Optimized polynucleotide for coding brain-derived neurotrophic factor and application of optimized polynucleotide

Abstract

The present invention relates to an optimized polynucleotide encoding a brain-derived neurotrophic factor (BDNF), a transgenic expression cassette comprising the polynucleotide, a recombinant vector, and a medicament useful for the prevention or treatment of neurodegenerative diseases, neurodevelopment-related diseases, stroke, chronic pain, nerve injury, mental disorders, and / or metabolic diseases. The polynucleotide disclosed by the invention can efficiently express the BDNF through codon optimization, and particularly, the expression quantity of the BDNF in nerve cells is remarkably improved.

Description

Technical Field

[0001] The present disclosure belongs to the field of genetic engineering technology. The present disclosure relates to an optimized polynucleotide encoding brain-derived neurotrophic factor (BDNF), a transgenic expression cassette comprising the polynucleotide, a recombinant vector, and a drug for preventing or treating neurodegenerative diseases, neurodevelopment-related diseases, stroke, chronic pain, nerve damage, mental illness, and / or metabolic diseases. Background Art

[0002] Neurological diseases include, but are not limited to, neurodegenerative diseases; neurodevelopmental diseases, such as mental retardation, autism spectrum disorder (ASD), Rett syndrome, attention deficit and hyperactivity disorder (ADHD), etc.; stroke; chronic pain; nerve damage; mental illness and metabolic diseases, etc. Among them, neurodegenerative diseases are degenerative diseases of the nervous system caused by neuronal degeneration and apoptosis. According to its clinical symptoms and pathological anatomy, it can be divided into: 1. Posture and movement abnormalities syndrome, such as Parkinson's disease (PD) and multiple system atrophy; 2. Progressive dementia syndrome, such as Alzheimer's disease (AD) and frontotemporal dementia; 3. Progressive dementia with other neurological abnormalities, such as Huntington's disease (HD) and corticobasal degeneration; 4. Progressive ataxia syndrome, such as spinocerebellar ataxia; 5. Slowly progressive muscle weakness and atrophy syndrome, such as motor neuron disease and amyotrophic lateral sclerosis (ALS); 6. Sensory and movement disorders, such as hereditary motor and sensory neuropathy (CMT); 7. Progressive blindness or extraocular muscle paralysis, such as hereditary optic neuropathy; 8. Syndromes characterized by neurosensory deafness, such as hereditary hearing loss with nervous system atrophy.

[0003] Due to the complexity of the nervous system itself and the physiological barriers that restrict drugs from entering the nervous system from the periphery, the efficacy of traditional drugs for such diseases is very limited. In fact, effective treatments for neurological diseases are rare, and patients with related diseases usually have to endure direct symptoms and complications for a long time, causing a heavy social burden. Therefore, it is urgent to develop new treatment strategies to meet such unmet medical needs.

[0004] Gene therapy is a type of treatment method that packages active DNA into a vector and delivers it to the target tissue to express therapeutic molecules. Its good safety and effectiveness have been demonstrated in many clinical trials, including neurodegenerative diseases. Among the effective gene delivery vectors, adeno-associated virus (AAV) has been the most widely used due to its advantages such as high safety, low immunogenicity, rich serotypes, and convenient modification and screening. For the nervous system, AAV can effectively infect non-dividing cells including neurons and achieve long-term expression of therapeutic genes in neurons. Certain specific types of AAV can also efficiently cross the blood-brain barrier and deliver therapeutic genes to the nervous system through peripheral administration. Therefore, the characteristics of AAV gene therapy that break through the blood-brain barrier and express therapeutic genes for a long time make it a very promising treatment for nervous system diseases.

