes, increased frequency of mutations in mtDNA, accumulation of mutant proteins, changes in mitochondrial membrane permeability, changes in redox potential and impaired oxidative phosphorylation, can lead to significant consequences for intracellular metabolism. In particular, muscle fibers and cells of neural origin are susceptible to mitochondrial damage and dysfunction. Oxidative stress and mitochondrial damage are implicated in the pathogenesis of several neurodegenerative diseases, as they are characterized by the overproduction of reactive oxygen species, which can cause mutations in mitochondrial DNA, reduce the efficiency of the mitochondrial respiratory chain, alter membrane permeability, and affect Ca ²⁺ homeostasis and mitochondrial defense systems.

Dysfunction of complex I of the respiratory chain, resulting from mutations in mtDNA (m.11778G>A in MT-ND4; m.3460G>A in MT-ND1; m.14484T>C in MT-ND6), leads to a decrease in the level of ATP synthesis. When hypoxia caused by such dysfunction occurs, the retinal ganglion cells, which are most sensitive to mitochondrial damage, die. The location and functional characteristics of retinal ganglion cells determine their unique energy requirements: the long axons of these cells in the retrobulbar region transition from an unmyelinated to a myelinated state. Axonal transport is caused by the motor proteins kinesin and dynein, which require large amounts of ATP to perform this function. Energy depletion that occurs with mitochondrial dysfunction impairs axonal transport [Chinnery P.F. et al. Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation? //American journal of medical genetics. 2001. T. 98. No. 3. - pp. 235-243]. In addition, apoptosis of retinal ganglion cells ultimately leads to optic nerve atrophy.

The role of reactive oxygen species (ROS) in the pathogenesis of LHON is complementary to the ATP deficiency hypothesis, since mitochondria are the site of oxidative phosphorylation, and complex I of the respiratory chain is the source, as by-products, of most cellular superoxides (O ₂ ^- ) [Bolisetty S., Jaimes E. A. Mitochondria and reactive oxygen species: physiology and pathophysiology //International journal of molecular sciences. 2013. T. 14. No. 3. C. 6306-6344], the predecessors of all AFCs [Juan S.A. et al. The chemistry of reactive oxygen species (ROS) revisited: outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies //International Journal of Molecular Sciences. - 2021. - T. 22. -No. 9. - P. 4642]. Although ROS play an intracellular signaling role in basic cellular processes [Starkov AA The role of mitochondria in reactive oxygen species metabolism and signaling //Annals of the New York Academy of Sciences. - 2008. - T. 1147. - No. 1. - P. 37-52], the range of ROS concentrations with beneficial physiological consequences is relatively narrow.

In recent years, gene replacement therapy has been considered as a potential solution to the problem of treating LHON. In gene replacement therapy, a copy of the gene that does not contain mutations is delivered to the target cell, which ensures the synthesis of a functional protein. However, gene therapy for mitochondrial DNA mutations has been challenging because the presence of a double membrane can interfere with the delivery of genetic material to the mitochondria. In addition, a single somatic cell can contain hundreds of mitochondria with a large number of copies of the genome, making the possibility of direct mitochondrial delivery of nucleic acids questionable. To circumvent this difficulty, Dr. Guy's group in Miami proposed allotopic expression technology in 2002. This technology uses a nuclear DNA (nDNA) transgene encoding the wild-type mitochondrial protein MT-ND4. Using an adeno-associated virus (AAV) vector, the transgene is introduced into the nucleus of retinal ganglion cells. Using a mitochondrial signal sequence, the messenger RNA is then translocated from the nucleus to the outer mitochondrial membrane, where the wild-type MT-ND4 protein is translated and incorporated into the mitochondrial respiratory chain complex [Guy J, Qi X, Pallotti F, et al. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol. 2002 Nov;52(5):534-42. doi: 10.1002/ana. 10354; Chi SC, Cheng HC, Wang AG. Leber Hereditary Optic Neuropathy: Molecular Pathophysiology and Updates on Gene Therapy. Biomedicines. 2022 Aug9;10(8): 1930. doi: 10.3390/biomedicines10081930; Artika I.M. Allotopic expression of mitochondrial genes: Basic strategy and progress // Genes Dis. Elsevier, 2020. Vol. 7, No. 4. P. 578-584].

In 2002, Guy et al used an adeno-associated viral vector (AAV) containing a codon-optimized sequence encoding the MT-ND4 protein and a sequence encoding a mitochondrial localization (MTS) peptide. The MTS was the N-terminal region of either (1) the P1 isoform of the human ATP synthase c subunit (ATS), containing the entire 61-amino acid MTS sequence along with the five amino acids of the mature P124 polypeptide, or (2) Aldh, containing the first 19-amino acid MTS Cybrids with the MT-ND4 gene mutation transduced with the described viruses produced three times more ATP than cybrids transduced with empty virus particles, which the authors interpreted as a successful restoration of complex I functions [Guy J, Qi X, Pallotti F, et al. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol. 2002 Nov;52(5):534-42. doi: 10.1002/ana. 10354].

In 2016, Feuer et al conducted the first clinical trial (CT) of the self-complementary AAV scAA-V2(Y444,500,730F)-P1ND4v2 in five patients with a mutation in the MT-ND4 gene who received gene therapy in one eye. scAA-V2(Y444,500,730F)-P1ND4v2 contains the MT-ND4 gene sequence optimized for nuclear transcription and the sequence encoding the mitochondrial localization peptide of the P1 isoform of the ATP synthase subunit. No patients experienced serious adverse events in response to therapy. A significant increase in visual acuity was observed in two patients. From an anatomical point of view, there were no significant differences in the thickness of the retinal layers [Feuer W.J. et al. Gene Therapy for Leber Hereditary Optic Neuropathy: Initial Results // Ophthalmology. Elsevier, 2016. Vol. 123, no. 3. P. 558-570].

In 2017, GenSight Biologics conducted a randomized, double-blind, placebo-controlled, multicenter Phase III clinical trial (CT). Patients were injected into one eye with GS010: a recombinant adeno-associated virus containing a modified cDNA encoding wild-type human mitochondrial protein MT-ND4 and MTS COX10. The other eye was given a sham injection. On average, patients experienced improvement in visual acuity. Similar improvement was reported in the other sham-injected eye. This raised suspicion of the transfer of viral vector DNA from the injected eye to the other eye [Yu-Wai-Man P. et al. Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy // Sci. Transl. Med. American Association for the Advancement of Science, 2020. Vol.12, No. 573]. In July 2019, phase III of the GS010 clinical trial was successfully completed.

In January 2022, the Chinese company Neurophth Biotechnology Limited received approval from the European Medicines Agency (EMA) for the drug NR082 (rAAV2-ND4) - a new recombinant adeno-associated viral vector of serotype 2 containing the mitochondrial codon-optimized MT-ND4 gene under control of the cytomegalovirus promoter and enhancer.

Also in 2014 at the Eye Institute named after. Bascom Palmer University of Miami has initiated a phase 1 clinical trial of scAAV2-P1ND4v2 for the treatment of ND4-associated LHON. Preliminary results reported evidence of efficacy in 2 patients in the chronic group and 4 patients in the acute group. No dose-dependent effect was observed in the study cohorts [Sahel J.A. et al. Gene Therapies for the Treatment of Leber Hereditary Optic Neuropathy // Int. Ophthalmol. Clin. Wolters Kluwer Health, 2021. Vol.61, No.4. P. 195].

Application WO2019241206A1 discloses the use of a recombinant vector encoding a human NADH dehydrogenase 4 (ND4) polypeptide and containing a nucleic acid sequence encoding the Cox10 MTS, a NADH dehydrogenase 4 (ND4) polypeptide, and a Cox10 3'UTR in the treatment of Leber's hereditary optic neuropathy (LHON). .

