APPLICATION OF proBDNF IN GENE THERAPY OF DISEASES AND INJURIES OF CENTRAL NERVOUS SYSTEM

Abstract

FIELD: gene therapy.SUBSTANCE: self-complementary adeno-associated viral vector based on the AAV9 serotype for expressing the precursor molecule of the brain-derived neurotrophic factor proBDNF for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system is described. Also a recombinant AAV virion containing said vector and a pharmaceutical composition containing said vector are described. A method for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system is also described, including the use of the said vector.EFFECT: ensuring physiological, stable and localized expression of the biologically active form of BDNF in the tissues of the central nervous system, with the aim of stimulating neuroregeneration, neuroprotection and reducing inflammation, while avoiding the limitations inherent in the use of the mature form of BDNF (mBDNF), such as low secretion efficiency during gene delivery.15 cl, 11 dwg, 2 tbl, 4 ex

Description

[0001] Technical field

[0002] The invention relates to the field of gene therapy for diseases and injuries of the central nervous system, more specifically to means and methods of therapy based on the expression of neurotrophic factors using recombinant adeno-associated viruses.

[0003] Technology Level

[0004] Neurotrophic factors (NTFs) are a family of secreted proteins that have been found in the nervous system. These growth factors have been shown to be involved in the regulation of neuronal survival, proliferation, differentiation, and apoptosis, myelination and axonal outgrowth, and synaptic plasticity. Most NTFs exert their function through interaction with specific receptors, including TrkA for nerve growth factor (NGF), TrkA and TrkC for neurotrophin-3 (NT-3), TrkB for brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), and RET for glial-derived neurotrophic factor (GDNF), among others [Stephen DS Neurotrophic factors: an overview / / Methods Mol Biol. 2018:1727:1-17]. Given that expression of both factors and receptors has been found in cells other than neurons, the effects of NTFs may extend to other cell types.For example, TrkB expression in astrocytes allows inflammatory processes to be modulated by BDNF [Colombo E. et al. Stimulation of the neurotrophin receptor TrkB on astrocytes drives nitric oxide production and neurodegeneration / / J Exp Med. 2012 Mar 12;209(3):521-535]. In addition, it has been described that BDNF can promote angiogenesis through TrkB activation on endothelial and smooth muscle cells [Kermani P. et al. BDNF: A Newly Described Mediator of Angiogenesis / / Trends Cardiovasc Med. 2007 May;17(4):140-143].

[0005] BDNF is one of the best-studied NTFs. It is synthesized as pre-proBDNF, consisting of a small signal peptide, a prodomain, and a mature domain. In the Golgi apparatus, pre-proBDNF is cleaved, after which the precursor molecule (proBDNF) is directed to secretory granules, namely, dense vesicles of the nucleus [Arévalo JC et al. Mechanisms Controlling the Expression and Secretion of BDNF / / Biomolecules. 2023 May 2;13(5):789]. The subsequent stage of BDNF maturation can occur both intracellularly and extracellularly. Intracellular cleavage of proBDNF occurs using furin and intracellular convertases, extracellularly - using plasmin and matrix metalloproteinases. Despite the maturation pathway, such cleavage is limited, and both the precursor molecule (proBDNF) and the mature molecule (mBDNF) are secreted into the extracellular environment, where they interact with receptors.Interestingly, different BDNF isoforms have different receptor affinities: while proBDNF binds sortilin, the mature molecule (mBDNF) preferentially activates TrkB, and both molecules interact with the sortilin-p75NTR heterodimer [Numakawa T. et al. Brain-Derived Neurotrophic Factor Signaling in the Pathophysiology of Alzheimer's Disease: Beneficial Effects of Flavonoids for Neuroprotection / / Int. J. Mol. Sci. 2021, 22(11), 5719]. This explains the diversity of BDNF functions: neuronal apoptosis and neuronal growth cone development are regulated by the interaction between sortilin-p75NTR and proBDNF, whereas synaptic plasticity, neuronal fiber growth, and other intracellular processes are controlled by mBDNF via TrkB.

[0006] Due to its key role in multiple processes of the nervous system, BDNF is a significant candidate for therapeutic use in various disorders. The list of disorders includes, but is not limited to, neurodegenerative diseases, psychiatric diseases, and some orphan diseases such as Rett syndrome and spinocerebellar ataxia. However, administration of BDNF protein has not shown therapeutic success, which is mainly due to its low ability to penetrate the blood-brain barrier when administered intravenously and the need for frequent administration of the drug due to rapid degradation of the protein [Poduslo J. et al. Permeability at the blood-brain and blood-nerve barriers of the neurotrophic factors: NGF, CNTF, NT-3, BDNF / / Brain Res Mol Brain Res 1996; 36:280-286; Beck M. et al. A Controlled Trial of Recombinant Methionyl Human BDNF in ALS / / Neurology, 1999; 52(7):1427-1433].

[0007] Gene therapy using recombinant viral vectors allows for the continuous production of therapeutic proteins after a single administration. Currently, various solutions aimed at conducting gene therapy using BDNF are known. Thus, in 2022, a phase I clinical trial was initiated to evaluate the safety, tolerability, and preliminary efficacy of BDNF in Alzheimer's disease and mild cognitive impairment (ClinicalTrials.gov ID: NCT05040217). The recombinant AAV2-BDNF vector is used to deliver BDNF to the entorhinal cortex of patients with early-stage Alzheimer's disease. According to the article by Nagahara AH et al. MR-guided delivery of AAV2-BDNF into the entorhinal cortex of non-human primates / / Gene Ther. 2018 Apr; 25(2):104-114, the mature form (mBDNF) without the prodomain is used. Clinical trials are currently ongoing. Other studies, described in the article by Osborne A. et al.Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons / / Hum Gene Ther. 2018 Jul 1;29(7):828-84 (see also patent RU2734678C2 of the same authors, published October 21, 2020), investigated the stimulation of nerve regeneration, including the optic nerve, using an adeno-associated viral vector encoding TrkB and mBDNF, combined via the 2A sequence in a single transgene. To accommodate the BDNF and TrkB coding sequences within the narrow frame of AAV vectors (4.7 kb), the coding region of the BDNF prodomain was removed, and the signal peptide sequence was modified to improve the production, intracellular transport, and secretion of mature BDNF (mBDNF). Despite the progress made, the small capacity of AAV vectors remains an important limiting factor, significantly influencing the choice and combination of regulatory elements.In a bicistronic cassette, it is also necessary to ensure the correct relative expression of the two proteins: an incorrect ratio can lead to excess ligand without sufficient receptor, or vice versa. Furthermore, dual-gene / complex constructs are more difficult to manufacture due to the need to ensure efficient spacer cleavage and the absence of recombinants. Furthermore, constructs designed to deliver mBDNF do not provide flexible tissue-specific regulation, which may reduce treatment efficacy and lead to side effects.

[0008] Despite various attempts to use gene therapy using neurotrophic factors to treat diseases and injuries of the central nervous system, to date, a universal and optimal solution has not been developed, which necessitates further search and development of new approaches.

[0009] Disclosure of invention

[0010] The created technical solution is aimed at developing an effective means and method for restoring damage to the central nervous system accompanied by neurodegeneration, inflammation and impaired regeneration, such as spinal cord injury, amyotrophic lateral sclerosis (ALS) and hemorrhagic stroke (HS).

[0011] The technical challenge is to ensure physiological, stable and localized expression of the biologically active form of BDNF in CNS tissues in order to stimulate neuroregeneration, neuroprotection and reduce inflammation, while avoiding the limitations inherent in the use of the mature form of BDNF (mBDNF), such as low secretion efficiency during gene delivery.