[0005] For humans, brain-derived neurotrophic factor (BDNF) is an important molecule that ensures the normal function of the brain. During development, BDNF can promote the survival and differentiation of peripheral and central nervous system neurons, and participate in the regulation of axon growth, pathfinding, and dendritic growth and morphology. In adulthood, BDNF is a major regulator of synaptic transmission and plasticity in many areas of the central nervous system [1,2]. In cells, BDNF is first synthesized as a 32 kDa precursor protein, which is then cleaved by different proteases to produce a 14 kDa mature form or a 28 kDa truncated form. The mature form of BDNF binds to and activates TrkB, a member of the Trk receptor tyrosine kinase family. Trk receptors activate three major signal transduction pathways: (1) the Ras-MAPK signaling pathway that promotes neuronal differentiation and neurite outgrowth; (2) the PI3 kinase-Akt signaling pathway that promotes neuronal survival and growth; and (3) the PLCγ1-PKC signaling pathway that promotes synaptic plasticity. Based on the above mechanisms of action, BDNF has great potential in reducing neuronal degeneration and stimulating the function of nervous system cells.

[0006] Previous studies have found that the level of BDNF in the brain or serum of AD patients is significantly reduced [3-5], and gene therapy targeting BDNF has shown therapeutic effects on AD animal models [6-8], including significantly reducing neuronal loss in AD model mice, increasing synaptic density, and improving their cognitive functions of learning and memory. These results indicate that the neuroprotective effect of BDNF may have preventive or therapeutic effects on neurodegenerative diseases, neurodevelopmental diseases, stroke, chronic pain, nerve damage, mental illness, and / or metabolic diseases including AD [9,10]. Therefore, the construction of a gene therapy vector that efficiently expresses BDNF is of great clinical significance for the treatment of the above-mentioned nervous system diseases.

[0007] References

[0008] 1. Kang H, Schuman EM. Long-lasting neurotrophin-induced enhancement of synaptic trans-mission in the adult hippocampus. Science. 1995; 267:1658–62.

[0010] 2. Lu B. BDNF and activity-dependent synaptic modulation. LearnMem. 2003; 10:86–98.

[0011] 3.Hock C, Heese K, Hulette C, Rosenberg C, Otten U. Region-specific neurotrophin imbalances in Alzheimer disease: decreased levels of brain-derived neurotrophic factor and increased levels of nerve growth factor in hippocampus and cortical areas. Arch Neurol. 2000; 57:846–51.

[0013] 4.Ted Kheng Siang Ng,Cyrus Su Hui Ho,Wilson Wai San Tam,Ee Heok Kua,and Roger Chun-Man Ho.Decreased Serum Brain-Derived Neurotrophic Factor(BDNF)Levels in Patientswith Alzheimer's Disease(AD):A Systematic Review and Meta-Int J Mol. Sci.2019:20(2):257.

[0015] 5.B Connor,D Young,Q Yan,RL Faull,B Synek,M Dragunow.Brain-derivedneurotrophic factor is reduced in Alzheimer's disease.Brain Res.Mol.BrainRes.1997;49,71–81.

[0016] 6.Iwasaki Y,Negishi T,Inoue M,Tashiro T,Tabira T,Kimura N.Sendaivirus vector-mediated brain-derived neurotrophic factor expressionameliorates memory deficits and synaptic degeneration in a transgenic mousemodel of Alzheimer's disease.J Neurosci Res.2018–2999:99.

[0017] 7.Nagahara AH,Merrill DA,Coppola G,Tsukada S,Schroeder BE,Shaked GM,Wang L,Blesch A,Kim A,Conner JM,Rockenstein E,Chao MV,Koo EH,Geschwind D,Masliah E,Chiba AA,Tuszynski MH.Neuroprotective effects of Brain-derivedneurotrophic factor in rodent and primate models of Alzheimer's disease.NatMed.2009:15:331-7.

[0018] 8. SS Jiao, LL Shen, C Zhu, XL Bu, YH Liu, CH Liu, XQ Yao, LL Zhang, HD Zhou, DG Walker, J Tan, J. Phys XF Zhou,YJ Wang.Brain-derived neurotrophic factor protects against tau-related neurodegeneration of Alzheimer's disease.Transl Psychiatry.2016:6(10):e907.