Patent CN109207520 B discloses a reading frame for intranuclear expression of the mitochondrial gene MT-ND4, which includes the nuclear CAM promoter, a fusion sequence encoding the mitochondrial localization peptide COX10 and the nuclear-expressed ND4 gene, as well as a sequence anchoring the mRNA to the mitochondrial surface.

Patent CN104450747 (Wuhan Neurophth Biological Technology Co., Ltd.), discloses the ND4 gene for the treatment of LHON, in combination with CAG, Cox10 MTS and UTR promoter sequences, which is 625 bp in length, for a total sequence length of 3824 bp . AAV carrying this sequence is administered intravitreally to treat LHON. Patent CN111073899 B of the same copyright holder discloses a nucleic acid fusion that encodes from the 5' end to the 3' end: MTS (COX10), ND4, UTR.

Patent CN110876269 B discloses various variants of the nucleic acid sequence encoding the ND4 protein, as well as various variants of the nucleotide sequence encoding MTS, such as the native and optimized MTS COX10, the native and optimized MTS OPA1. Optimized sequences have been designed to improve transcription and translation efficiency. The recombinant nucleic acid contained sequences coding for MTS and ND4, as well as the 3'UTR untranslated region. The synthetic recombinant nucleic acid was inserted into the AAV2 vector for further use when introduced into target cells. Another optimized ND4 sequence fused to the MTS coding sequence COX10 or OPA1 and/or 3'UTR is disclosed in patent CN110699367 B CN109970861B.

Patent EP1880008 B1 (Institut National de la Sante et de la Recherche Medicale INSERM) discloses an expression vector adapted for stable and efficient delivery of protein to mitochondria. The vector contains an MTS coding sequence, in particular COX10 (cytochrome c oxidase 10) or SOD2 (superoxide dismutase 2), a protein coding sequence, in particular ND4, and a sequence at the 3' end of these sequences, in particular the SV40 3'UTR or COX10 3'-UTR. This vector uses the post-translational import pathway, but does not use any other mitochondrial localization peptide sequences other than COX10 and SOD2.

However, existing analogues are not effective enough. For example, Gensight Biologics SA withdrew its marketing authorization application for Lumevoq on April 20, 2023, for the treatment of vision loss caused by the eye disease known as Leber's hereditary optic neuropathy. Study results showed no significant difference in vision between eyes treated with Lumevoq and eyes treated with sham or placebo. Additionally, studies have not provided sufficient evidence that administering Lumevoq to both eyes will benefit patients [https://www.ema.europa.eu/en/medicines/human/withdrawn-applications/lumevoq, accessed 08/09/2023] .

The technical problem to which the present invention is aimed is to solve at least one of the problems mentioned above in the prior art.

The essence of the invention

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

One of the possible technical problems that the present invention could be aimed at was expanding the arsenal of technical means aimed at reducing the consequences of mitochondrial dysfunction in cells with a mutation in the MT-ND4 gene. The invention can be used in gene therapy for Leber hereditary optic neuropathy.

The technical result achieved by implementing the present invention is to reduce the consequences of mitochondrial dysfunction in cells with a mutation in the MT-ND4 gene.

The problem can be solved by creating a nucleotide sequence for allotopic expression of the MT-ND4 gene in cells. The technical result can be achieved by creating a codon-optimized sequence ND4opt (Seq Id No: 1), encoding the normal MT-ND4 protein.

In one embodiment, the present invention provides a nucleic acid for allotopic expression of the MT-ND4 gene, containing the nucleotide sequence Seq Id No.: 1 and the sequence encoding the mitochondrial localization peptide of cytochrome c oxidase subunit 8 (Cox8 protein).

In one embodiment of the present invention, the mitochondrial localization peptide of the Cox8 protein contains the sequence Seq Id No.:6. In yet another embodiment of the invention, the sequence Seq Id No.:5 encodes the mitochondrial localization peptide of the Cox8 protein.

In another embodiment, the invention provides an expression vector for expression in eukaryotic cells containing a nucleic acid for allotopic expression of the MT-ND4 gene of the present invention.

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

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- a sequence encoding the sequence of SEQ Id No.:6 of the mitochondrial localization (MTS) peptide of the COX8 protein;

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- site of origin of replication for packaging into phage particles f1 ori;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

In yet another embodiment, the vector contains the sequence SEQ Id No.:5 as the sequence encoding the mitochondrial localization (MTS) peptide of the COX8 gene (SEQ Id No.:6).

In another embodiment of the invention, said vector is a viral expression vector, in particular an adeno-associated virus, in particular an adeno-associated virus of serotype 6.

In another embodiment of the invention, an adeno-associated virus of serotype 6, produced in HEK293 suspension cell cultures, is proposed as a viral expression vector.

Another embodiment of the invention provides the use of an expression vector of the present invention for the treatment of Leber hereditary optic neuropathy.

Introduction of the nucleic acid of the present invention into cells with a mutation in the MT-ND4 gene leads to a statistically significant decrease in the level of such markers of oxidative stress as the content of reactive oxygen species (ROS) in cells and calcium ions in mitochondria. This indicates a reduction in the consequences of mitochondrial dysfunction. A decrease in the level of oxidative stress markers also indirectly confirms the restoration of the function of the NADH dehydrogenase complex in cells with a mutation in the MT-ND4 gene. Thus, the technical result: reducing the consequences of mitochondrial dysfunction in cells with a mutation in the MT-ND4 gene can be achieved by implementing the present invention.

The invention is illustrated by the following graphic materials: FIG. Figure 1 shows a structural map of the recombinant plasmid expression vector pAAV-ND4opt expressing the optimized ND4opt sequence. DNA with the ND4opt nucleotide sequence was synthesized and inserted into the pAAV-MCS plasmid at restriction sites (BamHI and HindIII) under the control of the CMV promoter.

In FIG. Figure 2 shows peptides of mitochondrial localization of various proteins.

In FIG. Figure 3 shows a structural map of the recombinant plasmid expression vector pAAV-MTS4-ND4opt, expressing a codon-optimized variant of the MT-ND4 gene (ND4opt) with the sequence encoding MTS COX4. DNA with the sequence encoding the COX4 MTS fused to ND4opt was synthesized and inserted into the pAAV-MCS plasmid at restriction sites (BamHI and HindIII) under the control of the CMV promoter.

In FIG. Figure 4 shows a structural map of the recombinant plasmid expression vector pAAV-MTS8-ND4opt, expressing a codon-optimized variant of the MT-ND4 gene (ND4opt) with the sequence encoding the MTS COX8. DNA with the sequence encoding MTS COX8 fused to ND4opt was synthesized and inserted into the pAAV-MCS plasmid at restriction sites (BamHI and HindIII) under the control of the CMV promoter.

In FIG. Figure 5 shows a structural map of the recombinant plasmid expression vector pAAV-MTS8(N)-ND4opt, expressing a codon-optimized variant of the MT-ND4 gene (ND4opt) with the sequence encoding MTS COX8(N). DNA with the sequence encoding MTS COX8(N) fused to ND4opt was synthesized and inserted into the pAAV-MCS plasmid at restriction sites (BamHI and HindIII) under the control of the CMV promoter.

In FIG. Figure 6 shows a structural map of the recombinant plasmid expression vector pAAV-MTS10-ND4opt, expressing a codon-optimized variant of the MT-ND4 gene (ND4opt) with the sequence encoding MTS COX10. DNA with the sequence encoding MTS COX10 fused with ND4opt was synthesized and inserted into the pAAV-MCS plasmid at restriction sites (BamHI and HindIII) under the control of the CMV promoter.

In FIG. Figure 7 shows the results of fluorescence assessment after transfection of HEK293 cells with plasmid expression vectors: pHyper-dMito and a vector expressing the sequence encoding the MTS variant with ND4opt. The ordinate shows the number of fluorescent cells.

pHyper-dMito - transfection with the pHyper-dMito vector;

cox8k-ND4 - transfection with pHyper-dMito and pAAV-MTS8-ND4opt vectors;

cox8n-ND4 - transfection with pHyper-dMito and pAAV-MTS8(N)-ND4opt vectors;

cox10-ND4 - transfection with pHyper-dMito and pAAV-MTS10-ND4opt vectors;

cox4-ND4 - transfection with pHyper-dMito and pAAV-MTS4-ND4opt vectors;

pAAV-MCS - transfection with the original pAAV-MCS vector not expressing ND4opt.

