Construction and application of an inducible stable cell line that downregulates endogenous SMN

Abstract

The present disclosure relates to a polynucleotide for down-regulating endogenous SMN gene expression and a modified cell comprising the polynucleotide, wherein the modified cell is based on an inducible gene expression system (Tet-On 3G system). In addition, the present disclosure also relates to vectors and compositions comprising the polynucleotides described in the present disclosure, and uses thereof in preparing gene therapies for treating related diseases.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and in particular relates to a shRNA sequence capable of down-regulating an endogenous SMN gene and a stable cell line based on an inducible Tet-On 3G system comprising the shRNA sequence. Background Art

[0002] Spinal muscular atrophy (SMA) is a rare hereditary neuromuscular disease caused by a lack of a functional survival motor neuron 1 (SMN1) gene. In May 2019, Zolgensma was approved by the US FDA for the treatment of SMA children under 2 years old with biallelic mutations in the survival motor neuron 1 (SMN1) gene. Currently, many companies at home and abroad continue to participate in product research and development in this field.

[0003] Both the FDA and NMPA regulators require that the release items of the declared products must include functional (activity) testing of the products before they are put on the market. In fact, there are currently no reported good methods or commercially available stable cell lines for in vitro (cell line level) functional experimental studies of SMN. In addition, in the early stages of drug development, there is no good method to detect the function of the SMN target protein, which also hinders the progress of product research and development. Therefore, the present invention discloses a pharmacodynamic function detection cell line for in vitro evaluation of a series of gene therapy drug products for the treatment of SMA (gene therapy represented by gene replacement: supplementing exogenous normal SMN protein), which can stably, quickly and effectively detect the in vitro activity of gene replacement therapy or protein products for spinal muscular atrophy (SMA), thereby providing a basis for the release of the product or accelerating the progress of early drug development.

[0004] The Tet-On 3G system is an inducible gene expression system for mammalian cells. The Tet-On 3G system has two key components: the Tet-On 3G transactivator protein and the inducible TRE3G promoter ( P TRE3G ). The target cells are able to express the Tet-On 3G transactivator protein, and the TRE3G promoter is able to regulate the expression of the gene of interest (GOI), but the prerequisite is the presence of doxycycline (DOX) induction. Inducible promoter P TRE3G Provides very low basal expression and high maximal expression after induction. It consists of 7 repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter. Therefore, when DOX is not added, P TRE3GIt lacks binding sites for endogenous mammalian transcription factors, so it is actually silent in the absence of induction; when a certain concentration of DOX is added, DOX binds to the Tet-On 3G transactivator protein on the target cells, and then specifically binds to the TRE3G promoter to activate the transcription of the downstream GOI.

[0005] The present invention establishes a stable inducible cell line for knocking down endogenous SMN in mice, and can quickly and effectively detect the function of SMN protein in vitro, thus providing favorable conditions for drug development and product release. Summary of the invention

[0006] The present invention provides a method for down-regulating endogenous SMN A polynucleotide combination for gene expression, characterized in that it comprises two sequences, wherein the sequences respectively comprise the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3. In a specific embodiment, the present disclosure provides a polynucleotide combination for downregulating endogenous SMN gene expression, characterized in that it consists of the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

[0007] The present disclosure provides a vector comprising the polynucleotide combination as described above, wherein the sequences in the polynucleotide combination are in one vector or in different vectors. In a specific embodiment, when the sequences in the polynucleotide combination are in one vector, the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are in any order, and they can be in the same expression frame or in different expression frames.

[0008] In some embodiments, the vector is a viral vector, preferably, the viral vector is an AAV vector, an adenoviral vector, or a lentiviral vector.

[0009] In another aspect, the present disclosure provides a modified cell, wherein the modified cell comprises the polynucleotide combination or vector as described above.

[0010] In some embodiments, the modified cell comprises an inducible gene expression system, wherein the gene expression system is induced when an inducing agent is applied to the cell.

[0011] In some embodiments, the inducible gene expression system comprises a nucleic acid sequence encoding a transactivator and an inducible promoter operably linked to the polynucleotide as described above.

[0012] In some embodiments, the transactivator is a Tet-On 3G transactivator, and the Tet-On 3G transactivator is preferably a Tet-On 3G first generation or second generation transactivator.