[0012] The solution to the problem is achieved by developing a self-complementary adeno-associated viral vector (scAAV) based on the AAV9 serotype to express the precursor molecule of the brain-derived neurotrophic factor (proBDNF) to stimulate neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system,

[0013] containing an expression cassette comprising: a CMV promoter operably linked to a polynucleotide sequence encoding proBDNF, with a native signal peptide at its N-terminus, a Kozak sequence, a termination and polyadenylation signal sequence (polyA), wherein the expression cassette is flanked by inverted terminal repeats (ITRs) of an adeno-associated vector (AAV), one of which is mutated, wherein the mutated ITR includes a deletion of the D-region.

[0014] In particular embodiments of the invention, proBDNF with a native signal peptide at its N-terminus has the amino acid sequence of SEQ ID NO: 1.

[0015] In some embodiments of the invention, the sequence encoding proBDNF with a native signal peptide at its N-terminus is represented by SEQ ID NO: 2.

[0016] In particular embodiments of the invention, the CMV promoter has the sequence of SEQ ID NO: 3.

[0017] In some particular embodiments of the invention, the termination and polyadenylation signal sequence (polyA) is bGH polyA or SV40 polyA.

[0018] In some embodiments, the termination and polyadenylation (polyA) signal sequence is bGH polyA having the sequence of SEQ ID NO: 4.

[0019] In particular embodiments of the invention, the expression cassette further comprises at least one detectable label selected from a fluorescent label and / or a peptide epitope tag. In some particular embodiments of the invention, the detectable label has the sequence of SEQ ID NO: 5.

[0020] In particular embodiments of the invention, the expression cassette flanked by inverted terminal repeats (ITRs) has a nucleic acid sequence as set forth in SEQ ID NO: 6.

[0021] The solution to the problem is also achieved by developing a recombinant AAV virion for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, containing the specified scAAV vector and the capsid protein of AAV serotype 9.

[0022] Also, the solution to the stated problem is achieved by developing a pharmaceutical composition for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, containing an effective amount of the said scAAV vector or the said recombinant AAV virion and a pharmaceutically acceptable carrier, diluent, excipient and / or buffer.

[0023] In particular embodiments of the invention, stimulation of neuroregeneration and / or neuroprotection is aimed at preventing or reducing the intensity of a disease associated with a malfunction of the nervous system, such as a hereditary neurodegenerative disease, ischemic damage to the central nervous system, myelopathy, age-related changes in the nervous system, traumatic damage to the central nervous system.

[0024] In particular embodiments of the invention, the disease or injury of the central nervous system is a spinal cord injury, a brain injury, a hemorrhagic stroke, an ischemic stroke, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia.

[0025] The solution to the stated problem is also achieved by developing a method for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, including administering to the patient a therapeutically effective amount of the said scAAV vector, the said recombinant AAV virion or the said pharmaceutical composition.

[0026] In some embodiments of the invention, said scAAV vector, recombinant AAV virion or pharmaceutical composition is administered by intrathecal, suboccipital, intracerebroventricular or intravenous route, or a combination of administration routes.

[0027] Also, the solution to the stated problem is carried out by using the specified scAAV vector, recombinant AAV virion or pharmaceutical composition to stimulate neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system.

[0028] As a result of implementing the invention, the following technical results are achieved:

[0029] - a viral vector has been developed that ensures the effective delivery of the genetic sequence encoding proBDNF into target cells, its transcription, translation and subsequent secretion, the introduction of which ensures effective stimulation of neuroregeneration and neuroprotection in diseases and injuries of the central nervous system, while the developed design provides:

[0030] • efficient expression of proBDNF,

[0031] • sustained secretion and maturation of proBDNF to its active form in the CNS tissues,

[0032] • achieving a reproducible therapeutic effect in various CNS lesions,

[0033] • flexible delivery options depending on the location and stage of the pathological process,

[0034] • high degree of correlation between the restoration of morphological and functional parameters (ρ > 0.75 according to the results of Spearman correlation analysis),

[0035] • achieving a therapeutic effect without the need for multiple administrations or external control of the protein dose, due to physiological tissue-specific processing,

[0036] • absence of observed side effects associated with excess proBDNF when locally expressed from an AAV vector;

[0037] - the invention expands the arsenal of means for conducting gene therapy in the treatment of diseases and injuries of the central nervous system using neurotrophic factors.

[0038] Brief description of the drawings

[0039] Fig. 1 - Structure of the AAV9-proBDNF construct.

[0040] Fig. 2 - Schematic diagram of the AAV9-proBDNF expression vector.

[0041] Fig. 3 - A. Comparative design of two plasmids with promoter and expression elements. B. Western blot analysis of BDNF isoforms in HEK293F cell lysate and supernatant.

[0042] Fig.4 - Dynamics of the results of functional tests in the SCI model. A. Assessment of post-traumatic functional impairments (on a scale from 1 to 5); BD. Behavioral tests in the open field; E. Treadmill test; F. Rotator crank test.

[0043] Fig.5 - Comparison of the degree of neuroinflammation in different experimental groups in the SCI model.

[0044] Fig. 6 - Comparison of the depth of unmyelinated axon growth into the injury zone in different experimental groups in the SCI model.

[0045] Fig.7 - Comparison of cavity volume (mm 3 ) in different experimental groups when modeling SCI.

[0046] Fig.8 - Results of Spearman correlation analysis in SCI modeling.

[0047] Fig. 9 - Results of the assessment of neurological status according to the Garcia scale in different experimental groups during the modeling of hemorrhagic stroke.

[0048] Fig.10 - Life expectancy in different experimental groups in the modeling of amyotrophic lateral sclerosis.

[0049] Fig.11 - Life expectancy without symptoms in different experimental groups in the modeling of amyotrophic lateral sclerosis.

[0050] Terms and Definitions

[0051] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0052] In the documents of this invention, the terms "includes," "including," and the like, as well as "contains," "comprising," and the like, are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be construed as "consists only of."

[0053] The term "and / or" means one, more than one, or all of the listed elements.

[0054] It should be noted that the singular forms used in this description and the appended claims include the plural forms unless the context clearly dictates otherwise.

[0055] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0056] The term "optional" or "optional" or "optionally" or "optionally" as used herein means that the subsequently described event or circumstance may, but does not necessarily, occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0057] The term “approximately” (“essentially”), particularly with respect to a specified value, is understood to include a deviation of ±5%.

[0058] By "subject" ("patient") shall be meant a mammal, more preferably a human being.

[0059] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout this application, for example, if a particular nucleotide sequence is described as being located "upstream," "downstream," "3-prime (3')" or "5-prime (5')" relative to another sequence, it is to be understood that this means the position of the sequences in the "sense" or "coding" strand of the DNA molecule, which is standard in the art.

[0060] The "coding sequence" or sequence that "encodes" a selected polypeptide is a nucleic acid molecule whose transcription (in the case of DNA) and translation (in the case of mRNA) leads to the formation of a polypeptide in vivo when under the control of appropriate regulatory sequences. The boundaries of the coding sequence are defined by an initiation codon at the 5' end (N-terminus) and a translation termination codon at the 3' end (C-terminus). A transcription termination sequence may be located closer to the 3' end relative to the coding sequence.

[0061] The term "regulatory elements" ("DNA regulatory sequences") collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these regulatory sequences need always be present, provided that the selected coding sequence is capable of replication, transcription, and translation in the appropriate host cell.

