Nucleotide sequence for reducing motor neuron survival gene SMN expression and application thereof

Abstract

The invention provides RNA (Ribonucleic Acid), shRNA (Short Hairpin Ribonucleic Acid) and a DNA (Deoxyribose Nucleic Acid) coding sequence of the shRNA for reducing the expression of a motor neuron survival gene SMN, and a delivery vector or a cell containing the RNA, the shRNA and the DNA coding sequence of the shRNA. Furthermore, the invention successfully constructs a stable transfection cell line with SMN-targeted shRNA transcriptional expression regulated by a tet-on regulation system, expression of the target gene SMN is successfully knocked down through DOX induction, a cell viability decline phenotype appears, an in-vitro cell model of the SMA disease is successfully constructed, and a better platform is provided for high-throughput screening of SMA treatment drugs. Furthermore, the invention provides a method for detecting the biological activity of the SMN protein, the method is short in period, low in cost and simple to operate, and an important means for rapidly evaluating the biological activity is provided for drugs for expressing or promoting expression of the SMN protein, including gene therapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nucleotide sequence that reduces the expression of the motor neuron survival gene SMN and its application. Background Technology

[0002] Spinal muscular atrophy (SMA) is a rare, degenerative disease of motor neurons, characterized by degeneration of motor neurons in the anterior horn of the spinal cord, and sometimes also affecting motor neurons in the brainstem. Clinically, it mainly manifests as progressive, symmetrical muscle weakness and atrophy, with proximal muscles more severely affected than distal muscles, and lower limbs more severely affected than upper limbs. Due to muscle weakness, respiration, movement, and swallowing are all impaired.

[0003] SMA pathogenesis is associated with mutations in the survival motor neuron (SMN) gene on chromosome 5. SMN encodes the full-length SMN (SMN-fl), a protein containing 294 amino acids with a molecular weight of 38 kDa. SMN molecules are expressed in the cytoplasm and nucleus, primarily involved in transcriptional regulation. SMN forms a protein complex with molecules such as Gemins, which binds to the snRNP complex and enters the nucleus, forming the gem body (GB), which regulates intracellular gene transcription. The absence of SMN molecules in cells reduces transcription levels, particularly in motor neurons, leading to decreased motor neuron activity, reduced synaptic connections, and decreased muscle strength. There are two SMN alleles in the human body: SMN1, located near the telomere, and SMN2, located near the centromere. They are highly homologous, with the only difference in the coding region being a synonymous mutation in exon 7; all other differences are located in the non-coding region. 90% of the SMN1 gene expression product is functional SMN protein, while 90% of the SMN2 gene transcripts lack exon 7. Only 10% of the transcripts containing exon 7 express only about 10% of the functional SMN protein. SMA patients have a deleted SMN1 gene, and mutations in the 7th exon of the SMN2 gene lead to reduced SMN protein levels. Based on the number of mutated copies of the 7th exon in the SMN2 gene, patients can be divided into four types. Type I patients generally develop symptoms within 2 weeks to 3 months of age, with severe muscle weakness, making it difficult for the child to sit up, and usually die from respiratory failure around 2 years of age. Type II patients generally develop symptoms between 6 and 18 months of age, can sit up but have difficulty walking independently, and generally die in adulthood. Type III patients gradually lose the ability to walk with age, but have a normal lifespan. Type IV patients generally develop symptoms in adolescence, have mild motor impairment, and their lifespan is not affected.

[0004] The primary pathogenesis of SMA is SMN protein deficiency, caused by a defect in the SMN1 gene and the inability of the normal SMN2 gene to fully compensate. SMN-dependent treatment strategies mainly include SMN1 gene replacement therapy, enhancing SMN2 gene promoter activity, increasing the expression of the full-length SMN2 gene transcript, and stabilizing SMN. Specific treatment regimens generally fall into three categories: gene therapy, antisense oligonucleotides, and small molecule compounds. In addition, there are treatment strategies that do not rely on increasing SMN protein, such as stem cell therapy and neuromuscular protective drugs. The first SMA drug approved by the FDA was the antisense oligonucleotide drug (ISIS-SMNRx) developed by Biogen, which modifies the pre-messenger RNA of the SMN2 gene to produce normal SMN protein, primarily treating type II SMA patients. In May 2019, the FDA approved Novartis' gene therapy drug Zolgensma, which uses a non-replicating adeno-associated virus as a delivery vector for the human SMN functional gene to treat SMA patients, showing some therapeutic efficacy. In evaluating SMN protein activity in drugs, Novartis used an in vivo release method to detect median survival in a mouse SMA disease model. This method is time-consuming, costly, and requires significant human and material resources. Therefore, it is necessary to develop a rapid in vitro evaluation method that is short-cycle, low-cost, and easy to operate during the development of SMA drugs.

[0005] This invention provides a method for rapid in vitro detection of the bioactivity of SMN protein. This method is short in cycle, low in cost and simple to operate, and provides an important means for rapidly evaluating the bioactivity of drugs that express or promote the expression of SMN protein, including gene therapy drugs. Summary of the Invention

[0006] The purpose of this invention is to provide an RNA that reduces the expression of the motor neuron survival gene SMN, the RNA sequence of which is shown in SEQ ID NO.1, and the sequence shown in SEQ ID NO.1 is: CUCUUGGUACAUGAGUGGCUA.

[0007] The present invention also provides a double-stranded RNA formed by hybridization of the RNA sequence described above and its reverse complementary sequence.

[0008] The present invention also provides an RNA that can form a hairpin-like structure by covalently linking the RNA sequence as described above and its reverse complementary sequence with an intermediate non-complementary linker sequence.

[0009] The present invention also provides a double-stranded RNA formed by hybridization of an RNA sequence capable of forming a hairpin-like structure as described above and a sequence that is inversely complementary to it.