[0013] In some embodiments, the inducible promoter is a TRE3G promoter, a PminiCMV promoter, or an E2F1 promoter.

[0014] In some embodiments, the inducing agent is tetracycline or a derivative thereof.

[0015] In some embodiments, the inducing agent is doxycycline (DOX).

[0016] In some embodiments, the modified cell further comprises a reporter gene, preferably, the reporter gene is luciferase, GFP (green fluorescent protein), MCherry (red fluorescent protein) and the like.

[0017] In some embodiments, the cell is a NIH3T3 cell.

[0018] On the other hand, the present disclosure provides a method for preparing a cell line that stably downregulates endogenous SMN based on an inducible gene expression system.

[0019] 1) Construction and screening of cells that stably express inducible gene expression systems;

[0020] 2) transfecting the cells with a plasmid containing an inducible gene expression system of the polynucleotide as described above;

[0021] 3) Antibiotics were added for positive screening, and after WES identification, the cell line was determined to be a stable cell line.

[0022] In some embodiments, the cell is a NIH3T3 cell.

[0023] In some embodiments, the inducible gene expression system comprises a nucleic acid sequence encoding a transactivator and an inducible promoter operably linked to a polynucleotide as described above.

[0024] In some embodiments, the transactivator is a Tet-On 3G transactivator, and the Tet-On 3G transactivator is preferably a Tet-On 3G first generation or second generation transactivator.

[0025] In some embodiments, the inducible promoter is a TRE3G promoter, a PminiCMV promoter, or an E2F1 promoter.

[0026] In another aspect, the present disclosure provides a pharmaceutical composition, comprising the aforementioned vector, modified cells, and a pharmaceutically acceptable excipient.

[0027] In another aspect, the present disclosure provides use of the aforementioned polynucleotide, vector, modified cell or pharmaceutical composition in the preparation of a medicament for treating or preventing a disease in a subject.

[0028] In some embodiments, an effective amount of the vector, modified cell or pharmaceutical composition as described above is administered to a subject.

[0029] In some embodiments, the disease is spinal muscular atrophy (SMA).

[0030] In some embodiments, wherein the subject is a mammal, preferably, the subject is a human.

[0031] In another aspect, the present disclosure provides use of the modified cells as described above in preparing a kit for in vitro evaluation of pharmacodynamic function testing of gene drugs for treating SMA. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present disclosure can be more fully understood with reference to the following drawings.

[0033] Figure 1 A schematic diagram of the construction of an inducible stable cell line that downregulates endogenous SMN is shown.

[0034] Figure 2 The pTRE3G-GOI (pTRE3G-miR30-shRNA) plasmid map is shown.

[0035] Figure 3A and Figure 3B A summary of the results of SMN expression after transfection with different pSMN-shRNAs is shown.

[0036] Figure 4 The expression of endogenous SMN in different monoclonal cells detected by WES is shown. 4#, 5#, 17#, and 22# represent the monoclonal numbers; D0 represents no DOX induction, D4+, and D6+ represent DOX induction for 4 days and 6 days, respectively.

[0037] Figure 5 Shown is the dose response of AV9-101-01M2 in rescuing SMN.

[0038] Figure 6 A flow chart showing the detection of the biological activity of hSMN using an inducible stable cell line that downregulates endogenous SMN. DETAILED DESCRIPTION

[0039] The following description of the present disclosure is intended only to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0040] Polynucleotide

[0041] As used herein, the terms "nucleotide" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. In some embodiments, the term "polynucleotide" as used herein is a nucleotide sequence of an shRNA. In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0042] The term "promoter" as used herein means a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription of a coding sequence, such as a gene or an introduced gene. Promoters can be constitutive, inducible, repressible, or tissue-specific. In some embodiments, the promoter of the present invention is an inducible promoter, preferably a TRE3G promoter.

[0043] The term "vector" as used herein refers to a nucleic acid comprising, consisting essentially of, or consisting of a complete replicon, such that the vector can be replicated when placed into a cell by, for example, a transfection, infection, or transformation process. It is understood in the art that once inside a cell, the vector can replicate as an extrachromosomal (episomal) element, or can be integrated into a host cell chromosome. The vector may include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papillomaviruses, AAV viral vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like, preferably, the vector described herein is selected from an AAV vector, an adenoviral vector, or a lentiviral vector.