[0019] 9.Alan H Nagahara 1,Mark H Tuszynski.Potential therapeutic uses of BDNF in neurological and psychiatric disorders.Nat Rev Drug Discov.2011:10(3):209-1

[0020] 10. Peter Deng, Johnathon D Anderson, Abigail S Yu, Geralyn Annett, Kyle DFink, Jan A Nolta. Engineered BDNF producing cells as a potential treatment for neurologic disease. Expert Opin Biol Ther. 2016; 16(8): 1025-33. Summary of the invention

[0021] In view of the above-mentioned problems and needs in the art, in a first aspect, the present disclosure provides a polynucleotide encoding brain-derived neurotrophic factor (BDNF), whose nucleotide sequence has at least 85% identity with the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, preferably has at least 86%, 90%, 95%, 99% or 100% identity.

[0022] In one embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. In a preferred embodiment, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0023] The polynucleotides of the present disclosure have significantly higher BDNF expression levels compared to the native BDNF sequence (SEQ ID NO: 2).

[0024] In a second aspect, the present disclosure provides a transgenic expression cassette, comprising: a promoter, the polynucleotide according to the first aspect, and PolyA.

[0025] In one embodiment, the promoter is selected from: CMV promoter, EF1A promoter, CAG promoter, CB promoter, SV40 promoter, PGK promoter, UBC promoter, Camk2a promoter, hSyn promoter, Hb9 promoter, Th promoter, Thy1 promoter, NSE promoter, GFAP promoter. In a preferred embodiment, the promoter is CMV promoter. In a preferred embodiment, the promoter is hSyn promoter; preferably, the sequence of hSyn promoter is shown in SEQ ID NO: 9.

[0026] In one embodiment, the transgenic expression cassette further comprises an intron, preferably an SV40 intron, more preferably an artificially synthesized SV40 intron as shown in SEQ ID NO: 5; the intron is located between the promoter and the polynucleotide.

[0027] In one embodiment, the nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO:6 or SEQ ID NO:7.

[0028] In a third aspect, the present disclosure provides a recombinant vector comprising: a backbone vector and the polynucleotide according to the first aspect.

[0029] In one embodiment, the recombinant vector is a viral vector, such as an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a cytomegalovirus vector, a Sendai virus vector, an influenza virus vector, a measles virus vector. In one embodiment, the recombinant vector is a non-viral vector, such as a polymer, a lipid, an inorganic particle.

[0030] In one embodiment, the recombinant vector is an adeno-associated virus vector, such as AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-DJ, AAV-Retro, AAV-rh10 serotypes.

[0031] In a fourth aspect, the present disclosure provides use of the recombinant vector according to the third aspect in the preparation of a medicament for preventing or treating neurodegenerative diseases, neurodevelopmental-related diseases, stroke, chronic pain, nerve damage, mental illness and / or metabolic diseases.

[0032] In one embodiment, neurodegenerative diseases include, but are not limited to, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, motor neuron disease, Huntington's disease, corticobasal degeneration, spinocerebellar ataxia, sensory and motor disorders, progressive blindness, and neurosensory deafness.

[0033] In a fifth aspect, the present disclosure provides a medicine comprising: the recombinant vector according to the third aspect and an optional excipient.

[0034] In one embodiment, the medicament is an injection.

[0035] In a sixth aspect, the present disclosure provides a method for treating a neurodegenerative disease, comprising administering a therapeutically effective amount of the medicament according to the fifth aspect to a subject in need thereof.

[0036] In one embodiment, the drug is administered by a systemic or local route, such as oral, rectal, transmucosal, intranasal, inhalation, buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical contact, intralesional administration.

[0037] In a seventh aspect, the present disclosure provides a method for delivering a brain-derived neurotrophic factor gene into a target cell, comprising: 1) constructing the recombinant vector according to the third aspect; and 2) contacting the target cell with the recombinant vector.

[0038] In one embodiment, the target cell is an ex vivo cell. In one embodiment, the target cell is an in vivo cell.

[0039] In an eighth aspect, the present disclosure provides a host cell infected with the recombinant vector according to the third aspect.

[0040] In one embodiment, the host cell is a neural cell, including but not limited to a neuron, an astrocyte, an oligodendrocyte, a microglia, a Schwann cell.

