Target gene transcriptional activity determination method based on RNA editing
Through the RNA editing method, the sensor reporter vector is designed and mRNA editing is catalyzed using ADAR enzymes to solve the problem of difficult to accurately detect the transcriptional activity of the target gene in gene therapy products, and achieves high accuracy and reliability of transcriptional activity assays.
Patent Information
- Application Number
- CN202411786329.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-10-02
- Filing Date
- 2024-12-06
- Publication Date
- 2025-05-27
AI Technical Summary
In gene therapy products, it is difficult for the prior art to accurately detect the transcriptional activity of the target gene, especially when RNA in cells is prone to degradation, resulting in deviations in the detection results and difficulty in accurately determining the quantity.
Using the transcriptional activity determination method of the target gene based on RNA editing, the sensor reporter vector was designed, and mRNA editing was catalyzed by ADAR enzyme to activate the translation of downstream main reporter genes, thereby determining the transcriptional activity of the target gene.
Quantitative detection of mRNA of target genes without extracting RNA is achieved, which improves the accuracy and reliability of the detection, avoids deviations caused by RNA degradation, and can accurately measure the transcriptional activity of target genes in gene therapy products.
Smart Images
Figure CN120044008A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological activity detection of gene therapy products, and particularly relates to a new method for detecting the transcriptional activity of a target gene. Background Art
[0002] Gene therapy products are used to introduce therapeutic genes into cells or the human body through specific vectors to achieve the purpose of treating diseases. The vectors used include plasmids, mRNA, adeno-associated virus (AAV), adenovirus (AdV), lentivirus (LV), etc. The expression of the therapeutic gene (target gene) includes the mRNA level and the protein level. Usually, after introducing the gene therapy product into sensitive cells, total mRNA or total protein is extracted for detection. The detection of the protein level can be carried out by methods such as western blot, ELISA, and cell immunostaining, all of which require specific antibodies. Moreover, the therapeutic gene is usually a normal gene, and the background expression in cells is relatively high, which brings many challenges to the detection. The detection of the mRNA level has the following advantages: 1) It does not require specific antibodies; 2) The mRNA level can distinguish the codon-optimized therapeutic gene from the background gene. However, the mRNA detection also has technical challenges. It is necessary to extract total RNA for RT-PCR, which is cumbersome to operate, and RNA is easily degraded, which will cause deviation in the results and it is difficult to accurately quantify.
[0003] There is a class of RNA editing enzymes (Adenosine Deaminases Acting on RNA, ADAR) in human cells, which can recognize double-stranded RNA and catalyze the deamination reaction of A (Adenosine) to transform it into I (Inosine). During translation, I is regarded as G (Guanosine), realizing the mRNA editing of A-G. The RNA editing technology based on ADAR can be used to detect specific mRNA in living cells.
[0004] The present invention is completed under the support of the National Key Research and Development Program project "Quality Research and Quality System Construction of Viral Vectors" (2023YFC3403305) and the project of the basic scientific research business expenses of the Chinese Academy of Medical Sciences "Quality Evaluation and Related Mechanism Research of Gene Therapy Drugs and New Biological Products such as mRNA Vaccines" (2023-PT350-01). Summary of the Invention
[0005] The object of the present invention is to provide a method for measuring the transcriptional activity of a target gene based on RNA editing. This method is a method for quantitatively detecting the mRNA of a target gene based on RNA editing, which does not require RNA extraction, and measures the transcriptional activity of the target gene by detecting the expression of a reporter gene.
[0006] The object of the present invention is achieved as follows: A method for measuring the transcriptional activity of a target gene based on RNA editing, characterized in that: the method is based on mRNA editing and reporter gene technology, and its sensor reporter gene vector is composed of the following: Upstream internal reference reporter gene + T2A + sensor sequence + F2A + downstream main reporter gene, and the upstream and downstream reporter genes encode two different luciferases.