[0062] The term "promoter" is used herein in its usual sense to refer to a DNA nucleotide sequence recognized by RNA polymerase as the starting point for the initiation of transcription of a downstream (3'-direction) coding sequence. Transcriptional promoters may include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced using an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced using an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0063] "Operably linked" refers to an arrangement of elements where the components thus described are capable of performing their usual function. Thus, regulatory sequences operably linked to a coding sequence are capable of directing the expression of the coding sequence. Regulatory sequences are not necessarily adjacent to the coding sequence, as long as their function is to direct its expression. Thus, intervening untranslated but transcribed sequences may be present between the promoter sequence and the coding sequence, for example, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0064] An "expression cassette" is a functional genetic module that includes a coding sequence and regulatory elements.

[0065] The term "expression vector" in this invention refers to an AAV expression vector—a recombinant genetic construct containing an expression cassette sandwiched between the 5' and 3' ITR sequences of AAV, which facilitate packaging of the genetic material within the AAV capsid. The AAV vector includes elements that ensure its existence, stability, or packaging, as well as the transfer and expression of the expression cassette. The AAV vector does not contain viral replication genes and is not capable of autonomous replication. Self-complementary adeno-associated virus (scAAV) refers to a type of AAV vector used in gene therapy that is engineered to contain a double-stranded DNA genome instead of the usual single-stranded form, allowing for immediate formation of double-stranded DNA in the target cell, thereby ensuring faster and more efficient transgene expression and higher gene therapy efficacy.

[0066] The term "recombinant AAV virion" ("AAV virion") refers to a fully formed virus-like particle (virion) of an adeno-associated virus in which one or more genes have been replaced with another genetic material (transgene) for gene therapy (research). Thus, the virion retains the ability to infect cells but delivers the transgene instead of its own. The AAV virion comprises the AAV capsid, which determines its serotype and tropism, and genetic material in the form of an AAV vector, packaged within the capsid. The AAV virion is the physical form of the delivery agent and serves as a vehicle for transferring the genetic material into target cells.

[0067] A "native" polypeptide, such as a brain-derived neurotrophic factor (proBDNF) precursor molecule polypeptide or a native proBDNF signal peptide, refers to a polypeptide that has the same amino acid sequence as the corresponding molecule obtained from nature. Such native sequences can be isolated from nature or can be produced by recombinant or synthetic means. The term "native" sequence also encompasses naturally occurring allelic variants of the polypeptide. In particular embodiments of the invention, a brain-derived neurotrophic factor (proBDNF) precursor molecule with a native signal peptide at its N-terminus has the amino acid sequence of SEQ ID NO: 1.

[0068] The term "homology" refers to the percentage identity of two polynucleotide or two polypeptide fragments. Two DNA sequences or two polypeptide sequences are "substantially homologous" to each other when the sequences have at least about 80%, preferably at least about 85%, more preferably at least about 85-90%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a given molecule length. As used herein, the term "substantially homologous" also refers to a sequence that has complete identity with a specified DNA or polypeptide sequence.

[0069] The term "identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by directly comparing the sequence information for two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Easily available computer programs such as ALIGN and others can be used to facilitate this analysis.

[0070] The term "synonymous substitution" refers to a nucleotide sequence having a nucleotide sequence that may differ from a reference nucleic acid sequence by one or more substitutions that do not lead to a change in the amino acid sequence of the protein encoded by that nucleic acid molecule. Because the genetic code is degenerate, meaning triplets with different nucleotide sequences can encode the same amino acid, synonymous substitutions in the nucleotide sequence do not lead to a change in the amino acid sequence of the protein.

[0071] By "isolated," when referring to a nucleotide sequence, it is meant that the molecule is present substantially free of other biological macromolecules of the same type. Thus, an "isolated nucleic acid molecule that encodes a particular polypeptide" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide in question; however, the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristic of the composition.

[0072] The term "active" or "activity" for the purposes of the present invention refers to forms of a therapeutic protein that retain the biological activity of the corresponding native or naturally occurring polypeptide. The activity may be greater than, equal to, or lower than that observed in the corresponding native or naturally occurring polypeptide.

[0073] The present disclosure provides pharmaceutical compositions comprising: a) an scAAV vector, as described in detail below; and b) a pharmaceutically acceptable excipient (in particular, a carrier, diluent, and / or excipient). By "pharmaceutically acceptable" is meant a substance that is not biologically or otherwise undesirable, for example, a substance that can be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition containing a pharmaceutically acceptable excipient can be used, for example, in cell transfection ex vivo or in the administration of a viral particle or cell directly to a subject. In preferred embodiments, the pharmaceutically acceptable carrier, diluent, and excipient are suitable for use in humans.Pharmaceutically acceptable carriers and diluents include, but are not limited to, liquids such as a buffer (e.g., phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), or saline). Excipients that can be used include, for example, wetting or emulsifying agents. In specific embodiments, the present invention provides a pharmaceutical composition comprising an scAAV vector in a pharmaceutically acceptable carrier. For injection, the carrier will typically be a liquid such as sterile, pyrogen-free phosphate-buffered saline.

[0074] The terms "effective amount" or "therapeutically effective amount" of a composition or agent as used herein refer to a sufficient amount of the composition or agent to provide a desired response, such as preventing, delaying the onset, or reducing the intensity of symptoms in a subject, or to achieve a desired biological outcome, such as correcting a neurological pathology, for example, cellular pathology associated with disease or injury of the central nervous system. The term "therapeutic correction" refers to the degree of correction that leads to the prevention, delay in the onset, or reduction in the intensity of symptoms in a subject. The exact amount required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the condition being treated, the route of administration, etc.The appropriate "effective" amount in each individual case can be determined by a person skilled in the art using routine experimentation.

[0075] When performing treatment and / or prophylaxis, administration can be performed single-time. In some embodiments, more than one administration (e.g., two, three, four or more administrations) can be used to achieve the desired level of gene expression over a period of various intervals, such as daily, weekly, monthly, annually, etc. In some embodiments, administration can be performed using various delivery routes, including their sequential or combined use, such as initial local administration followed by intravenous administration, or other combinations of administration routes suitable for achieving the optimal level of gene expression.

[0076] The term "nervous system" includes both the central nervous system and the peripheral nervous system. The term "nervous system" includes, but is not limited to, nerve cells, glial cells, astrocytes, cells in the cerebrospinal fluid (CSF), cells in the interstitial space, cells in the protective membranes of the spinal cord, epidural cells (i.e., cells outside the dura mater), non-nervous tissue cells adjacent to, in contact with, or innervated by nervous tissue, cells in the epineurium, perineurium, endoneurium, funiculi, fasciculi, etc. The term "central nervous system" (CNS) includes all cells and tissues of the brain and spinal cord of vertebrates. The term "peripheral nervous system" refers to all cells and tissues of the part of the nervous system outside the brain and spinal cord.

[0077] "Treating" a specific disease includes: (1) preventing the disease, i.e., preventing the development of the disease or reducing the intensity of the disease in a subject who may be susceptible or predisposed to the disease but does not yet experience or exhibit symptoms of the disease, (2) suppressing the disease, i.e., stopping the development or reversing the condition of the disease, or (3) alleviating the symptoms of the disease, i.e., reducing the number of symptoms experienced by the subject as well as reversing the cellular pathology associated with the disease.

[0078] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0079] The nucleotide and amino acid sequence numbers cited in this application correspond to the numbers in the Sequence Listing (SEQ ID NO) according to Standard ST.26, which is part of this disclosure. In the event of a discrepancy in the sequence structure between the reference in the text of the description and the corresponding sequence in the Sequence Listing according to Standard ST.26, the data given in the text of the description shall prevail.