[0041] In a ninth aspect, the present disclosure provides a method for producing a recombinant AAV vector, comprising introducing the polynucleotide according to the first aspect into a mammalian host cell, wherein the mammalian host cell comprises an AAV rep gene, an AAV cap gene and a viral replication auxiliary gene.

[0042] The inventors obtained a BDNF gene fragment that is efficiently expressed in vivo by codon optimization of the brain-derived neurotrophic factor (BDNF) gene, thereby constructing a transgenic expression cassette, a recombinant vector, and a drug that can be used to prevent or treat neurodegenerative diseases, neurodevelopmental diseases, stroke, chronic pain, nerve damage, mental illness and / or metabolic diseases that efficiently express BDNF.

[0043] The above and other aspects of the present disclosure are set forth in more detail in the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Shown are the results of Western blotting of SY5Y cell lysates.

[0045] Figure 2 Shown are the results of Western blotting of SY5Y supernatants.

[0046] Figure 3 Shown are the results of Western blotting of 293 cell lysates.

[0047] Figure 4 Shown are the results of Western blotting of 293 supernatants.

[0048] Figure 5 Shown are the results of Western blotting of mouse hippocampal protein extracts 15 days after virus injection.

[0049] Figure 6 The plasmid map of CMV-BDNF-OG is shown.

[0050] Figure 7 The plasmid map of CMV-BDNF-N is shown.

[0051] Figure 8 The plasmid map of CMV-BDNF-P is shown.

[0052] Fig. 9 An optimized polynucleotide encoding BDNF 1 (BDNF-N) (SEQ ID NO: 3) is shown.

[0053] Fig.10 The optimized polynucleotide encoding BDNF 2 (BDNF-P) (SEQ ID NO: 4) is shown.

[0054] Fig.11 The sequence of a BDNF-N expression cassette according to one embodiment of the present disclosure is shown (SEQ ID NO: 6).

[0055] Fig.12 The sequence of the BDNF-β expression cassette according to one embodiment of the present disclosure is shown (SEQ ID NO: 7). DETAILED DESCRIPTION

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. The terms used in the description of the present disclosure herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0057] Unless otherwise indicated, nucleic acid or polynucleotide sequences listed herein are in single-stranded form, oriented from 5' to 3', from left to right. Nucleotides and amino acids provided herein use the format recommended by the IUPACIUB Biochemical Nomenclature Commission, using single-letter codes or three-letter codes for amino acids.

[0058] the term

[0059] As used herein, the term "polynucleotide" is a synonym for "nucleic acid" and refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or analogs. A polynucleotide may include modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs.

[0060] As used herein, the terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, meaning "including, but not limited to, ").

[0061] In this article, the term "codon optimization" refers to a polynucleotide sequence modified from its native form. Such modification results in the difference of one or more base pairs, with or without changes in its corresponding amino acid sequence, which may enhance or inhibit gene expression and / or cellular responses to the modified polynucleotide sequence.

[0062] As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism suffering from or susceptible to a condition that can be prevented or treated by administering the disclosed agents, and include humans and non-human animals (e.g., rodents or other mammals).

[0063] In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, wild birds, and game birds such as chickens, turkeys and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

[0064] As used herein, the term "treat" includes: (1) inhibiting a condition, disease or disorder, i.e., preventing, reducing or delaying the development of a disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) alleviating a disease, i.e., causing regression of a condition, disease or disorder or at least one of its clinical or subclinical symptoms.

[0065] Herein, the term "therapeutically effective amount" refers to a dose that produces the therapeutic effect for which it is administered. For example, a therapeutically effective amount of a drug suitable for treating an eye disease may be an amount that can prevent or improve one or more symptoms associated with the eye disease.

[0066] As used herein, the term "improvement" refers to an improvement in a symptom associated with a disease, and may refer to an improvement in at least one parameter that measures or quantifies the symptom.

[0067] As used herein, the term "preventing" a condition, disease or disorder includes preventing, delaying or reducing the incidence and / or likelihood of the occurrence of at least one clinical or subclinical symptom of a condition, disease or disorder developing in a subject who may have or be susceptible to the condition, disease or disorder but has not yet experienced or displayed the clinical or subclinical symptoms of the condition, disease or disorder.