[0010] The present invention also provides an shRNA that reduces the expression of the motor neuron survival gene SMN, comprising the RNA sequence as described above, wherein the RNA sequence is shown in SEQ ID NO.1; preferably, the shRNA sequence is shown in SEQ ID NO.2, wherein the sequence shown in SEQ ID NO.2 is:

[0011] CUCUUGGUACAUGAGUGGCUACUCGAGUAGCCACUCAUGUACCAAGAG.

[0012] The present invention also provides a DNA coding sequence for shRNA as described above, the DNA coding sequence being shown in SEQ ID NO.3, and the sequence shown in SEQ ID NO.3 is as follows:

[0013] CTCTTGGTACATGAGTGGCTACTCGAGTAGCCACTCATGTACCAAGAG.

[0014] The present invention also provides a delivery vector comprising a DNA coding sequence of RNA, shRNA or shRNA as described above.

[0015] In some embodiments, the delivery vector is selected from viral vectors and non-viral vectors; preferably, the non-viral vector is selected from the group consisting of liposomes, plasmid vectors, and phage vectors; preferably, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, and hybrid viral vectors; more preferably, the viral vector is selected from lentivirus vectors.

[0016] In some preferred embodiments, the lentiviral vector genome contains a tet-on regulatory element and the DNA coding sequence of the shRNA as described above, wherein the tet-on regulatory system regulates the transcriptional expression of the DNA coding sequence of the shRNA.

[0017] The present invention also provides a method for constructing a stable cell line, the method comprising the following steps: infecting cells with the lentiviral vector as described above, and obtaining a stable cell line after the cells are screened for resistance; preferably, the cells are any one of NSC34, MSCs, He1a, K562, HEK293, A549, MCF-7 or LNCap; preferably NSC34 cells.

[0018] In some embodiments, the lentiviral vector has an infection multiple of 2-10 (MOI); preferably, puromycin is used for resistance screening; more preferably, the working concentration for puromycin resistance screening is 5-10 μg / mL; in some specific embodiments, the infection multiple of 2 (MOI) is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and the working concentration for puromycin resistance screening is 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL.

[0019] In some embodiments, the expression level of the motor neuron survival gene SMN is reduced after DOX induction in the stabilized cell line; preferably, the concentration of DOX is 500-2000 ng / mL; preferably, the DOX induction time is 24 hours to 7 days. In some specific embodiments, the concentration of DOX is 500 ng / mL, 600 ng / mL, 800 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 1800 ng / mL, or 2000 ng / mL; the DOX induction time is 24h, 26h, 28h, 30h, 32h, 34h, 36h, 40h, 48h, 54h, 60h, 3d, 4d, 5d, 6d, or 7d.

[0020] The present invention also provides a stable cell line constructed according to the method described above, wherein the cell is any one of NSC34, MSCs, He1a, K562, HEK293, A549, MCF-7 or LNCap; preferably NSC34 cells.

[0021] The present invention also provides a method for detecting the biological activity of SMN protein, the method comprising the following steps: culturing a stable cell line as described above, inducing it with DOX, then seeding and culturing it, then adding the sample to be tested, and detecting the cell viability.

[0022] In some embodiments, the DOX induction time is 24 hours to 7 days; preferably, the DOX concentration is 500-2000 ng / mL. In some specific embodiments, the DOX induction time is 24h, 26h, 28h, 30h, 32h, 34h, 36h, 40h, 48h, 54h, 60h, 3d, 4d, 5d, 6d, or 7d, and the DOX concentration is 500 ng / mL, 600 ng / mL, 800 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 1800 ng / mL, or 2000 ng / mL.

[0023] In some embodiments, the seeding density of DOX-induced cells is 1000 cells / 100uL to 5000 cells / 100uL, preferably 2000 cells / 100uL. In some specific embodiments, the seeding density of DOX-induced cells is 1000 cells / 100uL, 1500 cells / 100uL, 1800 cells / 100uL, 2000 cells / 100uL, 2500 cells / 100uL, 3000 cells / 100uL, 3500 cells / 100uL, 4000 cells / 100uL, 4500 cells / 100uL, or 5000 cells / 100uL.

[0024] In some embodiments, the sample to be tested is a sample containing SMN protein or a sample that expresses or promotes the expression of SMN protein after entering the cell; preferably, the sample that expresses SMN protein after entering the cell is a recombinant vector carrying the SMN encoding gene; more preferably, the sample that expresses SMN protein after entering the cell is a recombinant AAV-SMN virus carrying the SMN encoding gene.

[0025] In some embodiments, the recombinant AAV-SMN virus infects DOX-induced cells, and the multiplicity of infection (MOI) of the recombinant AAV-SMN virus is 7000-15000, preferably 8000-11500. In some specific embodiments, the MOI of the recombinant AAV virus is 7000, 8000, 9000, 10000, 11000, 11500, 12000, 13000, 14000, or 15000.

[0026] In some embodiments, the recombinant AAV-SMN virus infects cells for 4-24 hours, preferably 4-8 hours. In some specific embodiments, the recombinant AAV virus infects cells for 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 22, or 24 hours.

[0027] In some embodiments, the method for detecting cell viability is the CCK-8 assay. Preferably, the specific steps are as follows: take cells after adding the sample to be tested, add CCK-8 reagent, incubate in an incubator, set the wavelength of the microplate reader to 450 nm, and detect the absorbance value of the cells.

[0028] In some embodiments, the method includes control groups, wherein the control groups are (1) a control group induced by DOX but without the test sample and (2) a control group not induced by DOX;

[0029] Preferably, the formula for calculating the bioactivity of the test sample is: (OD value of the experimental group induced by DOX and with the test sample added - OD value of the control group induced by DOX but without the test sample added) / (OD value of the control group without DOX induction - OD value of the control group induced by DOX but without the test sample added) × 100%.