In FIG. Figure 8 shows the results of fluorescence assessment during transfection of fibroblasts of the LHON cell model with a mutation in the ND4 gene with plasmid expression vectors: pHyper-dMito and a vector expressing the sequence encoding the MTS variant with ND4opt. The ordinate shows the number of fluorescent cells.

pHyper-dMito - transfection with the pHyper-dMito vector;

cox8k-ND4 - transfection with pHyper-dMito and pAAV-MTS8-ND4opt vectors;

cox8n-ND4 - transfection with pHyper-dMito and pAAV-MTS8(N)-ND4opt vectors;

cox10-ND4 - transfection with pHyper-dMito and pAAV-MTS10-ND4opt vectors;

cox4-ND4 - transfection with pHyper-dMito and pAAV-MTS4-ND4opt vectors;

pAAV-MCS - transfection with the original pAAV-MCS vector not expressing ND4opt.

In FIG. Figure 9 shows the results of fluorescence assessment during transfection of LHON cell model fibroblasts with a mutation in the ND4 gene with two plasmid expression vectors: pCase12-Mito and a vector expressing the sequence encoding the MTS variant with ND4opt. The y-axis is the number of fluorescent cells.

pCase12-Mito - transfection with the pCase12-Mito vector;

cox8k-ND4 transfection with pCase12-Mito and pAAV-MTS8-ND4opt vectors;

cox8n-ND4 transfection with pCase12-Mito and pAAV-MTS8(N)-ND4opt vectors;

cox10-ND4 - transfection with pCase12-Mito and pAAV-MTS10-ND4opt vectors;

cox4-ND4 - transfection with pCase12-Mito and pAAV-MTS4-ND4opt vectors;

pAAV-MCS - transfection with the original pAAV-MCS vector not expressing ND4opt.

In FIG. Figure 10 presents the results of assessing DCF fluorescence during transfection of LHON cell model fibroblasts with a mutation in the ND4 gene with plasmid expression vectors expressing the sequence encoding the MTS variant with ND4opt. The ordinate shows the fluorescence level in arbitrary units after normalization per 100 thousand cells.

Cells (K) - cells without transfection;

cox8k-ND4 transfection with pAAV-MTS8-ND4opt vector;

cox8n-ND4 -transfection with pAAV-MTS8(N)-ND4opt vector;

DNAJC30-ND4 - transfection with pAAV-MTS-DNAJC30-ND4opt vector;

cox10-ND4 transfection with pAAV-MTS 10-ND4opt vector;

cox4-ND4 -transfection with pAAV-MTS4-ND4opt vector;

pAAV - transfection with the original pAAV-MCS vector not expressing ND4opt.

In FIG. Figure 11 presents the results of assessing DCF fluorescence during transduction of fibroblasts from the LHON cell model with an adeno-associated viral vector expressing the sequence encoding the MTS variant with ND4opt. On the ordinate axis is the fluorescence level in arbitrary units after normalization per 100 thousand cells.

Cells (K) - cells without transduction;

AAV8k transduction with rAAV-MTS8-ND4 vector;

AAV10 - transduction with the rAAV-MTS10-ND4 vector.

Detailed description of the invention

Unless otherwise indicated, all terms, designations and other scientific terms used in this application are intended to have the meanings commonly understood by those skilled in the art to which the present invention relates. 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 specification should not be construed as substantially different in the meaning of the term from that generally understood in the art.

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

The present invention provides a nucleic acid for allotopic expression of the MT-ND4 gene, containing the nucleotide sequence Seq Id No.: 1 and the sequence encoding the mitochondrial localization peptide of cytochrome c oxidase subunit 8 (Cox8 protein).

The nucleotide sequence Seq Id No.: 1 encodes the normal human MT-ND4 protein and is codon optimized based on the wild-type MT-ND4 gene sequence. Based on the wild-type MT-ND4 gene sequence (NC 012920.1, Gene ID: 4538), codon optimization was performed to ensure allotopic expression of the mitochondrial MT-ND4 gene. Therefore, the codon optimized MT-ND4 gene sequence of the present invention, also referred to herein as ND4opt, encodes a normal MT-ND4 protein that is identical to the MT-ND4 protein synthesized in mitochondria. The codon-optimized ND4opt sequence is 73% homologous to the mitochondrial version of the ND4 gene, while the amino acid sequences encoded by both nucleotide sequences are identical.

The Cox8 protein is a subunit 8 of cytochrome c oxidase, its amino acid sequence contains a region responsible for mitochondrial localization - the mitochondrial localization peptide (MTS).

In one embodiment of the present invention, the mitochondrial localization peptide of the Cox8 protein contains the sequence Seq Id No.:6. In yet another embodiment of the invention, the sequence Seq Id No.:5 encodes the mitochondrial localization peptide of the Cox8 protein.

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

We optimized codons taking into account the representation of codons and tRNAs in human cells with a level of relative adaptability of at least 50%. The designed codon optimized sequence is only 73% identical to the human MT-ND4 sequence. Optimization of codons when creating the present invention was carried out taking into account the secondary structure of RNA and the stability of hairpins of the RNA molecule, to exclude stop codons and non-synonymous codons, and during optimization, the structural features of the ND4 protein were taken into account, in particular, an analysis of unstructured regions of the protein was carried out in order to select optimal regions for optimization using IUPred2 [https://iupred2a.elte.hu, accessed 06/16/2023].

As a result of codon optimization, several variants of the nucleotide sequence encoding ND4 were obtained, from which the nucleotide sequence Seq Id No.: 1 with optimal properties predicted in silico was selected. DNA with the nucleotide sequence of the present invention has been successfully synthesized and introduced into a pAAV-based plasmid expression vector and an adeno-associated virus-based viral expression vector. DNA with the nucleotide sequence of the present invention is stably expressed in both adhesion culture cells and suspension culture cells.

The term "codon optimization" refers to an experimental approach to improve the codon composition of a recombinant gene based on various criteria without changing the amino acid sequence. Codon optimization is possible due to the degenerate genetic code, meaning that most amino acids are encoded by more than one codon. Most codon optimization approaches involve avoiding rare codons. Also, the direction of codon optimization can be the identification of elements of mRNA instability, secondary structures of mRNA, sequence repeats, internal ribosome entry sites, promoter sequences, putative splicing sites, etc. examples of technologies for such optimization are written in the article [Gao, W. et al. (2004) UpGene: Application of a web-based DNA codon optimization algorithm. Biotechnol. Prog. 20, 443 448; Raab, D. et al. (2010) The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization. Syst. Synth. Biol. 4, 215-225; Gaspar, P. et al. (2012) EuGene: maximizing synthetic gene design for heterologous expression. Bioinformatics 28, 2683-2684; Fath, S. et al. (2011) Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression. PLoS ONE 6, e17596], the text of which is incorporated herein by reference. The frequency of occurrence of synonymous codons varies among different organisms and even among different cells of the same organism. They are decoded by ribosomes at different rates, since their corresponding tRNAs are also present in different cells in different quantities, 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 for successful expression of mitochondrial genes in the cytosol, codon optimization is used 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].

The term "allotopic expression" describes a method of mitochondrial gene expression in which the mitochondrial gene sequence is codon-optimized according to a universal code to allow transcription to occur in the nucleus using the eukaryotic transcription machinery. In this case, a sequence encoding a heterologous mitochondrial localization (MTS) peptide is usually added to the sequence of the mitochondrial gene of interest so that the protein resulting from allotopic expression is localized in the mitochondria. Nuclear gene sequences encoding mitochondrial proteins also encode MTS sequences.