[0068] As used herein, the term "topical administration" or "topical route" refers to administration that has a local effect.

[0069] As used herein, the terms "transduction", "transfection" and "transformation" refer to the process of delivering exogenous nucleic acid into host cells, followed by transcription and translation of the polynucleotide product, including the use of recombinant viruses to introduce exogenous polynucleotides into host cells.

[0070] As used herein, the term "gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification to produce the RNA or protein product of a gene.

[0071] In this article, the term "vector" refers to one or a series of macromolecules that encapsulate polynucleotides and facilitate the delivery of polynucleotides to target cells in vitro or in vivo. The types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vectors.

[0072] As used herein, the terms "expression cassette," "transgene cassette," and "transgene expression cassette" are used interchangeably to refer to a polynucleotide fragment encoding a specific protein, polypeptide, or RNAi element that can be cloned into a plasmid vector or packaged into a viral particle (such as AAV) to deliver the transgene product to target cells.

[0073] As used herein, the term "optional" or "optional" means that the subsequently described event or circumstance may occur but does not have to occur.

[0074] As used herein, the term "artificially recombinant viral genome" or "recombinant genome" refers to an artificially designed or artificially synthesized exogenous DNA sequence that replaces the natural AAV genome between ITRs. AAV containing an artificially recombinant viral genome is called "recombinant AAV". Among them, recombinant AAV can achieve different functions based on the expression of the recombinant genome contained therein.

[0075] As used herein, the term "excipient" refers to a natural or synthetic substance attached to the active ingredient in a drug, such as a solvent, a dispersion medium, a coating, an antibacterial or antifungal agent, an isotonic agent, and an absorption delaying agent. These excipients can help store the viral particles and administer them to a subject. Excipients can include any suitable component, such as, but not limited to, saline. Illustrative examples of saline include, but are not limited to, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, and polysorbate solution.

[0076] Polynucleotide encoding BDNF

[0077] The inventors optimized the nucleic acid sequence encoding BDNF, thereby obtaining a gene fragment that efficiently expresses BDNF. Specifically, the sequence fragments that affect gene expression were optimized, including but not limited to: codon usage preference, elimination of secondary structures that are not conducive to expression (such as hairpin structures), changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0078] In some embodiments, the inventors optimized the nucleotide sequence according to the human codon usage preference and changed the GC content, thereby obtaining the polynucleotide encoding BDNF disclosed in the present invention. The nucleotide sequence of the polynucleotide encoding BDNF disclosed in the present invention has at least 85% identity with the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. The polynucleotide encoding BDNF disclosed in the present invention is a biomacromolecule compound polymerized from multiple nucleotides, which can be a DNA molecule or an RNA molecule, which can be single-stranded or double-stranded, and can be linear or circular.

[0079] In some embodiments, the polynucleotide encoding BDNF is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0080] In some embodiments, the amino acid sequence encoded by the polynucleotide encoding BDNF is shown in SEQ ID NO:1.

[0081] In some embodiments, the amino acid sequence encoded by the polynucleotides encoding BDNF, BDNF-N (SEQ ID NO: 3) and BDNF-P (SEQ ID NO: 4), is as shown in SEQ ID NO: 1.

[0082] Transgene expression cassette

[0083] The transgenic expression cassette disclosed in the present invention comprises: a promoter, the polynucleotide according to the first aspect, and PolyA.

[0084] In some embodiments, the transgene expression cassette further comprises other regulatory elements to facilitate packaging of the transgene into the virus, such as ITRs.

[0085] In some embodiments, the transgenic expression cassette further comprises polynucleotide elements for controlling the expression of the protein product, such as enhancers, replication origins, polyadenylation signals, internal ribosome entry sites (IRES), 2A signals (e.g., P2A, T2A, F2A), poly A signals.

[0086] In some embodiments, the transgenic expression cassette comprises a CMV promoter, an intron as shown in SEQ ID NO: 5, a polynucleotide encoding BDNF, and Poly A. In one embodiment, the transgenic expression cassette consists of a CMV promoter, an intron as shown in SEQ ID NO: 5, a polynucleotide encoding BDNF, and Poly A.