[0080] Detailed description of the invention

[0081] The essence of the proposed technical solution lies in the development of a gene therapy agent and a method for its use aimed at stimulating neuroregeneration, enhancing neuroprotection and reducing inflammatory processes in diseases and injuries of the central nervous system.The proposed development can be used to prevent or mitigate diseases associated with dysfunction of the nervous system, such as hereditary neurodegenerative diseases, ischemic injuries of the central nervous system, myelopathies, age-related changes in the nervous system, and traumatic injuries of the central nervous system, particularly in pathological conditions such as spinal cord injury, traumatic brain injury, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, ischemic stroke, and hemorrhagic stroke, which are characterized by severe damage to nerve tissue, impaired trophic support for neurons, and the development of secondary neurodegenerative processes. A key element of the approach is a genetic construct that ensures the expression of the precursor molecule of brain-derived neurotrophic factor (proBDNF).The choice of a precursor molecule for this invention is based on the fact that its mature form (mBDNF), although possessing pronounced neurotropic and neuroprotective properties, exhibits extremely low secretion efficiency when expressed from gene vectors, even after targeted optimization of the delivery constructs (see Example 1). In contrast, expression of a precursor molecule ensures a more physiological process of intracellular processing and release of active forms, making it more promising for gene therapy.

[0082] For the delivery of genetic material into a cell, the present invention uses an AAV serotype 9 (AAV9) vector, which ensures efficient delivery of the construct to the CNS through the blood-brain barrier, efficient transduction of neurons, astrocytes, microglia and oligodendrocytes, as well as long-term and stable expression of proBDNF in CNS tissues.

[0083] The developed expression vector encoding the therapeutic protein proBDNF includes a complete set of regulatory elements necessary for the efficient delivery of the genetic sequence to target cells, its transcription, translation, and subsequent secretion (see Fig. 1). These elements include:

[0084] - a highly active CMV promoter that provides intense transcription in a wide range of cell types, including neurons; despite the lack of intrinsic tissue specificity of the CMV promoter, selectivity of expression in the central nervous system is achieved due to the natural tropism of the adeno-associated virus (AAV9), used as a delivery vehicle to CNS cells; in preferred embodiments of the invention, the CMV promoter has the sequence presented in SEQ ID NO: 3;

[0085] - Kozak consensus sequence located before the ATG start codon and enhancing translation initiation;

[0086] - transcription termination and polyadenylation signal (polyA), preferably bovine Growth Hormone polyA (bGH polyA) or SV40 polyA, which ensures correct transcription termination and transcript stabilization; in the most preferred embodiments of the invention, the termination and polyadenylation signal sequence is bGH polyA and has the sequence presented in SEQ ID NO: 4;

[0087] - additional regulatory elements that promote optimal expression and post-translational processing.

[0088] The expression cassette within the developed expression vector is flanked by inverted terminal repeats (ITRs) of the adeno-associated virus, which are necessary for packaging the genetic construct into viral particles and also ensure the vector's ability to efficiently replicate in target cells during delivery of recombinant AAV virions. The construct is implemented as a self-complementary AAV vector (scAAV), achieved by mutating one of the ITRs, which includes a deletion of the D region. This format significantly accelerates the onset of therapeutic gene expression, since after entering the target cell, the genome quickly forms double-stranded DNA without the need for cellular synthesis of the complementary strand.

[0089] The developed construct includes a native signal peptide (i.e., a signal sequence naturally associated with proBDNF). Using the native signal peptide in an expression cassette incorporated into the genome of a self-complementary AAV vector ensures increased secretion efficiency of the encoded proBDNF protein after delivery to target cells. In the context of scAAV, which forms double-stranded DNA immediately upon entering the cell, the native signal peptide facilitates the correct incorporation of the synthesized polypeptide into the secretory pathway, its optimal recognition by signal recognition particles (SRPs), and efficient translocation to the endoplasmic reticulum. This reduces the likelihood of misfolding and intracellular protein degradation, ensures natural post-translational processing, and the formation of a functionally active form.In particular embodiments of the invention, the precursor molecule of the brain-derived neurotrophic factor (proBDNF) with a native signal peptide at its N-terminus has the amino acid sequence presented in SEQ ID NO: 1, and the sequence encoding proBDNF with a native signal peptide at its N-terminus has the sequence presented in SEQ ID NO: 2. The use of a native signal peptide in the developed construct promotes a stable and predictable level of secretion of the therapeutic protein in vivo, which was successfully confirmed by experiments (see Examples 1-4).

[0090] If desired, fluorescent or epitope tags may be added to the construct for expression monitoring or subsequent biochemical detection. In particular, in some non-limiting embodiments of the invention, the HA tag sequence (YPYDVPDYA), represented in the expression cassette by the nucleotide sequence SEQ ID NO: 5, may be used as a detectable tag.

[0091] Thus, within the framework of the present invention, a self-complementary adeno-associated viral vector (scAAV) based on the AAV9 serotype for expressing a precursor molecule of the brain-derived neurotrophic factor (proBDNF) for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system is developed, comprising an expression cassette that includes a set of regulatory elements that ensure efficient delivery of the genetic sequence encoding proBDNF to target cells, its transcription, translation and subsequent secretion. In some preferred embodiments of the invention, the expression cassette flanked by inverted terminal repeats (ITRs) can be represented by the (isolated) sequence as indicated in SEQ ID NO: 6, or a sequence substantially homologous to SEQ ID NO: 6, as well as the sequence of SEQ ID NO: 6 having at least one synonymous substitution.A diagram of the AAV9-proBDNF expression vector developed within the framework of the present invention, in one of the preferred embodiments thereof, is shown in Fig. 2.

[0092] In other embodiments, the invention relates to a recombinant AAV virion comprising an scAAV vector as described above and an AAV serotype 9 (AAV9) capsid protein. In further embodiments, the invention relates to a pharmaceutical composition comprising an effective amount of an scAAV vector as described above or said recombinant AAV virion and a pharmaceutically acceptable carrier, diluent, excipient and / or buffer.

[0093] In additional embodiments, the invention relates to a method for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, comprising administering to the cells of a subject in need thereof a therapeutically effective amount of an scAAV vector as described above, said recombinant AAV virion, or said pharmaceutical composition. In certain embodiments, the composition is administered to the cells in vitro to transduce the cells, and the transduced cells are administered to the subject. In preferred embodiments, the compositions are administered in vivo.

[0094] According to the invention, various routes of administration of the developed construct comprising the AAV virion are possible: intrathecal, suboccipital, intracerebroventricular (intraventricular), intravenous (but not limited to) – depending on the pathophysiology of the disease and the goals of therapy. Both preventive (before the onset of the disease) and therapeutic (after its development) administration are possible. Combinations of both administration routes and administration times are also possible, depending on the various stages of the disease at which treatment is administered. Depending on the specific disease or nervous system injury, the administration route is selected taking into account the location of the lesion and the treatment regimen.The potential for flexible delivery depending on the location and stage of the pathological process, with the achievement of a reproducible therapeutic effect in various CNS lesions, as well as the absence of observed side effects associated with excess proBDNF, when locally expressed from an AAV vector, were demonstrated in in vivo experiments (see Examples 2-4).

[0095] After administration of scAAV9 encoding proBDNF to a patient, the virus penetrates CNS cells. The expressed protein is secreted and matures into mBDNF under the action of endogenous proteases (specifically, plasmin and MMP-7). The mature form activates the TrkB receptor, promoting neuronal survival, axonal growth, and synaptic regeneration. Uncleaved proBDNF interacts with the p75NTR / sortilin complex, modulating neuritogenesis and preventing excessive, chaotic growth. This promotes balanced regeneration of neurons and glial cells without pathological remodeling.