The nucleic acid of the present invention, as a result of expression in cells with a mutation of the MT-ND4 gene, leads to the localization of the normal MT-ND4 protein in the mitochondria, due to the presence of the mitochondrial localization peptide sequence of the COX8 protein, and replenishes the suppressed function of the autologous NADH dehydrogenase complex. As a result, the consequences of mitochondrial dysfunction in cells with a mutation in the MT-ND4 gene are reduced, which is reflected in a decrease in the level of oxidative stress markers. In this case, the nucleic acid of the present invention can be delivered into cells by any delivery and expression method known from the prior art, including by direct administration, as well as by delivery into the cell using expression vectors known from the prior art. The choice of method for delivering nucleic acid according to the present invention does not affect the achievement of the technical result.

Introduction into cells with a mutation in the MT-ND4 gene of a nucleic acid for allotopic expression of the normal MT-ND4 protein of the present invention reduced the amount of ROS in the cells by 55%. This suggests that the nucleic acid of the present invention is expressed in cells with a mutation in the MT-ND4 gene, leading to the accumulation of normal MT-ND4 protein and reducing the consequences of insufficient functioning of the NADH dehydrogenase complex. This effect is not limited to cells with a specific mutation and can extend to cells with any mutation in the MT-ND4 gene, since it replaces the mutant protein with a functional (healthy) one. The nucleic acid of the present invention can thus be used for gene replacement therapy for LHON.

A nucleic acid having a claimed sequence may be produced by any method known in the art, including, but not limited to, recombinant methods such as cloning nucleic acid sequences from a recombinant library or cell genome, using conventional cloning and PCR technology, and other methods. chemical synthesis.

The nucleic acid 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 leads to the expression of the heterologous MT-ND4 gene (ND4opt), while the level of reactive oxygen species (ROS) in the mitochondria of transfected cells is reduced by at least 2 times.

The term "vector" refers to a nucleic acid molecule used to transfer genetic material into a cell. The term "expression vector" means a vector containing 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 for exemplary purposes and do not limit the scope of the present invention. The term “gene expression” refers to the transformation of hereditary information encoded in the nucleotide sequence of a gene into a functional product - RNA or protein.

In one embodiment, the invention provides an expression vector for expression in eukaryotic cells containing a nucleic acid of the present invention. The nucleic acid of the present invention can be introduced into any vector known from the prior art, including plasmid, viral, including those based on the SV40 virus, adenoviruses, herpes viruses, 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 further used and does not limit the scope of the present invention; methods for obtaining vectors are known from the prior art; vectors containing the nucleic acid of the present invention can be obtained by specialists in the field of genetic engineering.

An embodiment of the invention provides a plasmid expression vector containing elements in accordance with the physical and genetic map shown in FIG. 4. The term “promoter” as used herein specifically refers to the sequence of DNA nucleotides recognized by RNA polymerase at which initiation of transcription of the element to which the promoter is operably linked occurs. A promoter may also be accompanied by an enhancer.

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

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

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- a sequence encoding the sequence of SEQ Id No.:6 of the mitochondrial localization (MTS) peptide of the COX8 protein;

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

In yet another embodiment, the vector contains the sequence SEQ Id No.:5 as the sequence encoding the mitochondrial localization (MTS) peptide of the COX8 protein (SEQ Id No.:6).

Transfection of cells with plasmid expression vectors of the present invention leads to stable expression of ND4opt and a decrease in ROS levels and mitochondrial calcium levels by at least 2 times.

In one embodiment, the invention provides a viral expression vector containing a nucleic acid of the present invention. The vector may be an adeno-associated virus (AAV) vector, an adenoviral vector, a retrovirus, or a lentiviral vector based on human immunodeficiency virus and other viruses. AAV and lentiviruses can provide long-term expression, while adenovirus can provide transient expression.

In one embodiment, the invention provides an adeno-associated virus containing a nucleic acid of the present invention as a viral expression vector. Examples of such AAV-based vectors can be found in the article: Peters, C.W., Maguire, S.A., & Hanlon, K.S. (2021). Delivering AAV to the Central Nervous and Sensory Systems. Trends in Pharmacological Sciences, 42(6), 461-474. AAV vectors are recognized as effective for gene therapy of neural tissue derivatives, including tissue from sensory organs such as the eye. AAV is a small, non-replicating, non-enveloped virus, approximately 20 nm in size. Many different AAV serotypes have been described in humans and primates. The genome of all known AAV serotypes is organized similarly: it is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in size. Inverted terminal repeats (ITRs) delimit unique nucleotide sequences encoding capsid proteins (Cap) and replication proteins (Rep). The Sar gene encodes the VP proteins (VP1, VP2 and VP3), which form the capsid. When a recombinant AAV vector is generated, an expression cassette containing no Rep genes and limited to ITRs is inserted into the AAV capsid. Recombinant AAV is incapable of replication and is currently recognized as one of the safest and most widely used viral expression vectors for in vivo gene transfer. AAV-based vectors can penetrate cells of various tissues, providing stable expression of a heterologous gene. These viruses are non-pathogenic and have low immunogenicity [High KA, Aubourg P. rAAV human trial experience. Methods Mol Biol. 2011;807:429-57]. The choice of AAV serotype is determined by the goals and objectives of the study, the type of cells for transduction of which the virus is being developed and does not limit the scope of the rights of the present invention.

In one embodiment, the invention provides an adeno-associated virus serotype 6 containing a nucleic acid of the present invention as a viral expression vector. All known serotypes of adeno-associated viruses can infect cells of many types of tissues. Tissue specificity is determined by the serotype of the capsid proteins, so vectors based on adeno-associated virus are constructed by specifying the required serotype. The use of a viral expression vector based on AAV serotype 6 containing the nucleic acid of the present invention leads to stable expression of ND4opt in transduced cells, while reducing the level of ROS by ~25%.

In one of the embodiments of the invention, an adeno-associated virus of serotype 6 containing the nucleic acid of the present invention, produced in HEK293 suspension cell cultures, is proposed as a viral expression vector. The introduction of a plasmid expression vector containing the nucleic acid of the present invention, together with the pHelper vector and the pRC2/6 vector, into HEK293 suspension cell culture cells leads to the production of viral expression vectors representing adeno-associated virus serotype 6. It has been shown that when cells are transduced with the resulting AAV vectors containing the nucleic acid of the present invention, the level of ROS in cells is reduced by ~25%.

In one embodiment, the invention provides the use of an expression vector of the present invention for the treatment of Leber hereditary optic neuropathy.

To model LHON in vitro and select treatment, you can use methods that assess the metabolic state of cells and factors that are a consequence of dysfunction of mitochondria, for example, the accumulation of intracellular calcium or reactive oxygen species, and as a method of correcting the disease, use the delivery of a therapeutic gene into the nucleus using well proven in clinical studies of AAV vectors and allotopic expression of the transgene through the use of a mitochondrial localization peptide. However, it is not obvious whether allotopic expression will have a positive effect on restoring the metabolic state of cells and whether this depends on the type of peptide of mitochondrial localization.

Expression vectors of the present invention demonstrate stable and high expression of ND4opt in cells. This suggests that the introduction of any of the claimed expression vectors of the present invention into eye tissues can lead to stable expression of ND4opt with the sequence Seq Id No.:1 and replenishment of the deficiency of MT-ND4 protein in the cells and tissues of the eye, and, consequently, restoration of its function and stabilization of vision in Leber hereditary optic neuropathy. Introduction of the plasmid and viral vectors of the present invention into cells with a natural mutation of the MT-ND4 gene leads to the accumulation of normal MT-ND4 protein and restoration of mitochondrial function, expressed in a decrease in the level of oxidative stress markers.

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

Example 1: Creation of plasmid expression vectors.

Example 1.1. Codon optimization of the nucleotide sequence of the MT-ND4 gene.