[0087] In one embodiment, the protein product (BDNF) encoded by the transgenic expression cassette is linked to an oligopeptide tag (eg, Flag, 6×His, 2×HA, Myc) that facilitates purification of the protein product. Those skilled in the art understand the techniques and procedures associated with protein purification.

[0088] Recombinant vector

[0089] In one embodiment, the recombinant vector is a viral vector, such as an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a cytomegalovirus vector, a Sendai virus vector, an influenza virus vector, a measles virus vector. In one embodiment, the recombinant vector is a non-viral vector, such as a polymer, a lipid, an inorganic particle.

[0090] In one embodiment, the recombinant vector is an adeno-associated virus (AAV) vector. In one embodiment, a DNA plasmid comprising 5'ITR, a recombinant genome and 3'ITR is used to produce an AAV viral vector, wherein 5'ITR and 3'ITR are located on both sides of the recombinant genome, respectively. AAV viral vectors can be produced by simultaneously introducing a DNA plasmid, a plasmid encoding the AAV cap / rep gene, and a helper plasmid provided by an adenovirus or herpes virus into a suitable host cell using known techniques, such as by transfection. The DNA plasmid can be expressed in a host cell and packaged into viral particles.

[0091] In some embodiments, the AAV capsid protein can be any AAV serotype capsid protein, including natural AAV capsid proteins (e.g., natural 1-11 types AAV, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV capsid proteins) and other artificially modified AAV capsid proteins (e.g., artificially modified 1-11 types AAV, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV capsid proteins). In a preferred embodiment, AAV is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-DJ, AAV-Retro, and AAV-rh10. The genomic sequences, ITR sequences, Rep, and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, such as the GenBank database. For example, the specific sequence of AAV-DJ has been described in WO 2021050970 A1; AAV-rh10 can be found in Choudhury SR et al., Mol Ther., 2016; AAV-PHP.B, AAV-PHP.eB and AAV-PHP.S can be found in Ken Y Chan et al., Nat Neurosci, 2017; AAV-Retro can be found in Tervo et al., Neuron, 2016.

[0092] drug

[0093] The effective amount of the drug may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the drug such as drug tissue distribution, bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc.

[0094] In one embodiment, the dosage form of the drug can be injection, tablet, capsule or powder.

[0095] In one embodiment, the drug may be delivered in a single dose or in multiple doses.

[0096] In one embodiment, more than one administration (eg, two, three, four, or more administrations) may be used to achieve desired levels of gene expression over various intervals of time (eg, daily, weekly, monthly, yearly, etc.).

[0097] In one embodiment, the drug may be presented in unit-dose or multi-dose containers, such as in sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid, such as saline or water for injection, immediately prior to use.

[0098] The present disclosure is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. The cells, reagents, etc. used in the examples can be obtained through commercial channels. The experimental methods in the examples without specifying specific conditions are operated according to conventional conditions known in the art or according to the conditions recommended by the manufacturer.

[0099] Example

[0100] Example 1: Optimization of BDNF encoding nucleic acid and expression in SY5Y neural cells

[0101] In order to improve the in vivo expression efficiency of the BDNF gene, the inventors designed multiple optimized BDNF coding sequences based on the amino acid sequence of the natural BDNF protein (SEQ ID NO: 1) and its natural coding sequence (SEQ ID NO: 2), and conducted experimental screening on the optimized BDNF coding sequences. As a result, two optimized sequences with particularly outstanding expression effects in the neural cell SY5Y were obtained: BDNF-N (SEQ ID NO: 3) and BDNF-P (SEQ ID NO: 4).