[0096] As a result of administration of the genetic construct of the invention to a patient, the following effects are observed at the cellular level: in neurons - maintenance of survival and axonal growth; in astrocytes - a decrease in the severity of gliosis; in microglia - weakening of pro-inflammatory activation; in oligodendrocytes - remyelination and restoration of conductivity. The use of the AAV9-proBDNF construct has demonstrated pronounced therapeutic effects in in vivo experiments. Thus, in a spinal cord injury model, restoration of motor function, axonal growth, and a decrease in gliosis were noted; in a model of amyotrophic lateral sclerosis, an increase in the lifespan of animals and a delay in symptoms; in a model of hemorrhagic stroke, restoration of functions even with delayed administration.Functional improvements statistically correlated with morphological indices of tissue repair (axonal growth depth, decreased volume of damaged tissue), with Spearman correlation coefficients exceeding 0.75. This confirms the structural-functional relationship between cellular changes and the restoration of post-traumatic functional impairments following AAV9-proBDNF administration and demonstrates the effectiveness of the developed viral vector in stimulating neuroregeneration and neuroprotection (see Example 2 and Fig. 8 for details). The therapeutic effect of proBDNF in the experiments was achieved without the need for multiple administrations or external control of protein dosage, due to physiological tissue-specific processing.

[0097] Thus, unlike known solutions, the proposed invention utilizes a construct containing a polynucleotide sequence encoding proBDNF with a native signal peptide at its N-terminus. This construct exhibits stable expression and secretion, as well as the ability to mature to an active form at the desired time and in the desired cell. For the first time, proBDNF has been demonstrated to provide a more pronounced and sustained therapeutic effect compared to mBDNF during gene delivery. The flexibility of the approach has been demonstrated: efficacy was achieved with various administration methods, both local and systemic. The proposed solution thus ensures effective and controlled delivery of a neurotrophic signal, creating the basis for restoring neuronal connections, reducing nerve tissue damage, and modulating inflammation in the affected area.

[0098] The possibility of objective manifestation of the technical result when using the invention is confirmed by reliable data given in examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

[0099] It should be understood that the examples given in the application materials are not limiting and are given only to illustrate the present invention.

[0100] Example 1. Construction of expression plasmid and production of viral vectors

[0101] AAV9-proBDNF and AAV9-mBDNF expression vectors were obtained by first-step assembly of an expression cassette comprising the CMV promoter, Kozak sequence, proBDNF gene (or mBDNF gene) with native signal peptide, and bGH polyA termination and polyadenylation signal sequence. A comparative design of the gene cassettes is shown in Fig. 3A. The cassettes were synthesized and cloned using restriction enzymes and ligase between the inverted terminal repeats (ITRs) of adeno-associated virus (one of which was mutated (the D region was deleted) to ensure subsequent packaging into the double-stranded (self-complementary) scAAV genome) into a plasmid vector based on the AAV9 serotype to obtain an expression plasmid. After assembly, the plasmid was verified by sequencing to confirm the correct insertion and orientation of the fragments. The resulting plasmid form of the AAV vector was used for the initial expression assessment.

[0102] A primary assessment of proBDNF and mBDNF gene expression was performed on the HEK293F cell line. For this purpose, transient transfection of HEK293F suspension cell lines with cell viability greater than 95% in serum-free medium was performed. Briefly, dilutions of plasmid DNA and polyethyleneimine (PEI 25K, Polysciences, 23966) in Opti-MEM medium (Thermo Fisher Scientific, 31985054) were incubated together at room temperature and then added to the cells. Transfected cells were cultured in a rotating incubator at 125 rpm, 37°C and 5% CO2 for 5 days. Transfection was assessed by flow cytometry using the eGFP control protein.

[0103] To assess protein secretion, the culture fluid was collected and centrifuged at 900 rpm and 4°C for 10 minutes, then at 13,400 rpm and 4°C for 10 minutes. The culture fluid of untransfected HEK293F cells was used as a negative control. To analyze intracellular protein production, the cells were lysed using a buffer containing 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, inhibitor cocktail, 1% Triton X-100, pH 7.4, at a rate of 1 ml of lysis buffer per 1.2 × 10 7 cells. The cells were then incubated on a vertical rotator (Biosan Multi Bio, RS-24) at 5 rpm and 4°C for 30 minutes. After incubation, cell debris was removed by two consecutive centrifugations at 21,000 g and 4°C for 15 minutes. A lysate of untransfected HEK293F cells was used as a negative control.

[0104] BDNF was detected in the obtained samples using Western blotting. For this, proteins were separated in denaturing 12% or 15% polyacrylamide gels (PAGE) under reducing conditions (samples were loaded with the addition of β-mercaptoethanol). Prestained Precision Plus Protein Dual Color Standards (Bio-Rad, 1610374) were used as a marker. After electrophoresis, the gel was either stained with Coomassie blue R-250, or the proteins were transferred from the separating gel to an NC 45 nitrocellulose membrane (Serva, 71208.01) using a Trans-Blot Turbo apparatus (Bio-Rad, 1704150). The membranes were then placed in a solution of 5% BSA in PBS to block free membrane binding sites. To detect BDNF, rabbit monoclonal antibodies against BDNF (ab108319, Abcam) were used.

[0105] As demonstrated by the conducted studies (see Fig. 3B), the target protein proBDNF was observed in the lysate of cells infected with a plasmid containing the proBDNF sequence, indicating its stable production. Analysis of the supernatant of these cells revealed the presence of both proBDNF and mBDNF, confirming successful secretion. The target protein was also observed in the lysate of cells infected with a plasmid containing the mBDNF sequence, indicating its production; however, no evidence of secretion was detected in the supernatant of these cells. Thus, despite protein production, no secretion was observed.

[0106] The results confirmed the viability of the expression cassette and the correctness of its design. Furthermore, it was demonstrated that the developed AAV9-proBDNF viral vector ensures efficient expression of proBDNF, as well as its sustained secretion and maturation of proBDNF to its active form (mBDNF) in CNS tissues.

[0107] Example 2. Study of the effectiveness of genetic constructs in the treatment of spinal cord injury

[0108] Animals and experimental design: The experiment was performed on male albino SD rats weighing 258.5 ± 19.7 g obtained from the bioresources collection of the Shemyakin and A.V. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. The rats were kept under a 12 / 12-hour light / dark cycle in a room with controlled temperature (20-26°C) with free access to food and water. All manipulations with animals were approved by the Institute of Agronomy and Bioorganic Chemistry of the Russian Academy of Sciences (protocol No. 934 / 23 dated 25.04.23). Animals were randomly divided into three groups: SCI model and PBS injections (n ​​= 4), SCI model and AAV9-mBDNF injections (n ​​= 7), SCI model and AAV9-proBDNF injections (n ​​= 7).

[0109] The spinal cord injury (SCI) model was conducted on day 0 of the experiment. Three days prior, animals were administered either an AAV-containing solution or a blank solution (PBS) intrathecally, according to the selected group. Seven days after the injury model was created, the solution was administered intravenously again. Post-traumatic functional impairments were assessed on days 7, 14, 21, and 28 post-injury, while functional tests were performed on days 7, 14, and 29. MRI was performed for each rat and examined on days 7 and 30 post-SCI.

[0110] To model spinal cord injury, we used cryoapplication scar modeling [Telegin G.B. et al. Surgical modeling of post-traumatic glial scar of the spinal cord in rats / / ACTA NATURAE, 2019, Vol. 11, No. 3 (42): 75-81]. Briefly, rats were anesthetized with 3% isoflurane under aseptic conditions, then, after unilateral laminectomy with a diamond dental bur at the Th13 level, a copper cryoconductor connected to liquid nitrogen was supplied to the spinal cord through the dura mater for one minute, with a temperature in the contact zone of the conductor with the spinal cord of -20 ° C, which made it possible to form a model of post-traumatic glial scar of the spinal cord.