[0044] Modified cells

[0045] The term "modified cell" as used herein refers to a cell line capable of downregulating endogenous SMN. In some embodiments, the modified cell provided by the present disclosure comprises a polynucleotide as shown in SEQ ID NO: 2 and / or SEQ ID NO: 3. In some embodiments, the modified cell comprises an inducible gene expression system, wherein the gene expression system is induced when an inducer is applied to the cell. In some embodiments, the inducible gene expression system comprises a nucleic acid sequence encoding a transactivator and an inducible promoter operably linked to a polynucleotide as shown in SEQ ID NO: 2 and / or SEQ ID NO: 3. In some embodiments, the transactivator is Tet-On 3G transactivator. In some embodiments, the inducible promoter is a TRE3G promoter. In some embodiments, the inducer is tetracycline or a derivative thereof, preferably doxycycline (DOX). In some embodiments, the modified cell further comprises a reporter gene, preferably, the reporter gene is luciferase, GFP (green fluorescent protein), MCherry (red fluorescent protein), etc. In some embodiments, the cell is a NIH3T3 cell.

[0046] In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0047] Example

[0048] Example 1: Construction and screening of mouse embryonic fibroblast (NIH / 3T3) cell lines stably expressing Tet-On 3G

[0049] The construction of the stable cell line disclosed in the present invention is based on the inducible Tet-On 3G system. Figure 1 The construction scheme of an inducible stable cell line that downregulates endogenous SMN is shown. First, the present disclosure screened mouse embryonic fibroblasts (NIH / 3T3) cells that can stably express Tet-On3G, and the specific steps are as follows:

[0050] 1. Add 5E+05 NIH / 3T3 cells (manufacturer: Chinese Academy of Sciences Cell Bank, catalog number: SCSP-515 / GNM 6) to each well of a 6-well plate, 2 ml of culture medium / well, and place at 37°C, 5% CO 2 Incubate overnight in an incubator.

[0051] 2. On the second day, when the cell confluence reaches about 80%, pCMV Tet-On 3G plasmid (manufacturer: Youbao Bio, catalog number: VT1613) was transfected. Lipo3000 (manufacturer: Thermo, catalog number: L3000015) was used as the transfection reagent. The transfection steps are as follows (refer to the Lipo3000 manual for detailed transfection steps):

[0052] a) Lipo3000 mix: Opti-MEM: 125 μl / well

[0053] i. Lipo3000: 3.75 μl / well

[0054] b) Plasmid mix: Opti-MEM: 125 μl / well

[0055] i. Plasmid: 2 μg / well

[0056] ii. P3000: 2 μl / μg plasmid / well

[0057] c) Gently mix the two mixes (Lipo3000 mix and Plasmid mix) separately, then add the Plasmid mix to the Lipo3000 mix, mix gently, and leave at room temperature for 10-15 minutes. Finally, add the prepared transfection system into a 6-well plate at 250 μl / well.

[0058] 3. 48 hours after plasmid transfection, trypsinize the cells, centrifuge, and plate the cells from each well of the 6-well plate in four 10 cm culture dishes for culture. Add 8 ml of complete culture medium to each dish (Geneticin™ selective antibiotic G418 (manufacturer: Gibco, catalog number: 10131027) is not added at this time).

[0059] 4. After 48 hours of transfer to a 10 cm culture dish, when the cell density is close to 50% (consistent with the initial cell density of the G418 Kill Curve), replace the culture medium with 500 μg / ml G418.

[0060] 5. Replace the complete medium containing 500 μg / ml G418 every 48 hours.

[0061] 6. After about 2 weeks, all NIH / 3T3 cells without Tet-On 3G integration will die. At this time, multiple independent clones will form on the 10 cm culture dish. Aspirate the culture medium, add 4 ml PBS and wash gently twice, discard the PBS, pick as many single clones as possible (≥24) and expand the culture in a 24-well plate for subsequent experimental verification. At the same time, you can take 1 to 2 culture dishes to digest all cells with trypsin, centrifuge and count, dilute the cells with culture medium according to the cell density, plate a single cell in a single well of a 96-well plate, place the culture plate in an incubator for culture, and transfer the single clones with larger growth and good cell status to a 24-well plate for expansion and culture in about 1 week for subsequent experimental verification. Note that when more than one clone grows in one well of a 96-well plate, it needs to be discarded.