[0030] The present invention also provides the application of the RNA, shRNA, DNA coding sequence of shRNA as described above, the delivery vector as described above, and the cell line as described above in the preparation of a spinal muscular atrophy cell model.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. This invention successfully constructed a stable cell line that regulates the transcriptional expression of shRNA targeting SMN by tet-on. After induction with doxycycline (DOX), the expression of the target gene SMN was successfully knocked down, and a phenotype of decreased cell viability was observed. This invention successfully constructed an in vitro cell model of SMA disease, providing a better platform for high-throughput screening of drugs for the treatment of SMA.

[0033] 2. This invention provides a method for detecting the bioactivity of SMN protein. This method is short in cycle, low in cost, simple to operate, and has high specificity, good reproducibility, precision and robustness. It provides an important means for rapidly evaluating the bioactivity of drugs that express or promote the expression of SMN protein, including gene therapy drugs.

[0034] Of course, it should be noted that the method in the embodiments of the present invention does not necessarily have to achieve all of the above advantages at the same time. Attached Figure Description

[0035] Figure 1 .pLKO-tet-on vector map.

[0036] Figure 2 Fluorescent image 24 hours after lentivirus packaging plasmid transfection.

[0037] Figure 3 .Puro resistance screening concentration results.

[0038] Figure 4 4A shows the identification results of SMN mRNA knockdown in the NSC 34-tet-on-shRNA1-SMN stable cell line after DOX induction; 4B shows the identification results of SMN mRNA knockdown in the NSC 34-tet-on-shRNA2-SMN stable cell line after DOX induction; and 4C shows the identification results of SMN mRNA knockdown in the NSC 34-tet-on-shRNA1-Con stable cell line after DOX induction.

[0039] Figure 5 The decrease in cell viability of NSC34-tet-on-shRNA1-SMN stable cell lines after DOX induction at different cell seeding densities.

[0040] Figure 6 The decrease in cell viability of NSC34-tet-on-shRNA2-SMN stable cell lines after DOX induction at different cell seeding densities. Detailed Implementation

[0041] To facilitate understanding of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0042] The "shRNA" mentioned in this article, or short hairpin RNA or small hairpin RNA, is an artificial RNA molecule with a hairpin structure. shRNA consists of two short inverted repeat sequences. It is currently known that shRNA mainly participates in RNA interference (RNAi), specifically regulating gene expression. After entering the cell, shRNA is unwound by RNA helicase in the host cell into a sense RNA chain and an antisense RNA chain. The antisense RNA chain binds to some enzymes in the body to form a silencing complex RISC, which recognizes and binds to mRNA containing its complementary sequence. At this point, the RISC functions as a nuclease, capable of cleaving and degrading the mRNA, thereby inhibiting the expression of the corresponding gene.

[0043] The Tet-on system described in this paper is a system for quantitatively and specifically controlling the expression of exogenous genes in eukaryotic cells using prokaryotic regulatory elements. It is based on the tetracycline operon principle in the *E. coli* Tn10 transposon. By inducing drugs (such as tetracycline and doxycycline) to alter the conformation of the regulatory protein, the expression of the target protein is regulated. The Tet-on system consists of a regulatory expression vector and a reactive expression vector. The regulatory expression vector contains a human cytomegalovirus (CMV) early promoter and a reverse tetracycline transcriptional activator (rtTA), while the reactive expression vector consists of a Tet-responsive element (TRE), a CMV promoter, and the target gene. The TRE is a set of seven repeating TetO sequences, and the rtTA is formed by fusing a reverse TetR (rTetR) with a transcriptional activation region at the C-terminus of the herpes simplex virus (HSV) VP16 protein. Under physiological conditions, rtTA does not bind to TRE, and because the CMV promoter lacks an enhancer, the target gene is not expressed. Upon administration of DOX, the DOX-rtTA binding complex binds to the TRE, thereby initiating gene expression. The Tet-on system possesses extremely tight on / off regulatory functions, enabling temporal regulation of the expression of introduced exogenous genes.

[0044] In this article, "viral vector" refers to a viral particle that lacks self-replication capability but has the ability to introduce nucleic acid molecules into the host. "Lentviral vector" refers to a viral vector derived from primate immunodeficiency virus (IFV). Lentiviral vectors contain the genetic information necessary for packaging, infection, and stable integration, and are a major component of the lentiviral vector system. Shuttle plasmids carrying foreign genes are packaged into infectious viral particles with the assistance of lentiviral packaging plasmids and cell lines. These particles then infect cells or living tissues, enabling the expression of the foreign gene. "Recombinant viral vector" refers to a viral vector constructed using gene recombination technology.

[0045] The lentiviral vectors described in this article are typically packaged using transient transfection or cell line packaging. For transient transfection, human cell lines used as packaging cells can be employed, including 293 cells, 293T cells, 293FT cells, 293LTV cells, 293EBNA cells, and other clones isolated from 293 cells; SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, TE671 cells, etc. Monkey cell lines, such as COS1 cells, COS7 cells, CV-1 cells, and BMT10 cells, are also commonly used. Furthermore, calcium phosphate and PEI transfection reagents are commonly used, and other transfection reagents such as Lipofectamine 2000, FuGENE, and S93fectin are also frequently used. Lentiviral packaging also utilizes various lentiviral packaging cell lines, such as stable cell lines derived from the most common Env glycoprotein, VSVG protein, or HIV-1 gag-pol protein.