Annotation of the coding sequence of the MT-ND4 gene was carried out using the NCBI/CCD service. To align nucleotide sequences, the UniPro UGENE tool was used [Okonechnikov K, Golosova O, Fursov M; UGENE team. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012 Apr 15;28(8): 1166-7. doi:10.1093/bioinformatics/bts091]. Translation of the nucleotide sequence into an amino acid sequence was carried out using the EMBOSS Transeq tool [https://www.ebi.ac.uk/Tools/st/emboss_transeq/, access date 08/08/2023]. Since there are coding differences between mitochondrial and nuclear genes, and the cloned transgene is expressed in the nuclear system, nucleotide sequence optimization was performed to eliminate stop codons and nonsynonymous 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, access date 06.16.2023]. A nucleic acid with an optimized MT-ND4 gene sequence (ND4opt, SEQ Id No.: 1), encoding the MT-ND4 protein sequence (SEQ Id No.: 2), was custom synthesized using a service from TopGenetech (Canada). Additionally, an analysis of unstructured protein regions was carried out in order to select optimal regions for optimization using IUPred2 [https://iupred2a.elte.hu, access date 06/16/2023].

Example 1.2. Preparation of an expression vector for allotopic expression of ND4opt

pAAV-MCS (cat#VPK-410, Cell biolabs) was used as a plasmid expression vector. DNA with an optimized sequence of the MT-ND4 gene (ND4opt) was used as a template for amplification; Q5 polymerase (New England Biolabs Inc., USA) was used for polymerase chain reaction. The reaction mixture for PCR was prepared according to the manufacturer's recommendations.

PCR was carried out with the following parameters:

preliminary DNA melting at 98°C for 1 minute, 25 amplification cycles, including melting for 10 seconds at 98°C, annealing for 15 seconds at 58°C and elongation at 72°C for 30 seconds, final elongation at 72°C for 3 minutes.

Conditions for PCR:

The ND4opt sequence was inserted according to the method recommended by the vector manufacturer, using BamHI and HindIII restriction endonucleases (New England Biolabs Inc., USA) and T4 DNA ligase according to standard protocols.

As a result, a recombinant plasmid expression vector pAAV-ND4opt was obtained (Fig. 1), containing, in accordance with the physical and genetic map presented in Fig. 1, the following items:

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

Example 1.3. Selection and cloning of sequences for targeted transport of MT-ND4 into mitochondria

Mitochondrial localization peptides (MTS or MLS) of cytochrome C oxidase are among the most well studied and frequently used to deliver proteins into mitochondria. MTS in cytochrome oxidases are usually short peptide sequences (25-30 amino acids) located in the N region of the polypeptide. We identified MTS, and also checked the mutational landscape and the efficiency of MTS processing in MitaViewer [http://132.216.58.173/MTSvieweRl/, access date 06/16/2023] and MitoFates [https://mitf.cbrc.pj.aist.go.jp /MitoFates/cgi-bin/top.cgi/, accessed 06/16/2023]. The results of the analysis are summarized in Table 1 and Fig. 2. For COX8, two variants of MTS were developed: with lysine (K) at position 25 of MTS and with the amino acid replacement of lysine (K) with asparagine (N) at position 25 of MTS. Position 25 in the COX8 MTS is terminal and critical for further protein processing.

Plasmid expression vectors pAAV-MTS-ND4opt, expressing ND4opt with one of the variants of the MTS coding sequence, were obtained by inserting the MTS-ND4opt sequence into the sequence of the pAAV plasmid vector for virus assembly. DNA with the MTS-ND4opt sequence was obtained by PCR with overlapping sequences (splicing by overlap extension / splicing by overhang extension (SOE) PCR, the principle of the method is described, for example, in the work: Horton RM, But SN, Pullen JK, et al. Gene splicing by overlap extension. Methods Enzymol. 1993;217:270-9. doi: 10.1016/0076-6879(93)17067-f).

DNA with the MTS sequence (MTS-COX8-OPT, MTS-COX-8-OPT N, MTS-COX-4-OPT, MTS-COX-8-OPT K, MTS-COX-10-OPT, Table 2) was combined with DNA with the ND4opt sequence obtained as described in Example 1.1 using sequence overlap PCR (SOE PCR) using the primers shown in Table 2. The primers contained a short MTS sequence from the 5' to the 3' end and a forward primer sequence .

The forward primers contained the short MTS sequence from the 5' to the 3' end and the forward primer sequence for ND4opt. At the first stage, forward primers MTS-COX-8-OPT_N, MTS-COX-8-OPT_K, MTS-COX-10-OPT, MTS-COX-4-ORT for the corresponding MTS fragment and reverse primer ND4_opt_rev were used; the previously obtained plasmid pAAV-ND4opt. Amplification program: preliminary DNA melting at 98°C for 30 seconds, 15 amplification cycles, including melting for 10 seconds at 98°C, annealing for 30 seconds at 55°C and elongation at 72°C for 30 seconds, final elongation at 72°C for 3 minutes.

At the second stage, forward primers MTS-COX8-BamHI_Fw, MTS-COX-4-OPT-Fw, MTS-COX-10-OPT-Fw for the corresponding MTS fragment and reverse primer ND4_opt_rev were used; the purified PCR product of stage 1 was used as a template. Amplification program: preliminary DNA melting at 98°C for 30 seconds, 30 amplification cycles, including melting for 10 seconds at 98°C, annealing for 30 seconds at 55°C and elongation at 72°C for 30 seconds, final elongation at 72°C for 3 minutes.

The resulting amplification fragments of MTS-COX-4-ND4, MTS-COX-10-ND4, MTS-COX-8K-ND4 and MTS-COX-8N-ND4 were inserted into the pAAV-CMV-MCS vector. The restriction reaction was carried out according to the method recommended by the manufacturer of pAAV-CMV-MCS using restriction endonucleases BamHI and HindIII (New England Biolabs Inc., USA). The ligation reaction of the produced inserts and the vector was carried out according to the manufacturer’s recommended protocol in a volume of 20 μl. For ligation, 10X T4 DNA Ligation Buffer and T4 DNA Ligase (New England Biolabs Inc., USA) were used; the reaction was carried out at 16°C for 16 hours (overnight). The enzyme was inactivated at 65°C for 10 minutes.

Cell transformation was carried out according to a standard protocol [Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular cloning. - M.: Mir, 1984. - 239-241 p.]. To perform transformation, an aliquot of chemically competent Escherichia coli DH5a cells was removed from the freezer (-80°C) and placed on ice to slowly thaw. 5 μl of the cooled ligase mixture was added to the thawed cells and incubated in ice for 30 min. Heat shock was performed for 30 sec at 42°C in a water bath, then the tube with cells was transferred to ice and incubated for 2 min. Then 1 ml of SOC nutrient medium preheated to 37°C was added to the cells and incubated in a thermostat for 1 hour at 37°C and stirring at a speed of 180-200 rpm. A suspension of transformed cells was seeded onto Petri dishes with LB agar and ampicillin, pre-dried in a thermostat at 37°C, 100 μl per dish. Cells were cultured in a thermostat at 37°C for 16-18 hours.

To analyze colonies of transformed cells, PCR screening was performed using vector-specific primers pAAV_For_seq2 and pAAV_Rev_seq2 (Table 2) and a ready-made mixture for PCR ScreenMix (Evrogen, Russia). The reaction mixture was prepared according to the manufacturer's recommendations. Thermolysates of individual bacterial colonies were used as a matrix. To do this, the cells of each of the 8 colonies were selected from the surface of the nutrient medium by pricking the tip of a micropipette and transferred to a test tube with 20 μl of water. Test tubes with a suspension of bacteria were heated at 95°C for 5 minutes. 2 μl of suspension was taken for PCR. PCR was carried out with the following parameters: DNA preliminary melting at 95°C for 3 minutes, 25 amplification cycles, including melting for 20 seconds at 95°C, annealing for 20 seconds at 55°C and elongation at 72°C for 2 minutes, final extension at 72°C for 5 minutes. Clones for which the presence of an insert of the correct size was confirmed by PCR were submitted for sequencing. Clones with the correct sequence were selected based on sequencing results.