[0062] Note: (When picking single clones, try to choose those with relatively independent positions, far away from adjacent cell clones and in good cell condition. After discarding the culture medium, immediately add 10μl of trypsin to the clones and quickly blow and then transfer the suspension to a 24-well plate. If the old culture medium is dried up and the cell clones are picked up, the subsequent state and growth rate will be greatly affected).

[0063] 7. When the cells in the 24-well plate are full, digest the cells in each well, count the cells of each clone, and take 2E+04 cells from each clone to be plated in a 96-well white bottom transparent culture plate, 6 to 8 replicates, and culture in an incubator overnight. The remaining cells are transferred to a 6-well plate for expansion culture. When the 6-well plate is full, the cells can be frozen, and 2 tubes are frozen for each clone.

[0064] 8. On the second day, use Lipo3000 to transfect pTRE 3G-Luc plasmid (manufacturer: Youbao Bio, catalog number: VT1615). The transfection steps are as follows (refer to the Lipo3000 manual for detailed transfection steps).

[0065] a) Lipo3000 mix: Opti-MEM: 5 μl / well

[0066] Lipo3000: 0.3 μl / well

[0067] b) Plasmid mix: Opti-MEM: 5 μl / well

[0068] Plasmid: 100 ng / well

[0069] P3000: 2 μl / μg plasmid / well

[0070] c) Gently mix the two mixes (Lipo3000 mix and Plasmid mix) separately, then add the Plasmid mix to the Lipo3000 mix, mix gently, and let it stand at room temperature for 10-15 minutes. Finally, add the prepared transfection system into a 96-well plate at 10 μl / well.

[0071] 9. While the plasmid is at room temperature for 15 minutes, replace half of the culture medium of each clone's cells with complete culture medium containing 10 ng / mL DOX, and keep the culture medium of the other half of the wells unchanged.

[0072] 10. After 48 hours, detect the LUC fluorescence intensity of each clone cell.

[0073] 11. Select the cell clone with the highest signal-to-noise ratio of LUC fluorescence intensity value after adding DOX with a final concentration of 10 ng / mL as the best candidate Tet-On 3G NIH / 3T3 cell line. Select the clone with high LUC signal-to-noise ratio, and revive the corresponding frozen cells in time for large-scale expansion culture and cryopreservation.

[0074] LUC signal-to-noise ratio (S / N) = LUC (DOX+) / LUC (DOX-)

[0075] The LUC signal-to-noise ratio (S / N) was calculated from the LUC values ​​before and after DOX induction. The S / N ratio (i.e., LUC signal-to-noise ratio) of the optimal clone should be large, and the N value (i.e., LUC (DOX-) The selection principle of smaller value was used to determine the 37# clone as the best Tet-On 3G NIH / 3T3 stable cell line. The results are shown in Table 1:

[0076] Table 1. Summary of LUC fluorescence intensity values ​​and LUC signal-to-noise ratios of different clones before and after DOX induction

[0077]

[0078] Example 2: Construction and screening of NIH / 3T3 cell lines stably expressing Tet-On 3G SMN-RNAi

[0079] The present invention is to construct an inducible down-regulation mouse endogenous SMN stable cell line, the GOI of the Tet-On 3G system is miR30-shRNA, and the vector also carries the Luciferase gene (Gene ID: 116160065), which is convenient for further cloning and screening. The constructed pTRE3G-miR30-shRNA (VB201120-1020ffc) plasmid map is shown in Figure 2 .

[0080] 1. Take NIH / 3T3 cell clone No. 37 of Tet-On 3G and plate it in a six-well plate, 4E+05 cells / well, and incubate at 37°C, 5% CO 2 Culture in incubator.

[0081] 2. On the second day, when the cell confluence reached about 80%, different SMN shRNA plasmids, namely pSMN-shRNA 1# (its shRNA sequence is shown in SEQ ID NO: 1), pSMN-shRNA 2# (its shRNA sequence is shown in SEQ ID NO: 2), and pSMN-shRNA 3# (its shRNA sequence is shown in SEQ ID NO: 3) were transfected. At the same time, blank control wells were set up without transfecting any plasmids. Lipo3000 was used as the transfection reagent, and the transfection steps were as follows (refer to the Lipo3000 manual for detailed transfection steps):

[0082] a) Lipo3000 mix: Opti-MEM: 125 μl / well

[0083] Lipo3000: 3.75 μl / well

[0084] b) Plasmid mix: Opti-MEM: 125 μl / well

[0085] Plasmid: 2 μg / well

[0086] P3000: 2 μl / μg plasmid / well

[0087] c) Gently mix the two mixes (Lipo3000 mix and Plasmid mix) separately, then add the Plasmid mix to the Lipo3000 mix, mix gently, and let it stand at room temperature for 10-15 minutes. Finally, add the prepared transfection system into a 6-well plate at 250 μl / well.