[0007] The object of the present invention can also be achieved as follows: the design principle of the sensor sequence is as follows: Screen the sequence containing the three bases CCA or TCA in the target gene sequence, make these three bases in the same codon, the sequence length is an integer multiple of 3, and the corresponding three bases in its complementary sequence are TGG or TGA. Replace the middle G with A to encode a stop codon, which is the sensor sequence, and the sensor sequence needs to ensure that there is no other stop codon in this reading frame.
[0008] A method for measuring the transcriptional activity of a target gene based on RNA editing, characterized in that: the sensor sequences designed for SMN, GFP and WPRE are as follows: SMN sensor1 sequence: 5’-tatgcttttatcagtgctgtatcatcccaaatgtcagaatcatcgctctAgcctgtgccgcgccggaacagcacggaatcctcctgctccgggacgccg-3’ SMN sensor2 sequence: 5’-ttaacatacttcccaaagcatcagcatcatcaagagaatctggacatatAggaggtggtgggggaattattggtggtccagaaggaaatggaggcagcc-3’ GFP sensor1 sequence: 5’-gtggcatcgccctcgccctcgccggacacgctgaacttgtAgccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtg-3’ GFP sensor2 sequence: 5’-tggtcggcgagctgcacgctgccgtcctcgatgttgtAgcggatcttgaagttcaccttgatgccgttcttctgcttgtcggcc-3’ GFP sensor3 sequence: 5’-cggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttAgggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggca-3’ WPRE sensor1 sequence: 5’-gatttatacaaggaggagaaaatgaaagccatacgggaagcaatagcatAatacaaaggcattaaagcagcgtatccacatagcgtaaaaggagcaaca -3’ WPRE sensor2 sequence: 5’-ccaacagccgagcccctgtccagcagcgggcaaggcaggcggcgatAagttccgccgtggcaagaactaaccaggatttatacaaggaggagaaaa-3’。
[0009] The initial cells for constructing the sensor reporter gene cells are cells with high ADAR expression, such as 293 or 293T cells.
[0010] The method for quantitatively determining the transcriptional activity of the target gene is as follows: Using the concentration or titer of the test sample as the dose, and the ratio of the main reporter gene signal to the internal reference reporter gene signal as the response, a four-parameter Logistic model is used for curve fitting, and the parallel line method is used to calculate the percentage content of the test sample relative to the reference product.
[0011] A method for determining the transcriptional activity of a target gene based on RNA editing is used for determining the transcriptional activity of the target gene of a gene therapy product.
[0012] The basic technical solution of the present invention is as follows: 1) Sensor design: Screen the sequences in the target gene sequence that contain the three-base CCA or TCA, with these three bases within the same codon, and the sequence length being an integer multiple of 3 (about 99 nt). The corresponding three bases in its complementary sequence are TGG or TGA. Replace the middle G with A (encoding a stop codon) to obtain the sensor sequence (ensure that there are no other stop codons within this reading frame).
[0013] 2) Construction of the Sensor reporter gene plasmid: Insert the sensor sequence, T2A, and P2A sequences into the dual-luciferase reporter gene vector. The order from the 5' end to the 3' end is: CMV promoter - internal reference reporter gene (firefly luciferase) - T2A - sensor sequence - P2A - main reporter gene (nano luciferase) - SV40 polyA, ensuring that all sequences are within the same reading frame (other types of reporter genes can also be selected).
[0014] 3) Establishment of mRNA reporter gene cells: Transfect 293 or 293T cells with the sensor dual-reporter gene vector, and obtain a stable cell line through hygromycin pressure screening. The internal reference reporter gene can be normally expressed, and the stop codon in the sensor sequence causes translation to be interrupted, and the downstream main reporter gene cannot be translated into a protein sequence.