As a result of cultivation of selected clones, recombinant plasmid expression vectors were obtained, each of which contained the ND4opt sequence and a nucleotide sequence encoding one of four different MTS variants (Fig. 3-6). The characteristics of the resulting vectors are presented below.

The recombinant plasmid expression vector pAAV-MTS4-ND4opt, expressing the codon-optimized sequence of the MT-ND4 gene (ND4opt, Seq Id No.: 1) with the nucleotide sequence encoding MTS COX4, contains, in accordance with the physical and genetic map presented in FIG. 3, the following items:

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- sequence SEQ Id No.:3 encoding MTS COX4 (SEQ Id No.:4)

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

The recombinant plasmid expression vector pAAV-MTS8-ND4opt, expressing the codon-optimized sequence of the MT-ND4 gene (ND4opt, Seq Id No.:1) with the nucleotide sequence encoding MTS COX8, contains, in accordance with the physical and genetic map presented in FIG. 4, the following items:

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- sequence Seq Id No.:5, encoding MTS COX8 (Seq Id No.:6);

- sequence ND4opt (Seq Id No.: 1);

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

The recombinant plasmid expression vector pAAV-MTS8(N)-ND4opt, expressing the codon-optimized sequence of the MT-ND4 gene (ND4opt, Seq Id No.: 1) with the nucleotide sequence encoding MTS COX8(N), contains in accordance with the physical and genetic map , presented in Fig. 5, the following items:

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- sequence SEQ Id No.: 7 encoding MTS COX8(N) (SEQ Id No.: 8);

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

The recombinant plasmid expression vector pAAV-MTS10-ND4opt, expressing the codon-optimized sequence of the MT-ND4 gene (ND4opt, Seq Id No.: 1) with the nucleotide sequence encoding MTS COX10, contains, in accordance with the physical and genetic map presented in FIG. 6, the following items:

- site of origin of replication ori;

- left inverted terminal repeat ITR;

- cytomegalovirus promoter CMV promoter;

- intron sequence of the human b-globin gene;

- sequence SEQ Id No.:9 encoding MTS COX10 (SEQ Id No.: 10);

- sequence ND4opt Seq Id No.: 1;

- polyadenylation signal sequence poly(A);

- right inverted terminal repeat ITR;

- ampicillin resistance gene promoter AmpR promoter;

- ampicillin antibiotic resistance gene AmpR.

Example 1.4. Assessing the impact of gene expression and coexpression after HEK293 transfection

Since mutations in genes encoding components of the electron transport chain can lead to an increase in the concentration of calcium ions and an increase in the production of ROS in mitochondria, to evaluate the activity obtained in Example 1.3. Expression vectors used 3 sensors that fluoresce in the green region of the spectrum.

1) pCase12-Mito (cat#FP992, Evrogen, Russia) a genetically encoded sensor for measuring calcium concentration in mitochondria;

2) pHyper-dMito - a genetically encoded sensor for measuring ROS inside mitochondria (cat#FP942, Evrogen, Russia);

3) H ₂ DCFDA dye to measure the amount of intracellular ROS.

There are several methods for _detecting ROS, including _H2O2 .

Direct detection methods include the method using the cell-permeable fluorogenic probe H ₂ DFCDA (2',7'-Dichloro-dihydro-fluorescein diacetate). Being cell-permeable, non-fluorescent _H2 DFCDA rapidly diffuses into cells, where it is later deacetylated by cellular esterases and oxidized by ROS to dichlorofluorescein (DFC). DFC is characterized by bright fluorescence in the green region of the spectrum and can be detected by microscopy, titration in microplates and cytometry (ex 485/em 535). The fluorescence intensity is proportional to the level of ROS in the cell [Woolley JF, Stanicka J., Cotter TG Recent advances in reactive oxygen species measurement in biological systems //Trends in biochemical sciences. 2013. T. 38. No. 11. P. 556-565].

Another approach uses genetically encoded biosensors to analyze changes in hydrogen peroxide, one of the main ROS produced in cells. Such biosensors can use, for example, reduction- and oxidation-sensitive green fluorescent protein (roGFP), which can exist in two different oxidation states: reduced dithiol or oxidized disulfide, each of which has different fluorescent properties [Hanson G.T. et al. Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators // Journal of Biological Chemistry. 2004. T. 279. No. 13. C. 13044-13053].

Vsevolod Belousov's group at the Institute of Bioorganic Chemistry named after. Shemyakin and Ovchinnikov in 2006 developed a genetically encoded biosensor based on a fusion protein - HyPer - a highly specific fluorescent biosensor for the detection of H ₂ O ₂ inside living cells. HyPer consists of cyclically permuted yellow fluorescent protein (cpYFP) integrated into the regulatory domain of the prokaryotic H ₂ O ₂ -sensing protein, OxyR. Unlike commonly used dichlorofluorescein (DCF) derivatives, HyPer has high specificity for H ₂ O ₂ , does not produce ROS when exposed to light and is not sensitive to superoxide, oxidized glutathione, nitric oxide and peroxynitrite [Belousov VV et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide //Nature methods. - 2006. T. 3. No. 4. - pp. 281-286].

For experimental work, we used the pHyPer-dMito vector (cat#FP942, Evrogen), designed for the expression of HyPer fused to MTS under the control of the constitutive CMV promoter in mammalian cells.

Damage to mitochondria causes a decrease in ATP production, which leads to the opening of the mitochondrial Ca2+-dependent pore (mPTP - Mitochondrial Permeability Transition pore). The mitochondrial pore (mPTP) is a non-selective channel that plays a significant role in calcium exchange between mitochondria and the environment. The opening of the mitochondrial pore affects the ionic homeostasis of the cell, the rate of formation of reactive oxygen species and the activation of cell death.

At the moment, fluorescent calcium probes have been developed based on the BARTA chelator, the fluorescence of which depends on the concentration of calcium ions in the medium (Fluo, Oregon Green, Fura Red). However, only UV-excited Fura 2 and Indo can be used for quantitative determination.

In 2007, a genetically encoded fluorescent Ca2+ ion sensor Case12 was developed, which has a high dynamic range for direct measurement of changes in intracellular Ca2+ under various physiological and pathological conditions [Souslova E.A. et al. Single fluorescent protein-based Ca2+ sensors with increased dynamic range //BMC biotechnology. 2007. T. 7. No. 1. P. 1-10]. This sensor is sensitive to changes in calcium concentration in the physiological range from hundreds of nanomoles to micromolar concentrations. The binding of Ca2+ is rapid and reversible, which makes it possible to monitor high-frequency vibrations of these ions. In response to an increase in Ca2+ concentration, an increase in fluorescence intensity up to 12 times is observed. Case12 fluorescence is characterized by single excitation/emission maxima with a maximum at 491/516 nm.

For experimental work, we used the pCase12-dMito vector, intended for expression under the control of the constitutive CMV promoter in mammalian cells Case12, fused with a sequence encoding a peptide of mitochondrial localization (cat#FP992, Evrogen, Russia).

In order to evaluate the effect of plasmids expressing ND4opt with a sequence encoding one of the MTS variants (Fig. 3-6) on the amount of ROS, HEK293TN cells (Bio-Cat, Germany) were simultaneously transfected with 2 plasmids:

1) pHyper-dMito sensor

2) one of the variants of plasmids expressing ND4opt with a sequence encoding one of the MTS variants, or the pAAV-MCS plasmid that does not contain a transgene.