[0088] Table 2. shRNA sequence information

[0089]

[0090] 3. After 6 hours of transfection, change the medium in one well with complete medium and in the other well with complete medium with the optimal DOX induction concentration.

[0091] 4. After plasmid transfection, cells were digested and total cell protein was extracted at 48 hours and 72 hours respectively. The protein concentration was determined by BCA method. The same amount of protein was taken for detection by fully automatic ultra-micro sample protein expression quantitative analysis system (WES) to determine the decrease level of SMN protein in cells after DOX induction. The shRNA with the most obvious SMN decrease after transfection of a certain shRNA plasmid was selected as the best SMN-shRNA.

[0092] After 72 hours of DOX addition, the SMN protein expression level in cells transfected with pSMN-shRNA2# and 3# was lower than that in the blank group and the group transfected with pSMN-shRNA1#, which means that pSMN-shRNA 2# and 3# had better SMN knockdown efficiency than pSMN-shRNA 1#, but the overall knockdown efficiency was still low ( Figure 3A and Figure 3B ), and then Tet-On 3G NIH / 3T3 cells were infected with lentivirus LV-SMN-shRNA1#, 2#, 3#, and SMN-shRNA2+3# co-infection, in order to achieve better knockdown efficiency of SMN.

[0093] 5. Take NIH / 3T3 cell clone No. 37 of Tet-On 3G and plate it in a 96-well plate, 2E+04 cells / well, set up 2 duplicate wells, and add LV-SMN-shRNA1#, LV-SMN-shRNA2#, LV-SMN-shRNA3# and LV-SMN-shRNA2+3# for joint infection after 3-4 hours of adherence in the incubator to improve the level of SMN-RNAi. The infection condition is 5 μl LV stock solution plus 8 μg / ml polybrene.

[0094] 6. After 48 hours, digest one well and count the cells. Dilute the cells according to the number of cells to ensure that one cell is planted in one 96-well plate. Each group is plated on 3 to 4 96-well plates. Add 1 μg / mL of puromycin to another well. After 72 hours, digest the cells and count them. Treat them the same as the group without puromycin and plate them on 3 to 4 96-well plates.

[0095] 7. After 1 to 2 weeks, pick the monoclones in good condition from the 96-well plate and expand the culture in the 24-well plate. The number of monoclones picked from each group should be ≥ 24.

[0096] 8. When the cells in the 24-well plate are full, digest the cells in each well, count the cells of each clone, take 1500 cells from each clone and spread them on a 96-well white bottom transparent culture plate, 500 cells on a 96-well transparent plate, 6-8 replicates, 100 μl / well, and take another 3000 cells and spread them on a 24-well plate, 2 replicates, 500 μl / well, take half of the replicates of the three wells and add the culture medium containing DOX, and the other half add the culture medium without DOX for culture. Transfer the remaining cells to a 6-well plate for expansion culture, and freeze the cells when the 6-well plate is full, and freeze 2 tubes for each clone.

[0097] 9. The culture medium containing DOX should be replaced every 2 to 3 days.

[0098] 10. After 48 hours, the LUC signal-to-noise ratio (S / N) of each group of clones was detected using the Bright-Glo™ Luciferase Assay System detection reagent. After the addition of DOX induction, the LV-SMN-shRNA with a high LUC signal-to-noise ratio (S / N) was selected as the best shRNA for screening Tet-On 3G SMN-RNAi NIH / 3T3 stable cell lines. The detection results of some LUC fluorescence intensity values ​​are summarized in Table 3 below:

[0099] Table 3 Summary of LUC fluorescence intensity and LUC signal-to-noise ratio after infection with different LV-SMN-shRNA

[0100]

[0101] As can be seen from the results in Table 3, after DOX induction, the signal-to-noise ratio of LUC fluorescence intensity D in the group co-infected with LV-SMN-shRNA2+3# was generally higher than that in the group infected with LV-SMN-shRNA1#, LV-SMN-shRNA2#, and LV-SMN-shRNA3#, respectively. Therefore, it was finally determined that the co-infection of LV-mSMN-shRNA2# and LV-mSMN-shRNA3# (i.e., LV-SMN-shRNA2+3#) was the best shRNA screened, and LV-SMN-shRNA2+3# was selected for the next step of screening of Tet-On 3G SMN-RNAi NIH / 3T3 stable cell lines.