[0046] For safety reasons, large-scale lentiviral vector systems employ a genome-splitting approach, where genes performing different auxiliary functions are located on different plasmids. Currently, there are four-plasmid systems (encoding the gag-pol gene, Rev gene, VSVG gene, and SIN transfer gene on four different plasmids), three-plasmid systems (the plasmid encoding the Rev gene is removed, and the gag-pol gene in the gag-pol plasmid uses a codon preferred in human cells), and two-plasmid systems (the auxiliary genes necessary for lentiviral vector packaging are located on the same plasmid; these auxiliary genes are single gene sequences; the other is a transgenic plasmid). Lentiviral packaging systems with more than four plasmids are also in use.

[0047] The recombinant lentiviral vectors described in this article can be completely purified. Purification methods include known purification / separation methods such as filtration, ion exchange chromatography, ultrafiltration, molecular sieving, multimode chromatography, nuclease digestion, and sterile filtration. High-speed centrifugation is also commonly used for small-scale preparations. For example, filtering the vector suspension through a 0.45 μm filter followed by centrifugation at 42500 × g for 90 minutes at 4 °C can precipitate and concentrate the vector.

[0048] The cell lines referred to in this document are cell populations obtained by amplification of one or more common ancestral cells, including but not limited to cell populations obtained by amplification of a single isolated cell.

[0049] The cell lines established as described herein refer to cell lines that have an indefinite proliferative potential when cultured under suitable conditions. These cell lines have undergone changes (e.g., transformations) in vitro compared to cells naturally occurring in an organism. A second cell line obtained by isolating single cells from a first cell line and then amplifying the isolated cells is sometimes referred to as a subclone of the first cell line.

[0050] The NSC34 cells described in this article refer to mouse neuronal cells, which are generated by fusing embryonic mouse spinal cord cells rich in motor neurons with mouse neuroblastoma. NSC34 cells possess the characteristics of motor neurons: generating action potentials, expressing neurofilaments, and synthesizing and releasing acetylcholine. In addition, NSC34 induces acetylcholine receptors in co-cultured muscle cells and undergoes the conversion of microwave proteins into neurofilaments during culture and maturation.

[0051] Those skilled in the art will understand that many known detection techniques exist that can be used in this invention. Exemplary techniques for detecting viruses include polymerase chain reaction (PCR), reverse transcription (RT), reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR combined with nested PCR, quantitative PCR (Q-PCR), RT-PCR combined with quantitative PCR (quantitative RT-PCR or RT-QPCR), various probe hybridization techniques, electron microscopy, and various antibody-based detection techniques known in the art (e.g., ELISA assays). Detection techniques also include, but are not limited to, plaque assays and cytopathic effect (CPE) observations, as well as bioinformatics techniques such as BLAST retrieval.

[0052] Those skilled in the art will understand that materials and conditions suitable for the growth and incubation of specific cell types are known in the art, and their sources are also known to those skilled in the art. These sources include, but are not limited to, cell culture manuals, commercial cell banks, or culture medium suppliers. Suitable cell culture conditions can also be readily determined using methods known in the art.

[0053] The present invention will be further illustrated below through specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] The techniques used in the following examples, including gene sequencing, PCR amplification and detection, cell transfection, cell culture and other biological techniques, are all conventional techniques known to those skilled in the art unless otherwise specified. The instruments, equipment and reagents used can be obtained by those skilled in the art through public channels unless otherwise specified in this specification.

[0055] Example 1: Synthesis of sh-SMN and sh-Control sequences

[0056] We designed the DNA coding sequence of shRNA targeting the mouse gene SMN, and added Age I and EcoRI restriction sites to both ends of it, respectively, to synthesize the following nucleotide sequence:

[0057] Justice Chain sh-SMN F1:

[0058] 5'-CCGGGAAGAATGCCACAACTCCC CTCGAG GGGAGTTGTGGCATTCTTCTTTTTG-3' (SEQ IDNO: 4)

[0059] antisense chain sh-SMN R1:

[0060] 5'-AATTCAAAAAGAAGAATGCCACAACTCCC CTCGAG GGGAGTTGTGGCATTCTTC-3' (SEQ IDNO: 5)

[0061] Justice Chain sh-SMN F2

[0062] 5'-CCGGCTCTTGGTACATGAGTGGCTA CTCGAG TAGCCACTCATGTACCAAGAGTTTTG-3'(SEQID NO: 6)

[0063] antisense chain sh-SMN R2:

[0064] 5'-AATTCAAAAACTCTTGGTACATGAGTGGCTA CTCGAG TAGCCACTCATGTACCAAGAG-3'(SEQID NO:7)

[0065] Justice Chain sh-Con F5'-CCGGCAACAAGATGAAGAGCACCAA CTCGAG TTGGTGCTCTTCATCTTGTTGTTTTTG-3' (SEQ ID NO: 8)

[0066] antisense chain sh-Con R:

[0067] 5'-AATTCAAAAACAACAAGATGAAGAGCACCCAA CTCGAG TTGGTGCTCTTCATCTTGTTG-3'(SEQID NO:9)

[0068] Note: Bold text indicates restriction enzyme sites, underlined text indicates stem-loop structures, and italic text indicates continuous U.

[0069] Example 2: Construction of recombinant lentiviral plasmids and packaging of lentiviruses

[0070] 1. Construction of recombinant lentiviral plasmids

[0071] The DNA coding sequence of shRNA obtained by primer annealing was ligated to a large fragment of pLKO-tet-on plasmid digested with AgeI and EcoRI (plasmid map shown). Figure 1 As shown in the figure, recombinant lentiviral plasmids pLKO-tet-on-shRNA1, pLKO-tet-on-shRNA2, and pLKO-tet-on-Control were obtained. Sequencing and DNAMAN software analysis confirmed that the sequences of the recombinant lentiviral plasmids were completely correct.