[0015] 4) Detection of the transcriptional activity of the target gene: Transfect or infect the reporter gene cells with the gene therapy product. The target mRNA transcribed by the target gene is complementary to the mRNA of the sensor sequence. The endogenous ADAR in the cells recognizes the double-stranded RNA and catalyzes the conversion of the non-paired A in the three bases to G, converting the TAG or TAA encoding the stop codon to TGG encoding tryptophan (both As in TAA are edited to G simultaneously), and the downstream main reporter gene is successfully translated into a protein sequence. The expression level of the main reporter gene is positively correlated with the expression level of the target mRNA, and thus the transcriptional activity of the target gene of the gene therapy product is measured. For the sensor reporter gene cells targeting the general sequence, they can be used as general cells to detect the transcriptional activity of the target gene containing this sequence. For example, the 3' UTR region of the lentiviral vector usually contains the WPRE sequence, and the WPRE mRNA reporter gene cells in the present invention can be used for the transcriptional activity detection of such vectors.
[0016] The present invention has the following beneficial effects: By using RNA editing and dual reporter gene technologies, the present invention constructs a sensor reporter gene cell line specific to the target gene. When a gene therapy product is transfected or infected into the reporter gene cells, the target mRNA transcribed from the target gene is complementary paired with the mRNA of the sensor sequence. The endogenous ADAR enzyme in the cells recognizes double-stranded RNA and catalyzes the conversion of TAG or TAA encoding the stop codon in the sensor mRNA into TGG encoding tryptophan, initiating the translation of the downstream main reporter gene. Moreover, the expression level of the main reporter gene is positively correlated with the expression level of the target gene mRNA, thereby measuring the transcriptional activity of the target gene. The technology established by the present invention can be used to measure the transcriptional activity of the target gene of different vectors such as plasmids, mRNAs, lentiviruses, and adeno-associated viruses. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 Dose-effect curve of SMN / GFP / WPRE sensor reporter gene cells against plasmid.
[0018] Figure 2 Dose-effect curve of GFP sensor reporter gene cells against mRNA.
[0019] Figure 3 Dose-effect curve of GFP and WPRE sensor reporter gene cells against lentivirus, a. GFP sensor. b. WPRE sensor.
[0020] Figure 4 Dose-effect curve of GFP sensor reporter gene cells against different serotypes of AAV.
[0021] Figure 5 4-PL parallel line method calculation model for quantitative determination of AAV transcriptional activity.
[0022] Figure 6 a. Dose-effect fitting curves of GFP sensor reporter gene cells against AAV2-GFP-WPRE control and sample 2. b. Dose-effect fitting curves of SMN sensor reporter gene cells against AAV2-SMN-GFP-WPRE control and sample 1. DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention will be further explained and illustrated by the following specific embodiments, which are only used to explain the present invention and should not be construed as a limitation to the present invention.
[0024] The present invention is based on mRNA editing and reporter gene technology. The composition of its sensor reporter gene vector is as follows: upstream internal reference reporter gene + T2A + sensor sequence + F2A + downstream main reporter gene. The upstream and downstream reporter genes encode two different luciferases. Among them, the sensor sequence can specifically recognize the mRNA expressed by the target gene. The sensor reporter gene vector is transfected into cells with high expression of ADAR to construct sensor reporter gene cells. After the sample to be tested acts on the cells, the expression of the main reporter gene can be activated in a dose-dependent manner. The method of the present invention can be used for the determination of the transcriptional activity of the target gene of different vectors such as plasmids, mRNAs, lentiviruses, and adeno-associated viruses.
[0025] A method for determining the transcriptional activity of a target gene based on RNA editing, characterized in that: the method is based on mRNA editing and reporter gene technology, and the composition of its sensor reporter gene vector is as follows: upstream (internal reference) reporter gene + T2A + sensor sequence + F2A + downstream (main) reporter gene. The upstream and downstream reporter genes encode two different luciferases.