[0102] Example 3: WES detection of SMN knockdown efficiency and determination of the final inducible stable cell line for downregulating endogenous mouse SMN

[0103] According to the above screening results, NIH / 3T3 cell clone No. 37 of Tet-On 3G was taken and infected with LV-SMN-shRNA2+3# again for the second round of monoclonal screening. Monoclonal clones were selected and cultured separately. The grown monoclones were induced with DOX respectively. The expression levels of SMN in different clones at different induction times were detected by WES, and the knockdown efficiency of each clone was obtained to determine the final inducible stable cell line for downregulating endogenous mouse SMN.

[0104] The WES of the Simple Western system was used to detect changes in the SMN protein levels of the clones. Wes uses micro-sample tubes, combined with inner coating technology, to separate and capture proteins from samples and quantify the target protein through subsequent immunoreaction and chemiluminescence detection. The samples and reagents were added to the pre-packed plates, and the program was set through the Compass software. The plate and capillary module instrument with the added samples were automatically completed to complete the detection of 25 or 13 samples, and the data was analyzed using the Compass software.

[0105] Different NIH / 3T3 RNAi monoclonal cell lines were induced with 100 ng / mL DOX. Cells were collected after 4 and 6 days of induction. 100-200 μl of cell lysis buffer was added according to the amount of cells. The cells were placed on ice for 30 minutes to fully lyse the cells. The supernatant was taken into another centrifuge tube and the protein concentration of the samples was determined by the BCA method. The WES experiment was performed according to the WES manual to detect the expression of endogenous SMN. The detection conditions were a final loading concentration of 0.5 μg / μL, the detection antibody SMN was diluted at 1:250, and the internal reference antibody was diluted at 1:2000. The four monoclonal cells (4#, 5#, 17#, and 22#) selected were detected. The results of WES detection and relative RNAi are shown in Figure 2. Figure 4 and Table 4.

[0106] The relative RNAi efficiency results are shown in Table 4 (5# clone without DOX induction, i.e. 5#D0, was used as a control):

[0107] Table 4. Summary of endogenous SMN expression and relative RNAi efficiency in different monoclonal cells detected by WES

[0108]

[0109] The results showed that the SMN knockdown efficiency of clone 22# was the highest after 4 and 6 days of DOX induction, both reaching more than 90%. Finally, the mouse NIH / 3T3 cell clone of Tet-On 3G No. 37# was selected to infect LV-SMN-shRNA2+3# and the resulting 22# monoclonal cell line was the final selected stable cell line for inducible downregulation of mouse endogenous SMN, named NIH / 3T3 SMNRNAi-001, which can induce the SMNRNAi effect of SMN-shRNA by adding DOX, thereby downregulating the expression of endogenous SMN protein in NIH / 3T3 cells. NIH / 3T3 SMNRNAi-001 cells will be used to verify the biological activity function of hSMN.

[0110] Example 4: Detection of biological activity of hSMN using a stable cell line that inducibly down-regulates endogenous mouse SMN