[0072] 2. Packaging and purification of recombinant lentiviruses

[0073] 293T cells were harvested and digested with 0.125% Trypsin-EDTA. The cell suspension was collected in centrifuge tubes and centrifuged at 800 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 10 ml of 10% DMEM medium and counted. Cells were seeded in culture flasks and cultured in an adherent incubator at 37°C with 5% CO2.

[0074] First, add 2×HBS buffer to the centrifuge tube. Then, add the recombinant lentiviral shuttle plasmid and packaging plasmids pLenti-P2A and pLenti-P2B constructed in step 1 to the centrifuge tube according to the concentrations in Table 1, and vortex to mix. Next, add CaCl2 to the centrifuge tube, vortex immediately, and let it stand at room temperature. Aspirate the original culture medium in the culture flask, wash the cells with an appropriate amount of Opti-MEM and aspirate, then add an appropriate amount of Opti-MEM. Add the plasmid mixture to the cell culture flask, gently shake to mix, and incubate at 37°C in a 5% CO2 incubator for 3 hours. Slowly add an appropriate amount of 30% DMEM complete culture medium, and incubate horizontally at 37°C in a CO2 incubator overnight.

[0075] Photographs were taken 24 hours after transfection. The culture medium in the flasks was discarded. 10 ml of pure DMEM was added to each flask to wash the cells, which was then discarded. An appropriate amount of DMEM medium containing 10 mM sodium butyrate was added, and the flasks were incubated at 37°C in a CO2 incubator. 48 hours after transfection, the supernatant of the recombinant lentivirus was harvested. A portion was used for titer detection. The remaining portion was filtered into sterile centrifuge tubes for virus concentration, and the virus was resuspended in an appropriate amount of PBS. The resuspended recombinant lentivirus was aliquoted and stored at -80°C.

[0076] Table 1. Plasmid details for the recombinant lentiviral packaging system

[0077]

[0078] 3. Rapid detection of lentivirus titers

[0079] Take the recombinant lentivirus titer rapid test card (purchased from Beijing Bio-Long Immunotherapy Co., Ltd., catalog number BF06202) and place it flat on the biosafety counter. Use a micropipette to draw 20 μL of recombinant lentivirus supernatant and add it to the sample window (S), then add 50 μL of buffer solution. After standing for 10-15 minutes, read the results.

[0080] 4. Experimental Results

[0081] During the packaging of the recombinant lentiviral vector, a photograph was taken 24 hours after plasmid transfection, and the results are as follows: Figure 2 As shown in the fluorescence image, the transfection efficiency of Lenti / V5-EGFP was around 80% 24 hours after transfection, indicating that there were no abnormalities during the experiment.

[0082] Rapid titer assays for recombinant lentiviruses showed that the titers of recombinant viruses Lenti-tet-on-sh1 SMN, Lenti-tet-on-sh2 SMN, Lenti-tet-on-sh2 Con, and Lenti / V5-EGFP all reached 1×10⁻⁶. 7 TU / ml. This indicates that the present invention has successfully packaged a qualified recombinant lentivirus, which can be used for subsequent experiments.

[0083] Example 3: Construction of NSC 34-tet-on-shRNA-SMN stable cell line

[0084] 1. Determination of the working concentration of puromycin

[0085] NSC 34 cells were seeded at a rate of 2E5 cells per well in 24-well plates and cultured overnight. When the cell density reached 70%-80%, the old culture medium in the wells was discarded. The cells were washed once with 1x PBS. Puro selection medium was added to the 24-well plates at concentrations of 1, 2.5, 3.5, 5, and 10 μg / mL; cells without the drug served as a negative control. Cell morphology and viability were observed daily. After 72 hours of continuous culture, the lowest concentration of Puro that effectively killed all cells was determined as the optimal working concentration for Puro resistance selection.

[0086] The results are as follows Figure 3 As shown, after 48 hours of treatment with Puro drug, the lowest Puro concentration that effectively killed all cells was 5 μg / mL. Therefore, the optimal working concentration for Puro resistance screening is 5 μg / mL.

[0087] 2. Establishment of NSC 34-tet-on-shRNA-SMN stable cell line

[0088] Healthy NSC 34 cells were seeded into 6-well plates at a density of 6E5 cells / well. After at least 12 hours of culture, three viruses (Lenti-tet-on-sh Con, Lenti-tet-on-sh1 SMN, and Lenti-tet-on-sh2 SMN) were mixed with Opti-MEM medium at an MOI of 5 and added to each well of the 6-well plate. A control group without viruses was also included. After 48 hours of culture, the cells were passaged. Once cells adhered, 5 μg / mL Puro selection medium was added, and the medium was replaced with fresh selection medium every 2-3 days for 10-14 days. Cell morphology and viability were observed daily. After 2 weeks of selection, positive cells were expanded and cryopreserved, thus establishing stable cell lines NSC 34-tet-on-shRNA-Con, NSC 34-tet-on-shRNA1-SMN, and NSC 34-tet-on-shRNA2-SMN.

[0089] Example 5: Identification of NSC 34-tet-on-shRNA-SMN stable cell line

[0090] To verify the successful construction of a stable cell line capable of knocking down the SMN gene, a DOX-induced cell line experiment was conducted. Cells induced for 48 hours with three different concentrations of DOX (500 ng / mL, 10000 ng / mL, and 2000 ng / mL) were used to extract RNA, and the transcriptional level of the target molecule, the SMN gene, was detected by qPCR.

[0091] 1. Extract total RNA from cells

[0092] After stable cell lines NSC 34-tet-on-shRNA-Con, NSC 34-tet-on-shRNA1-SMN and NSC 34-tet-on-shRNA2-SMN were induced with DOX at 500 ng / mL, 1000 ng / mL and 2000 ng / mL for 48 h, respectively, cellular RNA was extracted using a total RNA extraction kit (Trizol, purchased from Heyuan Liji (Shanghai) Biotechnology Co., Ltd., catalog number AN51L758).