To prepare the transfection mixture, DNA was added to the test tube at the rate of 250 ng of DNA per well of a 48-well plate for 80 thousand cells. Transfection reagent polyethylenimine (PEI-MAX, MW 40000, Polysciences, USA) was added to another tube at a DNA:PEI mass ratio of 1:5. OptiMEM medium was added to DNA and PEI to a volume of 15 μl of each reagent for each well. The contents of the two tubes were combined and incubated for 10-15 minutes. 30 μl of the transfection mixture was added to each well of a 48-well plate, and 300 μl of a cell suspension with 80 thousand cells in DMEM medium supplemented with 4.5 g/l glucose and 2% vol. was immediately added. fetal bovine serum (FBS). Incubated at 37°C, 5% CO ₂ . The nutrient medium was replaced after 24 hours with DMEM containing 10% vol. FBS in a volume of 300 µl per well. After completion of the transfection step, the culture plate was transferred to the IncuCyte S3 intravital cell analysis system with the following settings:

- The system has selected the scan type according to schedule (Scan on schedule).

- In the Create or Restore Vessel field, the corresponding type of vessel used and the channels for shooting (Brightfield and Green).

- In the Vessel Location field, the position in the system corresponding to the position of the scanned tablet was selected.

- In the Scan Pattern field, the wells of the plate to be analyzed and the number of microphotographs for each well of the plate (9) were selected.

- In the Vessel Notebook field, the name of the experiment in the system, as well as the cell type, was entered.

- In the Analysis Setup field, the following settings are selected: Basic Analyzer, GFP per cell.

- In the Scan Schedule field, a scanning schedule was generated for the analyzed wells of the plate.

The results obtained demonstrated the negative impact of the transfection process with two plasmids on the cell state and the amount of ROS, while a decrease in the genetic load (transfection with only one pHyper-Mito plasmid) did not lead to high values of the sensor signal.

In this case, the expression of ND4opt with a sequence encoding any of the various MTS variants compensated for the negative effect of transfection with two plasmids, reducing the level of mitochondrial ROS in HEK293 cells by the absolute value of the sensor signal by 2 or more times compared to cells transfected with the pHyper-Mito plasmid and the plasmid pAAV-MCS not expressing the transgene. The highest level of reduction in the amount of ROS was observed in the case of using the pAAV-MTS8-ND4opt plasmid - 57.85% (Fig. 7).

Example 1.5. Evaluation of the effect of ND4opt expression on the amount of reactive oxygen species using the pHyper-dMito biosensor in a model of primary fibroblasts of the LHON cell model.

As a relevant cellular model of LHON, we used primary fibroblasts from the biobank of the Federal State Budgetary Institution “Medical Genetic Research Center named after Academician N.P. Bochkova" carrying the most common mutation m. 11778G>A in the MT-ND4 gene. The presence of the mutation was confirmed by Sanger sequencing. Primary dermal fibroblast cells were cultured in DMEM with high glucose (4.5 g/L) and 10% fetal bovine serum containing L-Gln and nonessential amino acids. The culture medium was changed every 2-3 days. After the cells reached 80-90% confluency, the cells were frozen to form a working bank of cell lines. To do this, cells were trypsinized with a 0.25% trypsin-EDTA solution, washed from excess trypsin in phosphate-buffered saline, centrifuged at 200 g, washed cells were diluted in fetal bovine serum with 10% DMSO, aliquots of cell suspensions were transferred into cryoampoules in the amount of 1 million cells per ampoule, the ampoules were placed in a freezer with a temperature of -80°C. After freezing, cryoampoules with cells were transferred to a cryogenic storage facility with liquid nitrogen for storage. Cells were subsequently thawed as needed using standard protocols.

Cells were simultaneously transfected with two plasmids: pHyper-dMito and one of the plasmids expressing ND4opt with a sequence encoding one of the MTS variants (Fig. 3-6).

For transfection of fibroblasts, a transfection mixture was prepared. To do this, plasmid DNA was added to a centrifuge tube at the rate of 250 ng of DNA per well of a 48-well plate. Transfection reagent GenJect 39 (GenJect 39, Molecta, Russia) was added to another tube based on the DNA: GenJect 39 mass ratio of 1:2. DMEM medium was added to DNA and GenJect 39 to a total volume of 15 μl for each well of a 48-well plate. To obtain a transfection mixture, the contents of two tubes were combined and incubated for 10-15 minutes. To the cell sediment obtained after centrifugation at 200g, at the rate of 80 thousand cells per well, the transfection mixture was added in a volume of 30 μl per well and incubated for 20 minutes at 37°C. DMEM medium with 10% vol. was added to the cell suspension. FBS at 300 µl per well. 350 μl of the resulting cell suspension was transferred into the wells of a 48-well plate and incubated at 37°C, 5% CO ₂ . The nutrient medium was replaced after 24 hours with 300 μl DMEM medium containing 10% vol. FBS. After completing the transfection step, the culture plate was transferred to the IncuCyte S3 system for intravital cell analysis with the settings described previously in Example 1.4.

As a result of transfection of cells with the plasmid expression vector pAAV-MTS8-ND4opt, expressing ND4opt with the sequence encoding the MTS COX8, a significant decrease in the level of ROS is observed: by 50% compared to the control transfected with the plasmid expression vector pAAV-MCS (Fig. 8).

Example 1.6. Evaluation of the effect of ND4opt expression on mitochondrial calcium levels using the pCase12-Mito biosensor in a model of primary fibroblasts of the LHON cell model

Using the genetically encoded sensor pCase12-Mito, it is possible to determine the mitochondrial concentration of calcium ions: a vital mediator of mitochondrial energy metabolism and the initiation of cell death processes.

Fibroblast cells carrying the m.11778G>A mutation in the MT-ND4 gene were obtained as described above in Example 1.5. Cells were simultaneously transfected with two plasmids: pCase12-Mito and one of the plasmids expressing ND4opt with a sequence encoding one of the MTS variants (Fig. 3-6), as described above.

Fluorescence detection of the calcium sensor pCase12-Mito was carried out using the system for intravital analysis of IncuCyte S3 cells (experimental conditions are similar to those described previously in Example 1.4.).

As a result of transfection with the plasmid expression vector pAAV-MTS8-ND4opt, expressing ND4opt with the sequence encoding the MTS COX8, a decrease in the level of Ca ²⁺ cations in mitochondria by ~50% was observed (Fig. 9).

Example 1.7. Evaluation of the effect of ND4opt expression on the amount of reactive oxygen species using a dye in a model of primary fibroblasts of the LHON cell model

As described above, mutations in the MT-ND4 gene contribute to an increase in the amount of ROS in cells, and when the function of this gene is restored, a decrease in the amount of ROS is observed. In this regard, we used a method for assessing the level of ROS to test the functional activity of the ND4opt sequence in combination with the sequence encoding one of the MTS variants when introduced into cells as part of the plasmid expression vector pAAV-MTS-ND4opt. To estimate the amount of ROS, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was used as one of the widely used reagents for measuring the amount of ROS in living cells. H2DCFDA is a non-fluorescent derivative of fluorescein - its reduced acetylated form. The reagent begins to fluoresce only after the cleavage of acetyl groups and oxidation inside the cell under the influence of ROS, turning into 2',7'-dichlorofluorescein (DCF). The higher the level of ROS in cells, the more the fluorescent reaction product accumulates, which can be assessed using plate analyzers in a certain region of the spectrum.

Thus, to determine the functional activity of ND4opt with one of the sequences encoding MTS, we transfected primary fibroblasts with the defective MT-ND4 gene with the resulting expression vectors, after which we measured the level of ROS in the cells.

Fibroblast cell cultures were transfected with plasmid expression vectors described in Example 1.2, in the manner described previously in Example 1.5. Next, the culture plate was cultured at 37°C and 5% CO ₂ for 7 days. Actions with the dye were carried out in the dark. The cell monolayer in the wells of a 48-well plate was washed twice with 250 μl of phosphate-buffered saline (PBS). The remaining liquid was removed and 237.5 μl of PBS was added.

12.5 μl of H ₂ DCFDA dye solution was added to each well to a final concentration of 5 μM. The plate was incubated at 37°C for 2 hours.