[0111] This method uses the constructed NIH / 3T3 SMNRNAi-001 cell line. After DOX induction, the cell line can express the exogenously inserted SMN shRNA, recognize and bind to the endogenous SMN mRNA. Under the action of shRNA, the mRNA is degraded, and the endogenous SMN protein expression is reduced due to the reduction of the endogenous SMN mRNA content. There is a certain correlation between SMN protein expression and cell proliferation and survival: the decrease in endogenous SMN protein expression will lead to a slowdown or even stagnation of cell proliferation. Through DOX-induced NIH / 3T3 SMNRNAi-001 cells, the endogenous SMN protein expression is reduced, thereby reducing the cell proliferation rate. AV9-101-01M2 is a recombinant adeno-associated virus (AAV) vector expressing SMN protein. Different doses of AV9-101-01M2 virus infect DOX-induced cells, and the cells express different levels of SMN protein, so that the cells recover part / all of their proliferation ability. The number of cells can be detected using the CellTiter96 kit to evaluate its recovery ability. The principle is: MTS new tetrazolium salt can react with dehydrogenase in mitochondria to produce soluble brown formazan, which is directly released into the culture medium. The absorbance is detected at a wavelength of 490 nm. The OD490 nm value is proportional to the number of living cells. The higher the OD490 nm reading, the more cells there are, indicating that the rescue effect of AV9-101-01M2 is better.

[0112] The detection process is as follows Figure 6 As shown, the following steps are included:

[0113] 1. Prepare DMEM complete medium with a final DOX concentration of 1 μg / mL. When the cells proliferate to about 80% confluence, discard the medium, add 10 mL PBS, gently shake the culture flask to rinse the cells, and discard the PBS. Add 2 mL of 0.25% trypsin, shake the culture flask gently and place it in a cell culture incubator for about 30 seconds. After the cells become round and fall off, add 8 mL of complete culture medium immediately to terminate digestion. Use a pipette to blow the bottom of the culture flask gently to make all cells fall off completely. Divide the cell suspension into two equal parts and transfer them to two 15 mL centrifuge tubes. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add complete culture medium to one tube, resuspend the cells and count them, take 4E+05 cells and add them to a T75 culture flask containing DMEM complete culture medium, with a total volume of 15 mL; add complete culture medium containing 1 μg / mL DOX to the other tube, resuspend the cells and count them. According to the counting results, take 4E+05 cells and place them in a T75 culture flask containing DMEM complete culture medium containing 1 μg / mL DOX, with a total volume of 15 mL. Gently shake the culture flask to evenly distribute the cells on the bottom of the culture flask, and place it in a 37°C, 5% CO2 incubator for culture. The half-life of DOX is about 48 hours, so after 2 days of DOX-induced cell culture, the medium was replaced with DOX and culture was continued for 2 days, that is, DOX-induced culture was 4 days.

[0114] 2. Prepare DMEM complete medium with a final DOX concentration of 1 μg / mL. Remove the induced and uninduced cells, discard the medium, add 10 mL PBS, gently shake the culture flask to rinse the cells, and discard the PBS. Add 2 mL preheated 0.25% trypsin, gently shake the culture flask to evenly cover the cell layer with trypsin, and place it in a cell culture incubator for about 30 seconds. After the cells become round and fall off, immediately add 8 mL of complete medium to terminate digestion. Use a pipette to gently blow the bottom of the culture flask to completely fall off all cells, and transfer the cells to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and add new complete medium to resuspend the cells. Take 500 μL of the cell suspension and place it in a Vi-Cell cell counter for counting. Dilute the cells to 1E+04 cells / mL according to the counting results, mix well, and add 50 μL / well, that is, 500 cells per well, to a 96-well cell culture plate, so that the cells are evenly distributed on the bottom of the plate, and place it at 37°C, 5% CO 2 Incubate in an incubator for 20 to 24 hours.

[0115] 3. Observe the cell status in the 96-well plate under a microscope. After confirming that the cells are in good condition, take out the virus from the -80℃ refrigerator and melt it on ice. Prepare the infection system (see Table 5), slowly add 50 μL / well to the 96-well plate, and repeat three wells for each sample. Place the 96-well plate at 37℃ and 5% CO 2 Culture in an incubator.

[0116] Table 5. Test article infection system

[0117] Infection system Sample set PC group (DOX-) NC group (DOX+) Virus AV9-101-01M2 (MOI: 1E4 / 1E5 / 1E6 / 1E7) N / A N / A Infection-promoting agents 2.5 mM NaBu (final concentration) 2.5 mM NaBu (final concentration) 2.5 mM NaBu (final concentration) Culture medium Complete medium containing 1 μg / ml DOX Complete medium without DOX Complete medium containing 1 μg / ml DOX Total volume 50 μL / well 50 μL / well 50 μL / well

[0118] 4. After 12 to 16 hours, remove the 96-well plate, discard the culture medium in the wells, add 100 μL DMEM complete culture medium to each well of the PC group (positive control), and add 100 μL DMEM complete culture medium containing 1 μg / mL DOX to each well of the other groups. Place at 37°C, 5% CO 2 Culture in an incubator.