[0093] 2. Reverse transcription of RNA

[0094] (1) Reverse transcription was performed using a reverse transcription kit (purchased from TOYOBO, catalog number FSQ-201). The prepared reverse transcription system was placed into a 200 μL RNase-free EP tube according to the instructions in the kit. The system is shown in Table 2, and the reverse transcription program is shown in Table 3.

[0095] Table 2. RNA reverse transcription system

[0096] 5×RT Master Mix: 2μL RNA template: 1μg RNase-free water: XμL Overall system: 10μL

[0097] Table 3. RNA Reverse Transcription Procedure

[0098] 37℃： 15min 50℃： 5min 98℃： 5min 4℃： save

[0099] (2) After the reverse transcription process is complete, add 20 μL of water to the reaction system to dilute the cDNA. Store at -20°C for later use.

[0100] 3. Real-time PCR

[0101] (1) Using cDNA as a template, a real-time PCR reaction was performed using the real-time PCR enzyme from TOYOBO. A 10 μL PCR reaction system was prepared in a 96-well plate, as shown in Table 4.

[0102] Table 4. Real-time PCR reaction system

[0103]

[0104] The required primer sequences are shown in Table 5:

[0105] Table 5. Primer sequences required for Real-time PCR reactions

[0106] Primer name sequence Gapdh sense primer 5'-CTGCCACCCAGAAGACTG-3'(SEQ ID NO: 10) Gapdh antisense primer 5'-AACCTGGTCCTCAGTGTAG-3' (SEQ ID NO: 11) SMN Sense Primer 5'-TGTCGTGGTTTATACTGGA-3' (SEQ ID NO: 12) SMN antisense primer 5'-GGTGGAGGAAGAAATGAG-3' (SEQ ID NO: 13)

[0107] 4. Experimental Results

[0108] The identification results of the stable cell lines NSC 34-tet-on-shRNA1-SMN, NSC 34-tet-on-shRNA2-SMN, and NSC 34-tet-on-shRNA-Con are as follows: Figure 4 As shown in A, 4B, and 4C: After induction with three different concentrations of DOX (500 ng / mL, 1000 ng / mL, and 2000 ng / mL), the NSC34-tet-on-shRNA-Con cell line expressed irrelevant shRNA, which had no interference effect on SMN mRNA levels, and SMN mRNA levels remained normal (e.g., ...). Figure 4 (As shown in C). The expression of shRNA targeting SMN in the stable cell lines NSC34-tet-on-shRNA1-SMN and NSC34-tet-on-shRNA2-SMN was successfully interfered with, leading to a decrease in the mRNA level of the target molecule SMN (as shown in C). Figure 4 A, Figure 4As shown in B), the mRNA level of the target molecule SMN was decreased more significantly in the NSC34-tet-on-shRNA2-SMN cell line (e.g., Figure 4 (As shown in B). Therefore, this invention successfully constructed stable NSC 34-tet-on-shRNA-SMN cell lines NSC 34-tet-on-shRNA1-SMN and NSC 34-tet-on-shRNA2-SMN, which exhibit decreased mRNA levels targeting SMN after DOX induction.

[0109] Example 6: Detection of NSC 34-tet-on-shRNA-SMN cell line activity

[0110] In vitro SMA model studies revealed that decreased SMN expression levels in NSC 34 cells led to increased apoptosis. Therefore, the apoptotic phenotype of DOX-induced stable cell lines was examined.

[0111] 1. Experimental Procedure

[0112] The CCK-8 assay is a highly sensitive, non-radioactive colorimetric method for determining the number of viable cells in cell proliferation or toxicity experiments. The specific detection steps are as follows:

[0113] (1) Take NSC 34-tet-on-shRNA1-SMN and NSC 34-tet-on-shRNA2-SMN cells in the logarithmic growth phase and seed them in 96-well plates at 1000 cells / 100uL, 2000 cells / 100uL, 5000 cells / 100uL, 10000 cells / 100uL and 20000 cells / 100uL respectively. Set up 4 replicates for each group and then incubate them in a constant temperature incubator at 37℃ and 5% CO2.

[0114] (2) After overnight culture, change the medium to DOX(+) or DOX(-), with a DOX concentration of 500 ng / mL. After continuous induction for 5 days, take out one plate every 24 hours. During this period, change the medium every two days. The drug-treated group is continuously drug-treated. After changing to new medium, add 10 μL of CCK-8 reagent to each well and put it back into the incubator for 3 hours.

[0115] (3) Set the wavelength of the microplate reader to 450nm and detect the absorbance value of the cells.

[0116] 2. Experimental Results

[0117] The cell viability results of the stable cell lines NSC34-tet-on-shRNA1-SMN and NSC34-tet-on-shRNA2-SMN are as follows: Figure 5 and Figure 6 As shown, the cell viability of both stable transgenic cell lines, NSC34-tet-on-shRNA1-SMN and NSC34-tet-on-shRNA2-SMN, decreased after DOX induction. At five different cell plating densities (1000 cells / 100µL, 2000 cells / 100µL, and 5000 cells / 100µL), cell viability decreased after DOX induction, with the most significant decrease observed at 2000 cells / 100µL. Comparatively, at the same cell plating density, the decrease in cell viability of NSC34-tet-on-shRNA2-SMN was more pronounced than that of NSC34-tet-on-shRNA1-SMN, consistent with the decrease in mRNA levels.