[0111] For AAV injections, AAV-containing injection solutions were prepared in a volume of 75 μl. Solutions administered to experimental groups contained 1.0 x 10¹¹ viral vectors (AAV9-mBDNF or AAV9-proBDNF). Intrathecal injections were performed at the L2 vertebral level, and intravenous injections were administered into the tail vein.

[0112] Statistical analysis: Linear mixed models were used to evaluate the effects of treatment on functional test results and post-injury recovery. All models used test scores or scaled scores as dependent variables, and both treatment groups, time, and their interaction as fixed effects. Within-group variability in rats was used as a random effect. Between-group comparisons for quantitative morphological data were performed using the Wilcoxon test, and for categorical data, Fisher's exact test. Statistical analysis was performed in R version 4.4.1 using RStudio software.

[0113] Results:

[0114] A five-point scale was used to assess post-traumatic functional impairment. The scale evaluates the preservation of hind limb functions in the range from 1 to 5 points, where 1 means recovery of function, manifested in attempts to support the right hind limb, and 5 means paraplegia, manifested in the absence of movement in the hind limbs. According to the results of the functional tests, post-traumatic functional impairment was detected in all animals. Moreover, as the study showed, the recovery of animals from the AAV9-proBDNF administration group was better compared with the recovery of mice from the control group, and in the AAV9-mBDNF administration group, although a tendency towards recovery was observed, the effect was less pronounced (see Fig. 4A; curves are shown with 95% confidence interval (CI)). The estimate of the effect of AAV9-proBDNF on day 28 compared with day 7 was -1.29 (95% CI: -1.63, -0.94).For the AAV9-mBDNF group, this estimate was -0.57 (95% CI: -0.92, -0.23). In the control group, all values ​​were 0 at all time points, so the estimate was close to 0. To assess locomotor activity, the following variables were assessed for each animal: distance traveled, total active time, and vertical activity (each test was performed for a five-minute period). The maximum speed that the animals could reach (on an Exer 3 / 6 treadmill (Columbus Instruments, USA) and the coordination of the animals (using a rotarod test) were also assessed. In the functional tests of locomotor activity, similar results were obtained: animals from the AAV9-proBDNF administration group showed a gradual significant improvement in all functional parameters, including total active time (Fig. 4B), distance traveled (Fig. 4C), vertical activity (Fig. 4D), maximum speed on the treadmill (Fig.4E) and in rotarod tests (Fig. 4F). These data indicate that AAV9-proBDNF administration effectively improves motor behavior and recovery after spinal cord injury, while AAV9-mBDNF administration is ineffective.

[0115] Because neuroinflammation is a key factor in the recovery process after spinal cord injury, with the potential to both promote and hinder recovery, the degree of neuroinflammation was assessed at the cellular level. The assessment was performed on longitudinal histological sections of spinal cord segments at the level of vertebrae from Th11 to Th13. Measurements of the degree of neuroinflammation were included in the morphometric analysis on slides stained with hematoxylin and eosin and toluidine blue. The inflammatory response score on a scale from 0 (no inflammation) to 4 (severe inflammation) in the control group was distributed as follows: one case received a score of 2 (moderate inflammatory infiltration), two cases a score of 3 (high inflammatory infiltration), and one case a score of 4 (high inflammatory infiltration with lymphocyte infiltration), see Fig. 5.In contrast, the AAV9-proBDNF administration group showed a significant reduction in inflammatory activity. Specifically, three cases showed no inflammatory response (score 0), one case showed a score of 1 (low inflammatory infiltration), two cases showed a score of 2 (moderate inflammatory infiltration), and one case showed a score of 3 (high inflammatory infiltration). These results suggest that AAV9-proBDNF administration significantly suppresses neuroinflammation compared to the control group, potentially mitigating secondary damage associated with excessive inflammatory responses in spinal cord injury.

[0116] Replacement of the scar with new axons is a key part of motor function recovery after traumatic spinal cord injury. For its evaluation, we used transverse histological sections of spinal cord segments at the level of the Th11 to Th13 vertebrae, subjected to immunohistochemical staining with anti-MBP antibodies (1:5000, ab218011, Abcam) - antibodies to the myelin basic protein, which is a marker of oligodendrocytes responsible for the myelination of CNS axons; anti-GFAP antibodies (1:1000, ab7260, Abcam) - GFAP (glial fibrillary acidic protein) is a structural protein contained in CNS astrocytes and providing their mechanical support; and anti-Beta-3-tubulin antibodies (1:500, PHG56701, AntibodySystem) - β-3m tubulin is the main component of microtubules, expressed predominantly in neurons and used as a neuronal marker.Tissue samples from three animals in the control group and four animals in the AAV9-proBDNF group were available for evaluation. In the control group, the neurite ingrowth depth values ​​were 30%, 25%, and 40%, while in the AAV9-proBDNF group it was 100% in all cases. The two-tailed Wilcoxon signed-rank test confirmed the difference between groups (p-value = 0.032) (see Fig. 6). Thus, the results of the study showed that the ingrowth depth of neurons stained with antibodies to beta-3-tubulin was greater in mice from the AAV9-proBDNF treatment group. The same significant difference was obtained when comparing the edema volume in animals from different groups, obtained using MRI. Spinal cord images were acquired using a 7.0-T MRI scanner (MRS*DRYMAG 7017, MRSolutions, UK) with a universal coil for rats. Comparison of cavity volume between groups (Fig. 7) indicates significantly less edema in the AAV9-proBDNF group animals.The obtained data indicate that the administration of AAV9-proBDNF promotes accelerated recovery after injury, which is reflected in an increase in the number of sprouting unmyelinated axons and is accompanied by a decrease in edema.

[0117] To assess the degree of relationship between the obtained data on changes at the cellular level and the recovery of post-traumatic motor functional impairments in animals, Spearman's rank correlation analysis was used. The above-mentioned functional test data, MRI results, and histological analysis for all animals from the AAV9-proBDNF group and the control group were included in the analysis. The analysis was performed by assessing the correlation of the parameters, as shown in Fig. 8. A correlation coefficient > 0.75 indicates a strong positive relationship, in which an increase in one parameter is accompanied by an increase in the other. A correlation coefficient < -0.75 indicates a strong negative relationship, in which an increase in one parameter is associated with a decrease in the other. As can be seen in Fig.8, the analysis revealed a statistically significant correlation between motility time and axonal growth depth, functional recovery and cavity volume, axonal growth depth and functional recovery and cavity volume, and functional recovery and glial cell count. This confirms the existence of a structural-functional relationship between cellular changes and the recovery of post-traumatic functional impairments, indicating the effectiveness of the developed viral vector in stimulating neuroregeneration and neuroprotection in CNS injuries.

[0118] Example 3. Study of the effectiveness of genetic constructs in the treatment of hemorrhagic stroke

[0119] To evaluate the efficacy and safety of the developed genetic constructs in diseases or injuries of the central nervous system, an in vivo study was conducted on 3-4-month-old male CD1 mice weighing 30-35 g using a hemorrhagic stroke model. Autologous whole blood injection into the internal capsule of the right cerebral hemisphere was used.

[0120] Experimental animals were divided into 5 groups of 15 animals:

[0121] “1 PBS” - (negative control) 20 μl of PBS were injected into the lateral ventricles of the brain of the animals of this group, 4 weeks later hemorrhagic stroke (HS) was modeled;

[0122] «2 EGFP» - (control - AAV9 without therapeutic construct) animals of this group were administered 2*10 11 units AAV9-EGFP intracerebroventricularly 4 weeks before modeling hemorrhagic stroke;

[0123] "3 proBDNF i.v. before GI" - animals with simulated hemorrhagic stroke, with preliminary intravenous administration of 2*10 11 units AAV9-proBDNF 4 weeks before GI modeling;

[0124] "4 proBDNF i / g before GI" - animals with simulated hemorrhagic stroke, with preliminary intracerebroventricular (intraventricular) administration of 2*10 11 units AAV9-proBDNF 4 weeks before GI modeling;

[0125] “5 proBDNF intravenously on the 1st day” - the animals of this group were initially modeled with hemorrhagic stroke, after which, after 24 hours, they were given intravenous administration of 2*10 11 units AAV9-proBDNF.