[0102] Vector construction:

[0103] In the original pscAAV-BDNF-original vector (referred to as CMV-BDNF-OG, Figure 6 ; sequence as shown in SEQ ID NO: 8), two restriction sites AgeI and HindIII were added to obtain the vector backbone. Then, the optimized DNA fragment was amplified by PCR using the designed primers, and the fragment was also digested with AgeI and HindIII. The digested products were recovered and ligated with T4 DNA Ligase at 22°C for 2 hours. The ligated products were transformed into competent cells to obtain recombinant plasmids, namely CMV-BDNF-N ( Figure 7 )、CMV-BDNF-P( Figure 8 ). Single clones were selected for sequencing and ITR verification, and plasmids with correct sequences and no ITR loss were successfully obtained.

[0104] SY5Y cell transfection:

[0105] Take a 6-well plate and plate SY5Y cells at a cell number of 5E5 cells / well. One day later, transfect 4 μg of plasmids CMV-BDNF-OG, CMV-BDNF-N, CMV-BDNF-P, and CMV-GFP, respectively. After 48 hours of culture, extract the supernatant and cell proteins for Western Blot analysis.

[0106] Western blotting to detect the expression of BDNF protein:

[0107] After 48 hours of cell culture, 200 μL of supernatant was extracted, the cells were washed three times with PBS, and the cells were lysed with 200 μL of lysis buffer. The cell lysate was collected and 5× loading buffer was added. 20 μL of each sample was loaded for SDS-PAGE gel electrophoresis and protein immunoblot analysis, and chemiluminescence imaging was performed after incubation with primary and secondary antibodies.

[0108] Western blot results:

[0109] like Figure 1 and Figure 2 As shown, in the SY5Y neural cells, the BDNF expression level of cells transfected with CMV-BDNF-N and CMV-BDNF-P was significantly higher than that of CMV-BDNF-OG.

[0110] Example 2: Expression of optimized BDNF encoding nucleic acid in 293 cells

[0111] As described in Example 1, vector construction was performed, and HEK293 cells were transfected with the constructed plasmids CMV-BDNF-OG, CMV-BDNF-N, CMV-BDNF-P, and CMV-GFP.

[0112] The results showed that Figure 3 and Figure 4 As shown, in 293 cells, although the expression levels of CMV-BDNF-N and CMV-BDNF-P were slightly higher than those of CMV-BDNF-OG, the increased levels were not obvious.

[0113] Example 3: Expression of AAV virus containing optimized BDNF coding sequence in the hippocampus of C57 mice

[0114] The promoters of CMV-BDNF-OG, CMV-BDNF-N, CMV-BDNF-P, and CMV-GFP were replaced with the neuronal cell-specific promoter hSyn (SEQ ID NO: 9).

[0115] Vector construction: Add two restriction sites, KpnI and HpaI, to the constructed CMV-BDNF-OG, CMV-BDNF-N, CMV-BDNF-P, and CMV-GFP vectors to obtain the vector backbone. Then, the hSyn promoter fragment was amplified by PCR using the designed primers and digested with KpnI and HpaI. The digestion products were recovered and connected with T4 DNALigase at 22°C for 2 hours. The connected products were transformed into competent cells to obtain recombinant plasmids, namely hSyn-BDNF-OG, hSyn-BDNF-N, hSyn-BDNF-P, and hSyn-GFP. Single clones were selected for sequencing and ITR verification, and plasmids with correct sequences and no ITR loss were successfully obtained.

[0116] The capsid protein 9P31 described in "Mathieu Nonnenmacher et al., Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning, Molecular Therapy: Methods & Clinical Development, Issue 20, March 2021" was used to package hSyn-BDNF-OG, hSyn-BDNF-N, hSyn-BDNF-P, and hSyn-GFP expression cassettes into AAV viruses according to the method described in the article. The virus codes are AAV-hSyn-BDNF-OG, AAV-hSyn-BDNF-N, AAV-hSyn-BDNF-P, and AAV-hSyn-GFP, respectively. The virus was injected into 6-8 week old C57 mice by tail vein injection, and the virus injection dose was 5E12 vg / kg. Fifteen days after the injection, the mouse hippocampus tissue was taken to extract protein for protein immunoblotting (Western Blot) analysis.

[0117] The results showed that Figure 7 As shown, in the mouse hippocampus, the level of BDNF expressed by AAV-hSyn-BDNF-N and AAV-hSyn-BDNF-P viruses was significantly higher than that by AAV-hSyn-BDNF-OG.