[0118] In summary, the stable cell lines NSC34-tet-on-shRNA1-SMN and NSC34-tet-on-shRNA2-SMN constructed in this invention, after DOX induction, target the shRNA expression of SMN, successfully interfering with the mRNA of the target molecule SMN, causing a decrease in mRNA levels, which in turn leads to a decrease in SMN protein expression levels, resulting in decreased cell viability. This successfully simulates the pathogenesis of SMA disease and also constructs an in vitro cell model of SMA disease, which also indicates that the biological activity of SMN protein can be detected using this cell model.

[0119] Example 7: Detection of the bioactivity of AAV-SMN using NSC34-tet-on-shRNA2-SMN stable cell line

[0120] This embodiment assesses the bioactivity of recombinant AAV-SMN by evaluating its rescue efficiency against DOX-induced cell viability decline in the NSC34-tet-on-shRNA2-SMN cell line. The recombinant AAV-SMN used in this embodiment was prepared according to the method for constructing the candidate drug L-orS1 as described in patent CN113755524A.

[0121] Detection principle:

[0122] The aforementioned NSC34-tet-on-shRNA2-SMN stable cell line can be induced by DOX to express shRNA2, which targets SMN, generating siRNA that interferes with the expression of endogenous SMN protein mRNA, leading to a decrease in endogenous SMN protein expression and ultimately cell death. Recombinant AAV-SMN gene therapy drug, after infecting cells, overexpresses SMN protein, replenishing the endogenous SMN protein reduced by siRNA, thereby rescuing cell viability after DOX addition, thus reflecting the biological activity of recombinant AAV-SMN in replenishing SMN protein after cell infection.

[0123] Detection method:

[0124] 1. DOX-induced

[0125] In healthy NSC34-tet-on-shRNA2-SMN cells, DOX was added to a final concentration of 500 ng / mL for 7 days for induction.

[0126] 2. Inoculating cells

[0127] Cells induced in step 1 were seeded at a rate of 2000 cells / 100 μL in 96-well plates. NSC34-tet-on-shRNA2-SMN cells, which had not undergone DOX induction, were seeded in parallel as a control. Cells were cultured overnight in DMEM containing 10% FBS. The cell condition in the wells was observed before the experiment the following day.

[0128] 3. Recombinant AAV-SMN infection

[0129] Recombinant AAV-SMN was prepared with sample solution at MOI = 8000. The experiment included a control group 1 without DOX induction, a control group 2 induced by DOX but not infected with recombinant AAV-SMN, and an experimental group induced by DOX and infected with recombinant AAV-SMN. Each group had four replicates. All cell wells were washed once with Opti-MEM. 50 μL of the prepared sample was added to each well. 50 μL of Opti-MEM was added to both the control group 1 without DOX induction and the control group 2 induced by DOX but not infected with recombinant AAV-SMN. The cells were incubated at 37°C and 5% CO2 for 6 hours. After 6 hours, 50 μL of Opti-MEM was added. 48 hours post-infection, the Opti-MEM was replaced with complete medium (DMEM with 10% FBS), and the cells were incubated for 5 days.

[0130] Five days later, after replacing the culture medium, add 10 μL of CCK-8 reagent to each well and incubate again for 3 hours. Use a microplate reader with a wavelength of 450 nm to measure the absorbance of the cells.

[0131] 4. Data Processing

[0132] Bioactivity of recombinant SMN-AAV = (OD value of the experimental group induced by DOX and infected with recombinant AAV-SMN - OD value of the control group 2 induced by DOX but not infected with recombinant AAV-SMN) / (OD value of the control group 1 without DOX induction - OD value of the control group 2 induced by DOX but not infected with recombinant AAV-SMN) × 100%

[0133] The calculated bioactivity of the recombinant AAV-SMN was 65%.

[0134] Example 8, Method Validation

[0135] 1. Exclusivity

[0136] To investigate whether the solvent, excipient solution, and cell culture medium affect the detection, specificity verification was performed using these components. The solvent, excipient solution, and SF9 cell culture medium (Vigor-S101 serum-free insect cell culture medium, catalog number TP0102201) were used as test samples, and the detection was performed according to the reaction conditions in Example 7, repeated three times. If the activity of the solvent, excipient solution, and cell culture medium were all zero, the specificity of this method was considered to meet the requirements.

[0137] The test results showed that the activity of the injection solvent, excipient solution and cell culture medium was all zero, and the CV% was less than 30%, indicating that the method has good specificity for detecting the biological activity of recombinant SMN-AAV.

[0138] 2. Repeatability

[0139] Samples were taken, and the sample processing methods, experimental conditions and methods were as described above. The same experimenter performed the tests, and each sample was repeated 6 times to detect the bioactivity of recombinant AAV-SMN. The RSD values ​​of the 6 experimental results were calculated.

[0140] The results showed that the RSD of the 6 replicates of the sample in this experiment was less than 30%, indicating that the method has good reproducibility in detecting the biological activity of recombinant SMN-AAV.

[0141] 3. Intermediate precision

[0142] Two samples were taken and tested by two lab technicians. The test was repeated three times, and the RSD values ​​of the six test results were calculated.

[0143] The results showed that the RSD value of the 6 results was less than 30%, indicating that the intermediate precision of the method for detecting the biological activity of recombinant SMN-AAV was good.

[0144] 4. Durability

[0145] (1) Referring to the detection method of Example 7, detection was performed at MOIs of 8500, 10000 and 11500 for the recombinant AAV-SMN, and the RSD values ​​of the three detection results were calculated.

[0146] The results show that the RSD value is 2.14% under different MOI conditions (8500, 10000, 11500), indicating that the method has good robustness.

[0147] (2) Referring to the detection method in Example 7, the tests were performed according to the conditions of infection time of 4h, 8h and overnight, and the RSD values ​​of the three results were calculated.

[0148] The results showed that the RSD value was 8.21% under different infection times (4h, 8h, overnight), indicating that the method is robust to detecting the biological activity of recombinant SMN-AAV.