[0026] The design principle of the sensor sequence is as follows: Screen the sequence containing the three-base CCA or TCA in the target gene sequence, make this three-base in the same codon, the sequence length is an integer multiple of 3 (about 99 nt), and the corresponding three-base in its complementary sequence is TGG or TGA. Replace the middle G with A (encoding a stop codon), which is the sensor sequence (ensure that there is no other stop codon in this reading frame).
[0027] The sensor sequences designed for SMN, GFP, and WPRE are as follows: SMN sensor1 sequence: 5’-tatgcttttatcagtgctgtatcatcccaaatgtcagaatcatcgctctAgcctgtgccgcgccggaacagcacggaatcctcctgctccgggacgccg-3’ SMN sensor2 sequence: 5’-ttaacatacttcccaaagcatcagcatcatcaagagaatctggacatatAggaggtggtgggggaattattggtggtccagaaggaaatggaggcagcc-3’ GFP sensor1 sequence: 5’-gtggcatcgccctcgccctcgccggacacgctgaacttgtAgccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtg-3’ GFP sensor2 sequence: 5’-tggtcggcgagctgcacgctgccgtcctcgatgttgtAgcggatcttgaagttcaccttgatgccgttcttctgcttgtcggcc-3’ GFP sensor3 sequence: 5’-cggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttAgggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggca-3’ WPRE sensor1 sequence: 5’-gatttatacaaggaggagaaaatgaaagccatacgggaagcaatagcatAatacaaaggcattaaagcagcgtatccacatagcgtaaaaggagcaaca -3’ WPRE sensor2 sequence: 5’-ccaacagccgagcccctgtccagcagcgggcaaggcaggcggcgatAagttccgccgtggcaagaactaaccaggatttatacaaggaggagaaaa-3’.
[0028] The initial cells for constructing sensor reporter gene cells are 293 or 293T cells with high ADAR expression.
[0029] The method for quantitatively determining the transcriptional activity of the target gene is as follows: taking the concentration or titer of the test sample as the dose, taking the ratio of the main reporter gene signal to the internal reference reporter gene signal as the response, using a four-parameter Logistic model for curve fitting, and using the parallel line method to calculate the percentage content of the test sample relative to the reference product.
[0030] A method for determining the transcriptional activity of a target gene based on RNA editing, which is applied to the determination of the transcriptional activity of the target gene of gene therapy products.
[0031] The technical solution of the present invention is described in detail below: 1. Construction of a sensor dual - reporter gene vector In the present invention, dual - reporter gene vectors targeting the target gene SMN (Homo sapiens survival of motor neuron1), the tag sequence GFP, and the WPRE sequence in the 3’UTR region were constructed respectively. The sensor sequences are as follows: SMN sensor1 sequence: 5’-tatgcttttatcagtgctgtatcatcccaaatgtcagaatcatcgctctAgcctgtgccgcgccggaacagcacggaatcctcctgctccgggacgccg - 3’ SMN sensor2 sequence: 5’-ttaacatacttcccaaagcatcagcatcatcaagagaatctggacatatAggaggtggtgggggaattattggtggtccagaaggaaatggaggcagcc - 3’ GFP sensor1 sequence: 5’-gtggcatcgccctcgccctcgccggacacgctgaacttgtAgccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtg - 3’ GFP sensor2 sequence: 5’-tggtcggcgagctgcacgctgccgtcctcgatgttgtAgcggatcttgaagttcaccttgatgccgttcttctgcttgtcggcc - 3’ GFP sensor3 sequence: 5’-cggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttAgggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggca - 3’ WPRE sensor1 sequence: 5’-gatttatacaaggaggagaaaatgaaagccatacgggaagcaatagcatAatacaaaggcattaaagcagcgtatccacatagcgtaaaaggagcaaca - 3’ WPRE sensor2 sequence: 5’-ccaacagccgagcccctgtccagcagcgggcaaggcaggcggcgatAagttccgccgtggcaagaactaaccaggatttatacaaggaggagaaaa-3’ Insert the sensor sequence, T2A, and P2A sequences into the dual-luciferase reporter gene vector. The order from the 5' end to the 3' end is: CMV promoter - firefly luciferase - T2A - sensor sequence - P2A - nano luciferase - SV40 polyA. The eukaryotic selection tag of the vector is hygromycin resistance.