[0118] The results of Examples 1 to 3 above indicate that the optimized BDNF-N and BDNF-P sequences specifically and significantly increase the expression level of BDNF in nerve cells.

[0119] Although the present disclosure has been illustrated and described with reference to certain preferred embodiments of the present disclosure, it should be understood by those skilled in the art that the above contents are further detailed descriptions of the present disclosure in combination with specific embodiments, and it cannot be determined that the specific implementation of the present disclosure is limited to these descriptions. Those skilled in the art may make various changes in form and details, including making several simple deductions or substitutions, without departing from the spirit and scope of the present disclosure.

Claims

1. A polynucleotide encoding brain-derived neurotrophic factor, wherein: The nucleotide sequence of the polynucleotide has at least 85% identity with the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, preferably at least 86%, 90%, 95%, 99% or 100% identity.

2. The polynucleotide according to claim 1, wherein The polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4. Preferably, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 3 or SEQ ID NO:

4.

3. A transgenic expression cassette comprising: Promoter, the polynucleotide according to claim 1 or 2, and PolyA.

4. The transgenic expression cassette according to claim 3, wherein: The promoter is selected from: CMV promoter, EF1A promoter, CAG promoter, CB promoter, SV40 promoter, PGK promoter, UBC promoter, Camk2a promoter, hSyn promoter, Hb9 promoter, Th promoter, Thy1 promoter, NSE promoter, GFAP promoter; preferably, the promoter is CMV promoter.

5. The transgenic expression cassette according to claim 3 or 4, wherein: The transgenic expression cassette further comprises an intron, preferably an SV40 intron, more preferably an artificially synthesized SV40 intron as shown in SEQ ID NO: 5; the intron is located between the promoter and the polynucleotide.

6. The transgenic expression cassette according to any one of claims 3 to 5, wherein The nucleotide sequence of the transgenic expression cassette is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

7. A recombinant vector comprising: a backbone vector and the polynucleotide according to claim 1 or 2.

8. The recombinant vector according to claim 7, wherein The recombinant vector is a viral vector or a non-viral vector. The viral vector is, for example, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a cytomegalovirus vector, a Sendai virus vector, an influenza virus vector, or a measles virus vector. The non-viral vector is, for example, a polymer, a lipid, or an inorganic particle. Preferably, the recombinant vector is an adeno-associated virus vector.

9. Use of the recombinant vector according to claim 7 or 8 in the preparation of a medicament for preventing or treating neurodegenerative diseases, neurodevelopmental diseases, stroke, chronic pain, nerve damage, mental illness and / or metabolic diseases.

10. The use according to claim 9, wherein: The neurodegenerative diseases include, but are not limited to, Alzheimer's disease, frontotemporal dementia, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, motor neuron disease, Huntington's disease, cortical basal degeneration, spinocerebellar ataxia, sensory and motor disorders, progressive blindness, and neurosensory deafness.

11. A medicine comprising: the recombinant vector according to claim 7 or 8 and an optional excipient.

12. The drug according to claim 11, wherein The drug is administered by a systemic or local route, such as oral, rectal, transmucosal, intranasal, inhalation, buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical contact, and intralesional administration.

13. The drug according to claim 11 or 12, wherein The medicine is an injection.

14. A method for delivering a brain-derived neurotrophic factor gene into a target cell, comprising: 1) constructing the recombinant vector according to claim 7 or 8; as well as 2) contacting the target cell with the recombinant vector.

15. The method according to claim 14, wherein: The target cell is an ex vivo cell.

16. A host cell, wherein The host cell is infected with the recombinant vector according to claim 7 or 8.

17. The host cell according to claim 16, wherein The host cell is a neural cell, including but not limited to neurons, astrocytes, oligodendrocytes, microglia, and Schwann cells.

18. A method for producing a recombinant AAV vector, comprising introducing the polynucleotide of claim 1 or 2 into a mammalian host cell, wherein the mammalian host cell comprises an AAV rep gene, an AAV cap gene, and a viral replication accessory gene.

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