[0149] (3) Referring to the detection method in Example 7, the tests were performed at post-infection culture times of 44h, 48h, and 52h, and the RSD values ​​of the three results were calculated.

[0150] The results showed that the RSD value was 1.23% under different post-infection culture times (44h, 48h, 52h), indicating that the method was robust to detecting the bioactivity of recombinant SMN-AAV.

[0151] The above results demonstrate that the proposed detection method exhibits high specificity, good repeatability, good precision, and good robustness. The method of this invention can accurately and rapidly detect the biological activity of drugs that express or promote the expression of SMN proteins.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An RNA that reduces the expression of the motor neuron survival gene SMN, characterized in that, The RNA sequence is shown in SEQ ID NO.

1.

2. The double-stranded RNA formed by hybridization of the RNA sequence of claim 1 and its reverse complementary sequence.

3. The RNA that can form a hairpin-like structure obtained by covalently linking the RNA sequence of claim 1 and its reverse complementary sequence with an intermediate non-complementary linker sequence.

4. The double-stranded RNA formed by hybridization of the RNA sequence capable of forming a hairpin-like structure as described in claim 3 and its reverse complementary sequence.

5. An shRNA that reduces the expression of the motor neuron survival gene SMN, comprising the RNA sequence of claim 1; preferably, the sequence of the shRNA is shown in SEQ ID NO.

2.

6. A DNA coding sequence of the shRNA of claim 5, wherein the DNA coding sequence is shown in SEQ ID NO.

3.

7. A delivery vector comprising the DNA coding sequence of the RNA of any one of claims 1-4, the shRNA of claim 5, or the shRNA of claim 6.

8. The delivery carrier according to claim 7, characterized in that, The delivery vector is selected from viral vectors and non-viral vectors; preferably, the non-viral vector is selected from the group consisting of liposomes, plasmid vectors, and phage vectors; preferably, the viral vector is selected from the group consisting of adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, and hybrid viral vectors; more preferably, the viral vector is selected from lentivirus vectors.

9. The delivery carrier according to claim 8, characterized in that, The lentiviral vector genome contains a tet-on regulatory element and the DNA coding sequence of the shRNA of claim 6, wherein the tet-on regulatory system regulates the transcriptional expression of the DNA coding sequence of the shRNA of claim 6.

10. A method for constructing a stable cell line, characterized in that, The method includes the following steps: infecting cells with the lentiviral vector of claim 9, and obtaining stable cell lines after the cells are screened for resistance; preferably, the cells are any one of NSC34, MSCs, He1a, K562, HEK293, A549, MCF-7 or LNCap; preferably NSC34 cells.

11. The method according to claim 10, characterized in that, The lentiviral vector has an infection multiplicity (MOI) of 2-10; preferably, puromycin is used for resistance screening, and more preferably, the working concentration of puromycin for resistance screening is 5-10 μg / mL.

12. The method according to claim 10 or 11, characterized in that, The stable cell line showed a decrease in the expression level of the motor neuron survival gene SMN after DOX induction; preferably, the concentration of DOX was 500-2000 ng / mL; preferably, the DOX induction time was 24 hours to 7 days.

13. A stable cell line obtained by the method according to any one of claims 10-12, wherein, The cells are any one of NSC34, MSCs, He1a, K562, HEK293, A549, MCF-7 or LNCap; preferably NSC34 cells.

14. A method for detecting the biological activity of SMN protein, characterized in that, The method includes the following steps: culturing the stable cell line described in claim 13, adding DOX for induction, inoculating and culturing, adding the sample to be tested, and detecting the cell viability.

15. The method according to claim 14, characterized in that, The DOX induction time is 24 hours to 7 days; preferably, the concentration of DOX is 500-2000 ng / mL.

16. The method according to claim 14 or 15, characterized in that, The seeding density of DOX-induced cells is 1000 cells / 100uL to 5000 cells / 100uL, preferably 2000 cells / 100uL.

17. The method according to any one of claims 14-16, characterized in that, The sample to be tested is a sample containing SMN protein or a sample that expresses or promotes the expression of SMN protein after entering the cell; preferably, the sample that expresses SMN protein after entering the cell is a recombinant vector carrying the SMN encoding gene; more preferably, the sample that expresses SMN protein after entering the cell is a recombinant AAV-SMN virus carrying the SMN encoding gene.

18. The method according to claim 17, characterized in that, The recombinant AAV-SMN virus infects DOX-induced cells, and the multiplicity of infection (MOI) of the recombinant AAV-SMN virus is 7000-15000, preferably 8000-11500.

19. The method according to claim 18, characterized in that, The time for infecting cells with the recombinant AAV-SMN virus is 4-24 hours, preferably 4-8 hours.

20. The method according to any one of claims 14-19, characterized in that, The method for detecting cell viability is the CCK-8 assay. Preferably, the specific steps are as follows: take cells after adding the sample to be tested, add CCK-8 reagent, incubate in an incubator, set the wavelength of the microplate reader to 450nm, and detect the absorbance value of the cells.

21. The method according to any one of claims 14-20, characterized in that, The method sets up control groups, wherein the control groups are (1) a control group induced by DOX but without the sample to be tested and (2) a control group not induced by DOX; Preferably, the formula for calculating the bioactivity of the test sample is: (OD value of the experimental group induced by DOX and with the test sample added - OD value of the control group induced by DOX but without the test sample added) / (OD value of the control group without DOX induction - OD value of the control group induced by DOX but without the test sample added) × 100%.

22. The use of the RNA of any one of claims 1-4, the shRNA of claim 5, the DNA coding sequence of the shRNA of claim 6, the delivery vector of any one of claims 7-9, and the cell line of claim 13 in the preparation of a spinal muscular atrophy cell model.

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