[0119] 5. After 12 to 16 hours, remove the 96-well plate, discard the culture medium in the wells, add 100 μL DMEM complete culture medium to each well of the PC group, and add 100 μL DMEM complete culture medium containing 1 μg / mL DOX to each well of the other groups. Place at 37°C, 5% CO 2 Culture in an incubator and replace the medium with DOX every 2 days.

[0120] 6. On the 8th day after infection, take out the aliquoted CellTiter96 detection reagent (stored at -20℃) and place it in a 37℃ incubator for about 15 minutes to melt it. Take out the 96-well cell culture plate to be tested from the incubator, add 20 μL CellTiter96 detection reagent to each well, and place it at 37℃, 5% CO in a dark environment. 2 Incubate in an incubator for 3 to 4 hours and read the OD value at 490 nm using a multifunctional microplate reader.

[0121] 7. Calculation of rescue rate (%):

[0122] The rescue rate (%) of the DOX+ group was set to 0, and the rescue rate (%) of the DOX- group was set to 100%;

[0123] Calculation formula: rescue rate (%) = x 100%

[0124] AV9-101-01M2 can express SMN protein and effectively rescue the proliferation of NIH / 3T3 SMNRNAi-001 cells, and it shows a significant dose effect ( Figure 5 ). This inducible stable cell line that downregulates endogenous mouse SMN can be used to detect the biological activity of exogenous hSMN.

[0125] Incorporated by Reference

[0126] Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0127] Equivalence

[0128] The present disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the above description, and all changes within the equivalent meaning and scope of the claims are intended to be covered therein.

Claims

1. Downregulation of endogenous SMN A polynucleotide combination for gene expression, characterized in that It consists of two nucleotide sequences, namely SEQ ID NO: 2 and SEQ ID NO:

3.

2. A vector comprising the polynucleotide combination according to claim 1, wherein the sequences in the polynucleotide combination are in one vector or in different vectors.

3. The vector of claim 2, wherein the vector is a viral vector.

4. The vector of claim 3, wherein the viral vector is an AAV vector, an adenoviral vector, or a lentiviral vector.

5. A modified cell, wherein the modified cell comprises the polynucleotide combination according to claim 1 or the vector according to claim 2 or 3.

6. The modified cell according to claim 5, comprising an inducible gene expression system, wherein the gene expression system will be induced when an inducer is applied to the cell, the inducible gene expression system comprising a nucleic acid sequence encoding a transactivator and an inducible promoter operably linked to the polynucleotide combination according to claim 1, the transactivator is Tet-On 3G transactivator, the inducible promoter is TRE3G promoter, PminiCMV promoter or E2F1 promoter, and the inducer is doxycycline (DOX), tetracycline or its derivatives.

7. The modified cell according to claim 5 or 6, further comprising a reporter gene.

8. The modified cell according to claim 7, wherein the reporter gene is luciferase, green fluorescent protein (GFP) or red fluorescent protein (MCherry).

9. The modified cell of any one of claims 5 to 8, wherein the cell is a NIH3T3 cell.

10. A method for preparing a cell line that stably downregulates endogenous SMN based on an inducible gene expression system, characterized in that: 1) Construction and screening of cells that stably express inducible gene expression systems; 2) transfecting the cell with a plasmid containing an inducible gene expression system of the polynucleotide combination according to claim 1; 3) adding antibiotics for positive screening, and after WES identification, determining it as a stable cell line, the inducible gene expression system comprises a nucleic acid sequence encoding a transactivator protein and an inducible promoter operably linked to the polynucleotide of claim 1, the transactivator protein is Tet-On 3G transactivator protein, and the inducible promoter is TRE3G promoter, PminiCMV promoter or E2F1 promoter. The preparation method according to claim 10 , wherein the cells are NIH3T3 cells.

12. A pharmaceutical composition comprising the vector according to any one of claims 2 to 4, or the modified cell according to any one of claims 5 to 8, and a pharmaceutically acceptable excipient.

13. Use of the modified cell according to any one of claims 5 to 8 in the preparation of a kit for in vitro evaluation of the pharmacodynamic function of a gene drug for the treatment of SMA.

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