[0032] 2. Construct mRNA reporter gene stably transfected cells Transfect the above-mentioned sensor reporter gene vector into 293 or 293T cells, and obtain a stably transfected mRNA reporter gene cell line through hygromycin pressure screening.
[0033] 3. Reactivity of different gene therapy vectors 3.1 Dose-response to plasmid Dose-response of SMN sensor1 / 2, GFP sensor3, and WPRE sensor1 stably transfected 293T cells to pAAV-SMN-GFP-WPRE plasmid (see attachment Figure 1 ) 3.2 Dose-response to mRNA Dose-response of GFP sensor3 and WPRE sensor1 stably transfected 293T cells to circular GFP mRNA (see attachment Figure 2 ) 3.3 Dose-response to lentivirus Dose-response of GFP sensor3 and WPRE sensor1 stably transfected 293T cells to LV-GFP (see attachment Figure 3 ) 3.4 Dose-response to adenovirus Dose-response of GFP sensor3 and WPRE sensor1 transfected A549 cells to GFP adenovirus (see attachment Figure 4 ) 3.5 Dose-response to adeno-associated virus Dose-response of GFP sensor3 and WPRE sensor1 stably transfected 293T cells to different serotypes of AAV-GFP (see attachment Figure 5 ) 4. AAV transcriptional activity assay 4.1 Test method 1) Cell seeding: For mRNA reporter gene 293T cells (for adenovirus, A549 cells are used), add 7E04 / 100 μL / well to a 96-well white culture plate and culture for 24 hours; 2) Sample transfection / infection: Plasmid: Perform serial dilution with TE buffer, add to Opti-MEM and mix well, then add transfection reagent, incubate at room temperature for 5 - 20 min (according to the instruction manual), add 10 μL / well to the cell plate, and culture for 48 - 72 h for luciferase activity detection; mRNA: Perform serial dilution with mRNA diluent, add mRNA transfection reagent, incubate at room temperature for 5 - 10 min (according to the instruction manual), add 10 μL / well to the cell plate, and culture for 48 - 72 h for luciferase activity detection; Viral sample: Perform serial dilution with Opti-MEM, discard the cell supernatant, add 50 μL / well of virus diluent, after culturing for 12 - 16 h, supplement 150 μL / well of complete medium, and perform luciferase activity detection after infection for 72 - 96 h.
[0034] 3) Luciferase activity detection: Refer to the kit instruction manual.
[0035] 4.2 Quantification method Take the concentration (or titer) of the sample to be measured as the dose, and the ratio of the main reporter gene signal to the internal reference reporter gene signal as the response. Use the four-parameter Logistic model for curve fitting to reduce the interference caused by high-concentration (titer) samples to be measured on the cell background, and use the parallel line method to calculate the percentage content of the sample to be measured relative to the reference substance (see attachment Figure 6 ).
[0036] 4.3 Linearity, accuracy and repeatability Using the method established in the present invention, detect the transcriptional activity of AAV samples. The AAV2-GFP sample is tested with GFP sensor reporter gene cells, and the AAV2-SMN-GFP sample is tested with SMN sensor reporter gene cells.
[0037] The RSD of the results of five repeated tests is less than 15% (Table 1), the linear test R 2 is greater than 0.99, the slope is close to 1.0, and the recovery rates at each concentration point are between 90% and 120% (Table 2).