[0126] To exclude the influence of artifacts from the AAV delivery procedure on the interpretation of the results and to assess the safety of the constructs, animals underwent a neurological examination using a battery of behavioral tests before modeling hemorrhagic stroke. One week before modeling hemorrhagic stroke (on the 21st day after AAV / PBS injection in groups 1-4), neurological deficits were assessed using the Garcia scale. Six days before (on the 22nd day after AAV / PBS injection in groups 1-4), the characteristics of orientation-exploratory behavior were assessed in a test.

[0127] Twenty-four hours after the HI simulation, as well as on days 3, 7, 14, and 21, the neurological status of the experimental animals was assessed using the Garcia scale. Animals were humanely withdrawn from the experiment on day 28 after the pathology simulation by an overdose of anesthesia.

[0128] The Garcia scale is used to quantify neurological deficits in rodents after stroke. It assesses several parameters: spontaneous activity, symmetry of limb and body movements, response to touching the whiskers on both sides, tail suspension, and coordination of movements during walking and turning. Each parameter is scored, with a maximum total score of 21 points, which represents a normal state. A decrease in the overall score reflects the severity of sensorimotor impairments.

[0129] To analyze the Garcia scale data, intergroup comparisons were performed using the nonparametric Mann-Whitney U test (for non-normal distributions) and analysis of variance (ANOVA) with a Bonferroni post-hoc test (for normal distributions). Differences were considered statistically significant at p<0.05.

[0130] All studied AAV9-proBDNF administration regimens (prophylactic intravenous, prophylactic intraventricular, and therapeutic intravenous on day 1) demonstrated statistically significant superiority over the 1 PBS and 2 EGFP control groups in improving functional outcomes at the critical time points (day 21), demonstrating the potential of AAV9-proBDNF to restore specific sensorimotor deficits after intracerebral hemorrhage (see Fig. 9). Moreover, according to the dynamics of the neurological status assessment according to the Garcia scale, the most pronounced and persistent reparative effect was observed with the delayed intravenous administration of proBDNF on day 1, while prophylactic intravenous administration provided an earlier, and intraventricular administration provided a more moderate, but also reliable improvement in neurological indicators (Fig. 9).Within-group analysis showed a significant improvement in neurological parameters compared to the first day after stroke: by the third day (p<0.05) with prophylactic intravenous administration of AAV9-proBDNF, by the seventh day (p<0.01) with intraventricular administration, and by the 14th day (p<0.01) with therapeutic administration on the first day, with the effect persisting up to the 28th day. In the control groups, no significant recovery was observed until the end of the experiment. Thus, in the group of animals that received the proBDNF construct, a more rapid recovery of neurological deficit, assessed using the Garcia scale, was noted.

[0131] A study of the effectiveness of AAV9-proBDNF genetic constructs in the treatment of hemorrhagic stroke demonstrated their high efficiency in stimulating neuroregeneration and neuroprotection in CNS diseases.

[0132] Example 4. Study of the effectiveness of genetic constructs in the treatment of amyotrophic lateral sclerosis

[0133] The study was conducted on 60 transgenic male mice of the tg_hFUS[1-359] line expressing a truncated form of the FUS protein (1-359) with a high level of aggregation in the cytoplasm. A form of amyotrophic lateral sclerosis associated with a mutation in the FUS gene leading to the loss of the nuclear localization domain was modeled using a transgene cassette integrated into chromosome 11, consisting of the cDNA of the mutant version of the human FUS gene. Animals were divided into 3 groups depending on the administered substance: PBS (negative control), AAV9-EGFP (control - AAV9 without the therapeutic construct) and AAV9-proBDNF intravenously (i.v.). Confirmation of human proBDNF expression was performed using reverse transcription PCR performed on RNA isolated from the brain tissue of three animals from each group.

[0134] Amyotrophic lateral sclerosis (ALS), a degenerative disease of the central nervous system, is characterized by progressive motor neuron damage, accompanied by limb paralysis (paresis) and muscle atrophy, including respiratory muscle atrophy. The effectiveness of ALS treatment was assessed by the lifespan of model animals, as well as by the duration of symptom-free survival. The results of the studies, characterizing the mean lifespan (i.e., the arithmetic mean of the lifespans of animals in a group) and the median lifespan (the exact lifespan at which half the animals lived less than this lifespan, and half lived more than this lifespan) of model animals in different groups, are shown in Fig. 10 and Table 1.

[0135] Table 1. Life expectancy of ALS model animals

[0136] Group Average life expectancy Median life expectancy FUS + PBS 129.11 123.5 FUS + AAV9-EGFP 119.66 123.5 FUS + AAV9-proBDNF (IV) 135.11 127

[0137] Furthermore, the symptom-free lifespan and median symptom-free lifespan of the model animals were evaluated, which are shown in Fig. 11 and Table 2.

[0138] Table 2. Asymptomatic survival time of ALS animal models

[0139] Group Average duration without symptoms Median duration without symptoms FUS + PBS 127.46 120 FUS + AAV9-EGFP 109.4 112 FUS + AAV9-proBDNF (IV) 129.75 119.5

[0140] When assessing survival, it was found that the mean and median survival time of animals after intravenous administration of the AAV9-proBDNF constructs was longer than in the control groups, which were administered PBS or the EGFP construct. The duration of the symptom-free period was longer than in the AAV9-EGFP group, but comparable to that of the control group.

[0141] The study demonstrated that administration of AAV9-proBDNF contributes to an increase in survival in both the pre-symptomatic and symptomatic periods, indicating not only an increase in lifespan but also an improvement in quality of life. The study of the efficacy of AAV9-proBDNF genetic constructs in the treatment of ALS demonstrates their high effectiveness in stimulating neuroregeneration and neuroprotection in CNS diseases.

[0142] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific cases described in detail are given merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention.

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[0236] < / INSDSeq_feature-table>

[0237] <INSDSeq_sequence>tagttattaatagtaatcaattacggggtcattagttcatagccc

[0238] atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacga

[0239] cccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttcca

[0240] ttgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcat

[0241] atgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagt

[0242] acatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccat

[0243] ggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaa

[0244] gtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaa

[0245] tgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatat

[0246] aagcagagctggtttagtgaaccgtcagatc< / INSDSeq_sequence>

[0247] < / insdseq>

[0248] < / sequencedata>

[0249] <sequencedata sequenceidnumber="4">

[0250] <insdseq>

[0251] <INSDSeq_length>208< / INSDSeq_length>

[0252] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0253] <INSDSeq_division>PAT< / INSDSeq_division>

[0254] <INSDSeq_feature-table>

[0255] <insdfeature>

[0256] <INSDFeature_key>source< / INSDFeature_key>

[0257] <INSDFeature_location>1..208< / INSDFeature_location>

[0258] <INSDFeature_quals>

[0259] <insdqualifier>

[0260] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0261] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0262] < / insdqualifier>

[0263] <insdqualifier id="q8">

[0264] <INSDQualifier_name>organism< / INSDQualifier_name>

[0265] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0266] < / insdqualifier>