After completion of incubation with H ₂ DCFDA dye, the culture plate was transferred to the ClarioStar-Plus system. The following scanning type was selected in the system: measurement at an absorption maximum at λ 483 nm, fluorescence maximum at λ 530 nm. After measurement, the cell monolayer was trypsinized and cells were counted on a Countess II counter according to the manufacturer's protocol.

The results of the experiment (Fig. 10) showed that the expression of the sequence according to the present invention ND4opt is able to compensate for the contribution of transfection to the excessive accumulation of ROS, as well as reduce the level of ROS by 2 or more times during long-term incubation (7 days) compared to fibroblasts transfected with the pAAV plasmid -MCS without ND4opt (equivalent genetic load to simulate stress conditions).

It was shown that transfection of primary fibroblasts of the LHON cell model with plasmid expression vectors expressing ND4opt with one of the MTS coding sequences leads to a decrease in the level of fluorescence in the cells, which indicates a decrease in ROS in the cells compared to untransfected cells and cells transfected with the original plasmid expression vector pAAV-MCS, which does not contain the ND4opt sequence and the nucleotide sequence encoding MTS. Of the studied variants of plasmid expression vectors expressing ND4opt with the nucleotide sequence encoding MTS, the variant pAAV-MTS8-ND4opt contributed most to reducing the level of ROS (Fig. 10). The use of the pAAV-MTS8-ND4opt vector led to a decrease in the level of fluorescence caused by ROS by at least 55% and 40.2%, the pAAV-MTS8(N)-ND4opt vector - by 35% and 13.2%, and the pAAV- MTS10-ND4opt - to a decrease by 21% and an increase by 5.1% and the pAAV-MTS4-ND4opt vector - to a decrease by 23% and an increase by 3.2% relative to cells transfected with the original pAAV-MCS vector and untransfected cells, respectively.

Example 1.8. Assessment of the effect of ND4opt sequence expression on markers of oxidative stress after transduction in viral vectors

To obtain viral AAV vectors, we used plasmid expression vectors pAAV-MTS8-ND4opt, pAAV-MTS10-ND4opt, as well as commercially available helper plasmid (pHelper) and packaging plasmid for AAV6 pAAV-RC6 (cat#VPK-426, Cell Biolabs Inc, USA ).

HEK293 suspension cell line cells were thawed and cultured according to standard operating procedures and techniques. At the time of receipt, the calculated density of the cell culture was 10 ⁶ cells/ml, the volume of the cell culture was 30 ml. Transfection was carried out in 500 ml Erlenmeyer flasks (working volume 175 ml). The seeding dose is 87.5⋅10 ⁶ cells. Transfection with plasmids was carried out 12 hours after seeding. Transfection conditions: cell concentration during transfection 10 ⁶ cells/ml, viability 85-90%, DNA weight 1.5 μg/1 million cell biomass. The DNA:PEI ratio is 1:5, the volume of the transfection mixture is 5% of the cell culture volume. After transfection, cells were incubated at 37°C, 5% CO ₂ , 75% humidity, 100 rpm for 120 h.

To lyse the cells, Tween-20 was added to the flask to a concentration of 0.05% and incubated for 1 hour). Next, endonuclease (Dia-M, Russia) was added to 20 IU/ml and MgCl ₂ to 1-2 mM and 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 the removal 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 an AAVX sorbent (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s recommendations.

The viral titer of the resulting AAV samples was assessed using real-time PCR (qPCR), as well as the physical titer and the presence of impurities (low molecular weight or high molecular weight aggregates) using the dynamic light scattering (DLS) method. In all samples, particles with a size of predominantly 20-25 nm and a minor amount of high-molecular impurities (aggregates) were found.

Fibroblasts obtained as described in Example 1.5 were transduced with viral particles at a dose of 10,000 MOI (the ratio of the number of viral particles to the number of target cells, or multiplicity of infection). Untransduced cells were used as a negative fluorescence control. Fluorescence analysis was carried out under the conditions described above in Example 1.2.

Transduction of primary LHON cell model fibroblasts with AAV6 viral vectors expressing ND4opt with the COX8 MTS coding sequence has been shown to result in a ~25% decrease in fluorescence levels, indicating a reduction in ROS in these cells (Figure 11).

Thus, the invention makes it possible to restore the function of NADH dehydrogenase in cells, which is expressed in a decrease in reactive oxygen species and the concentration of calcium ions in mitochondria, in other words, there is a decrease in the consequences of impaired mitochondrial function in cells with a mutation in the MT-ND4 gene.

--->

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<INSDSeq_length>25</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..25</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q26">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatgtccgtcctga</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>107</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..107</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q30">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatgtccgtcctgacgccgctgctgctgcggggcttga

caggctcggcccggcggctcccagtgccgcgcgccaacctgaaactgatcgtgccaac</INSDSeq_se

quence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>98</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..98</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q32">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatgttggctaccagggtatttagcctagttggcaagc

gagcaatttccacctctgtgtgtgtacgactgaaactgatcgtgccaac</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>107</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..107</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q34">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatgtccgtcctgacgccgctgctgctgcggggcttga

caggctcggcccggcggctcccagtgccgcgcgccaagctgaaactgatcgtgccaac</INSDSeq_se

quence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>116</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..116</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q36">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatggccgcatctccgcacactctctcctcacgcctcc

tgacaggttgcgtaggaggctctgtctggtatcttgaaagaagaactctgaaactgatcgtgccaac</I

NSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>32</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..32</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q38">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatggccgccatgcgctggcg</INSDSeq_sequenc

e>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>42</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..42</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q40">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gttggcacgatcagtttcaggtaggtgcgggcgcccaggccc</INSDS

eq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>26</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..26</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q42">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatgttggctaccag</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q44">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatatggatccatggccgcatc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="22">

<INSDSeq>

<INSDSeq_length>37</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..37</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q46">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atatataagctttcattaggagctaaatcctgtgatg</INSDSeq_se

quence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="23">

<INSDSeq>

<INSDSeq_length>42</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..42</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q50">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gttggcacgatcagtttcaggtaggtgcgggcgcccaggccc</INSDS

eq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="24">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q52">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcttcctcccacagctcctg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="25">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q54">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ttccagggccaggagaggca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (21)

1. Nucleic acid for allotopic expression of the MT-ND4 gene, containing the nucleotide sequence SEQ ID No. 1 and the sequence encoding the mitochondrial localization peptide of cytochrome c oxidase subunit 8 (Cox8 protein). 2. Nucleic acid according to claim 1, in which the peptide of the mitochondrial localization of the Cox8 protein contains the sequence SEQ ID No. 6. 3. The nucleic acid according to claim 1 or 2, wherein the sequence of SEQ ID No. 5 encodes the mitochondrial localization peptide of the Cox8 protein. 4. An expression vector for expression in eukaryotic cells, containing a nucleic acid according to any one of paragraphs. 1-3. 5. The vector according to claim 4, which is a plasmid expression vector containing, in accordance with the physical and genetic map presented in FIG. 4, the following items: - site of origin of replication ori; - left inverted terminal repeat ITR; - cytomegalovirus promoter CMV promoter; - intron sequence of the human b-globin gene; - a sequence encoding the sequence SEQ ID No. 6 mitochondrial localization peptide (MTS) of the COX8 protein; - sequence ND4opt SEQ ID No. 1; - polyadenylation signal sequence poly(A); - right inverted terminal repeat ITR; - ampicillin resistance gene promoter AmpR promoter; - ampicillin antibiotic resistance gene AmpR. 6. The vector according to claim 5, containing the sequence SEQ ID No. 5 as the coding sequence of SEQ ID No. 6. 7. Vector according to any one of paragraphs. 4-6, which is a viral expression vector. 8. The vector according to claim 7, which is an adeno-associated virus. 9. The vector according to claim 8, which is an adeno-associated virus of serotype 6. 10. Vector according to claim 9, produced in suspension cell cultures HEK293. 11. Application of the vector according to any one of paragraphs. 4-10 for the treatment of Leber hereditary optic neuropathy.