[0038] Table 1. Repeatability of the determination of AAV sample transcriptional activity
[0039] Table 2. Linearity and accuracy of the determination of AAV sample transcriptional activity 。
Claims
1. A method for measuring the transcriptional activity of a target gene based on RNA editing, characterized in that: The method is based on mRNA editing and reporter gene technology, and its sensor reporter gene vector is composed of the following: Upstream internal reference reporter gene + T2A + sensor sequence + F2A + downstream main reporter gene, the upstream and downstream reporter genes encode two different luciferases.
2. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 1, characterized in that: The design principles of the sensor sequence are as follows: The target gene sequence is screened for sequences containing three bases CCA or TCA, so that these three bases are located in the same codon, the sequence length is an integer multiple of 3, and the corresponding three bases in the complementary sequence are TGG or TGA. The middle G is replaced with A to encode a stop codon, which is the sensor sequence, and the sensor sequence must ensure that there are no other stop codons in the reading frame.
3. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 1 or 2, characterized in that: The sensor sequences designed for SMN, GFP and WPRE are as follows: SMN sensor1 sequence: 5'-tatgcttttatcagtgctgtatcatcccaaatgtcagaatcatcgctctAgcctgtgccgcgccggaacagcacggaatcctcctgctccgggacgccg-3' SMN sensor2 sequence: 5'-ttaacatacttcccaaagcatcagcatcatcaagagaatctggacatatAggaggtggtgggggaattattggtggtccagaaggaaatggaggcagcc-3' GFP sensor1 sequence: 5'-gtggcatcgccctcgccctcgccggacacgctgaacttgtAgccgtttacgtcgccgtccagctcgaccaggatgggcaccaccccggtg-3' GFP sensor2 sequence: 5'-tggtcggcgagctgcacgctgccgtcctcgatgttgtAgcggatcttgaagttcaccttgatgccgttcttctgcttgtcggcc-3' GFP sensor3 sequence: 5'-cggcggcggtcacgaactccagcaggaccatgtgatcgcgcttctcgttAgggtctttgctcagggcggactgggtgctcaggtagtggttgtcgggca-3' WPRE sensor1 sequence: 5'-gatttatacaaggaggagaaaatgaaagccatacgggaagcaatagcatAatacaaaggcattaaagcagcgtatccacatagcgtaaaaggagcaaca -3' WPRE sensor2 sequence: 5'-ccaacagccgagcccctgtccagcagcgggcaaggcaggcggcgatAagttccgccgtggcaagaactaaccaggatttatacaaggaggagaaaa-3'.
4. A method for determining the transcriptional activity of a target gene based on RNA editing according to claim 1 or 2, characterized in that: The initial cells for constructing sensor reporter gene cells are cells with high expression of ADAR.
5. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 4, characterized in that: The cells with high ADAR expression are 293 or 293T cells.
6. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 1, 2 or 3, characterized in that: The quantitative method of target gene transcription activity is: The concentration or titer of the sample to be tested is taken as the dose, and the ratio of the main reporter gene signal to the internal reference reporter gene signal is taken as the response. The four-parameter Logistic model is used for curve fitting, and the parallel line method is used to calculate the percentage of the sample to be tested relative to the reference.
7. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 4, characterized in that: The quantitative method of target gene transcription activity is: The concentration or titer of the sample to be tested is taken as the dose, and the ratio of the main reporter gene signal to the internal reference reporter gene signal is taken as the response. The four-parameter Logistic model is used for curve fitting, and the parallel line method is used to calculate the percentage of the sample to be tested relative to the reference.
8. A method for measuring the transcriptional activity of a target gene based on RNA editing according to claim 5, characterized in that: The quantitative method of target gene transcription activity is: The concentration or titer of the sample to be tested is taken as the dose, and the ratio of the main reporter gene signal to the internal reference reporter gene signal is taken as the response. The four-parameter Logistic model is used for curve fitting, and the parallel line method is used to calculate the percentage of the sample to be tested relative to the reference.
9. The method according to any one of claims 1 to 8 is used for determining the transcriptional activity of the target gene of a gene therapy product.