[0267] < / INSDFeature_quals>

[0268] < / insdfeature>

[0269] < / INSDSeq_feature-table>

[0270] <INSDSeq_sequence>ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtg

[0271] ccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcg

[0272] cattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggatt

[0273] gggaagagaatagcaggcatgctgggga< / INSDSeq_sequence>

[0274] < / insdseq>

[0275] < / sequencedata>

[0276] <sequencedata sequenceidnumber="5">

[0277] <insdseq>

[0278] <INSDSeq_length>30< / INSDSeq_length>

[0279] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0280] <INSDSeq_division>PAT< / INSDSeq_division>

[0281] <INSDSeq_feature-table>

[0282] <insdfeature>

[0283] <INSDFeature_key>source< / INSDFeature_key>

[0284] <INSDFeature_location>1..30< / INSDFeature_location>

[0285] <INSDFeature_quals>

[0286] <insdqualifier>

[0287] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0288] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0289] < / insdqualifier>

[0290] <insdqualifier id="q10">

[0291] <INSDQualifier_name>organism< / INSDQualifier_name>

[0292] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0293] < / insdqualifier>

[0294] < / INSDFeature_quals>

[0295] < / insdfeature>

[0296] < / INSDSeq_feature-table>

[0297] <INSDSeq_sequence>tacccatacgatgttccagattacgcttag< / INSDSeq_sequence>

[0298] < / insdseq>

[0299] < / sequencedata>

[0300] <sequencedata sequenceidnumber="6">

[0301] <insdseq>

[0302] <INSDSeq_length>1929< / INSDSeq_length>

[0303] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0304] <INSDSeq_division>PAT< / INSDSeq_division>

[0305] <INSDSeq_feature-table>

[0306] <insdfeature>

[0307] <INSDFeature_key>source< / INSDFeature_key>

[0308] <INSDFeature_location>1..1929< / INSDFeature_location>

[0309] <INSDFeature_quals>

[0310] <insdqualifier>

[0311] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0312] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0313] < / insdqualifier>

[0314] <insdqualifier id="q12">

[0315] <INSDQualifier_name>organism< / INSDQualifier_name>

[0316] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0317] < / insdqualifier>

[0318] < / INSDFeature_quals>

[0319] < / insdfeature>

[0320] < / INSDSeq_feature-table>

[0321] <INSDSeq_sequence>ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccggg

[0322] cgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggcc

[0323] aactccatcactaggggttccttctagacaactttgtatagaaaagttgtagttattaatagt

[0324] aatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacgg

[0325] taaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatg

[0326] ttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaa

[0327] ctgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatg

[0328] acggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggc

[0329] agtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaat

[0330] gggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatggg

[0331] agtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattg

[0332] acgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaacc

[0333] gtcagatccaagtttgtacaaaaaagcaggctgccaccatgaccatccttttccttactatggtt

[0334] atttcatactttggttgcatgaaggctgcccccatgaaagaagcaaacatccgaggacaaaggtgg

[0335] cttggcctacccaggtgtgcggacccatgggactctggagagcgtgaatgggcccaaggcaggtt

[0336] caagaggcttgacatcattggctgacactttcgaacacgtgatagaagagctgttggatgaggacc

[0337] agaaagttcggccccaatgaaaaaacaataaggacgcagacttgtacacgtccagggtgatgctca

[0338] gtagtcaagtgcctttggagcctcctcttctctttctgctggaggaatacaaaaaattacctagatg

[0339] ctgcaaacatgtccatgagggtccggcgccactctgaccctgcccgccgagggagctgagcgtgtt

[0340] tgacagtattagtgagtgggtaacggcggcagacaaaaaagactgcagtggacatgtcgggcgggac

[0341] ggtcacagtccttgaaaaggtccctgtatcaaaaggccaactgaagcaatacttctacgagaccaa

[0342] gtgcaatcccatgggttacacaaaagaaggctgcaggggcatagacaaaaggcattggaactccca

[0343] gtgccgaactacccagtcgtacgtgcgggcccttaccatggatagcaaaaagagaattggctggcga

[0344] ttcataaggatagacacttcttgtgtatgtacattgaccattaaaaggggaagatacccatacgat

[0345] gttccagattacgcttagacccagctttcttgtacaaagtgggaattcctagagctcgctgatcag

[0346] cctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccc

[0347] tggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagta

[0348] ggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaata

[0349] gcaggcatgctggggagggccgcccactccctctctgcgcgctcgctcgctcactgaggccgggcga

[0350] ccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagaga

[0351] ggga< / INSDSeq_sequence>

[0352] < / insdseq>

[0353] < / sequencedata>

[0354] < / st26sequencelisting>

Claims

1. A self-complementary adeno-associated viral vector (scAAV) based on the AAV9 serotype for the expression of the precursor molecule of the brain-derived neurotrophic factor (proBDNF) to stimulate neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, comprising an expression cassette comprising: a CMV promoter operably linked to a polynucleotide sequence encoding proBDNF with a native signal peptide at its N-terminus, a Kozak sequence, a termination and polyadenylation signal sequence (polyA), wherein the expression cassette is flanked by inverted terminal repeats (ITRs) of an adeno-associated vector (AAV), one of which is mutated, wherein the mutated ITR includes a deletion of the D-region.

2. The scAAV vector of claim 1, wherein the proBDNF with the native signal peptide at its N-terminus has the amino acid sequence of SEQ ID NO:

1.

3. The scAAV vector of claim 1, wherein the sequence encoding proBDNF with the native signal peptide at its N-terminus is represented by SEQ ID NO:

2.

4. The scAAV vector according to any one of the preceding claims, wherein the CMV promoter has the sequence of SEQ ID NO:

3.

5. The scAAV vector of any one of the preceding claims, wherein the termination and polyadenylation (polyA) signal sequence is bGH polyA or SV40 polyA.

6. The scAAV vector of any one of the preceding claims, wherein the termination and polyadenylation (polyA) signal sequence is bGH polyA having the sequence of SEQ ID NO:

4.

7. The scAAV vector according to any one of the preceding claims, wherein the expression cassette further comprises at least one detectable label selected from a fluorescent label and / or a peptide epitope tag.

8. The scAAV vector of claim 7, wherein the detectable label has the sequence SEQ ID NO:

5.

9. The scAAV vector according to any one of the preceding claims, wherein the expression cassette flanked by inverted terminal repeats (ITRs) has a nucleic acid sequence as set forth in SEQ ID NO:

6.

10. A recombinant AAV virion for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, comprising a scAAV vector according to any one of claims 1-9 and a capsid protein of AAV serotype 9.

11. A pharmaceutical composition for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, containing an effective amount of an scAAV vector according to any one of claims 1-9 or a recombinant AAV virion according to claim 10 and a pharmaceutically acceptable carrier, diluent, excipient and / or buffer.

12. The pharmaceutical composition according to claim 11, wherein stimulation of neuroregeneration and / or neuroprotection is aimed at preventing or reducing the intensity of a disease associated with a disorder of the nervous system, such as a hereditary neurodegenerative disease, ischemic injury of the central nervous system, myelopathy, age-related changes in the nervous system, traumatic injury of the central nervous system.

13. The pharmaceutical composition according to any one of claims 11-12, wherein the disease or injury of the central nervous system is a spinal cord injury, a brain injury, a hemorrhagic stroke, an ischemic stroke, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia.

14. A method for stimulating neuroregeneration and / or neuroprotection in diseases or injuries of the central nervous system, comprising administering to a patient a therapeutically effective amount of a scAAV vector according to any one of claims 1-9, a recombinant AAV virion according to claim 10, or a pharmaceutical composition according to any one of claims 11-13.

15. The method according to claim 14, wherein the scAAV vector, recombinant AAV virion or pharmaceutical composition is administered by intrathecal, suboccipital, intracerebroventricular or intravenous routes, or by a combination of administration routes.

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