Regulatory element capable of regulating gene expression, and use

Abstract

Provided are a regulatory element capable of regulating gene expression, and a use. The sequence of the regulatory element is any one or more of sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. By means of innovative bidirectional symmetrical design, the regulatory element can function in two directions at the same time. The regulatory element can be applied to lentiviral vectors, can prevent enhancers / promoters carried by the vectors from affecting downstream genes and prevent uncontrolled and unintended expression caused by enhancers / promoters of host genes at insertion sites, and thus can improve the safety of vector applications.

Description

Regulatory element capable of regulating gene expression and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a regulatory element capable of regulating gene expression and application. BACKGROUND

[0002] Gene therapy is mainly used for the treatment of tumors or genetic diseases. The concept of gene therapy was born in the 1970s. The principle is to change the biological characteristics of cells by modifying or manipulating the expression of genes, so as to improve or treat diseases. In the early stage of gene therapy, gene delivery was a major challenge. Until the invention of viral vectors in the 1980s, they were used as effective tools to introduce foreign genes into human cells. In the 1990s, it began to gradually enter the clinical stage. Up to now, there are many gene therapy products on the market as drugs.

[0003] With the rapid development of the field of gene therapy, its safety as an issue that cannot be ignored has gradually attracted the attention of regulatory departments and clinicians. In 1999, an 18-year-old teenager died of a severe inflammatory reaction to the adenovirus vector used. The following year, in 2000, the use of gamma retrovirus vectors to treat X-linked severe combined immunodeficiency (SCID-X1) led to T-cell leukemia in nearly half of the patients. These setbacks announced the entry of gene therapy into a "dark age". Although in the past 20 years, viral vectors have been further improved by scientists, greatly improving safety, but there is still a great risk. On January 19, 2024, the US Food and Drug Administration issued a notice requiring all CAR-T cell therapy products that have been approved to update their product labels, adding "about the possibility of secondary T lymphoma after treatment" to the black box warning. This safety label change notice is not sudden. Previously, the FDA has mentioned in several announcements that CAR-T cell therapy products targeting BCMA or CD19 have potential risks. On July 9, 2023, in the FDA's "Potential Signals of Serious Risks Identified by the FDA Adverse Event Reporting System (2023 July-September)", it was reported that all 6 products that have been approved for marketing have occurred after treatment T lymphoma events, and the necessity of taking regulatory action is being evaluated, which suggests that gene therapy using viral vectors may have risks, and the safety risk may come from the risk of gene integration, that is, the integration of foreign genes into the host genome may induce the activation of oncogenes, etc.

[0004] The above problems suggest that the safety of the vector is the cornerstone of gene therapy. In recent years, the viral vectors used in the field of gene therapy mainly include adenovirus vectors, vaccinia virus vectors, herpes simplex virus vectors, adeno-associated virus vectors, lentivirus vectors, and retrovirus vectors, all of which have the risk of gene integration. The main safety problem of most viral vectors is that the promoter carried by the vector or its own enhancer activates the oncogene or suppresses the tumor suppressor gene in the host genome, causing uncontrolled cell proliferation and ultimately leading to tumor formation. For example, retrovirus vectors such as gamma retrovirus (GV) vectors and lentivirus (LV) vectors have a powerful enhancer in the U3 region of their long terminal repeat (LTR). Due to the non-random integration of the transcription start site of the proto-oncogene in the host cell, this can cause activation of the proto-oncogene. This is also known as insertional activation (Maruggi et al., Mol. Ther. 17: 851-6, 2009). Non-viral DNA-mediated vectors such as transposon vectors and plasmid delivery vectors also have the safety risk of integration mutation.

[0005] The integration of the vector-delivered gene into the host genome is a random insertion, which can be inserted into the CDS region of the host genome. The inserted sequence is affected by the enhancer / promoter in the host gene, and uncontrolled unintended expression also poses a safety risk.

[0006] In addition, the overexpression of the delivered gene can have a significant impact on the therapeutic effect and even safety. Therefore, in recent years, scientists have added elements for regulating and silencing gene expression to the vector to increase the safety effect.

[0007] Therefore, in recent years, scientists have added insulator sequences to the vector used for gene therapy, which can block the enhancer and act as a heterochromatin barrier through its two features (Wang et al., Chin. J. Biochem. Mol. Biol. 2011, Vol. 27, Issue (6), 493-498), increasing the safety and long-term maintenance of the therapeutic effect of the gene therapy vector.

[0008] Insulators are a class of boundary DNA sequences composed of about several hundred base pairs (bp), which are cis-acting elements that can establish independent transcriptionally active domains within the genome and prevent adjacent promoters, enhancers, or silencers from regulating the genes defined by them.

[0009] Insulators are a class of boundary DNA sequences composed of about several hundred base pairs (bp), which are cis-acting elements that can establish independent transcriptionally active domains within the genome and prevent adjacent promoters, enhancers, or silencers from regulating the genes defined by them.

[0010] The first identified insulator of vertebrate genes is the 5' boundary sequence of chicken beta-globin locus 5'HS4, the core of which is a 250bp GC-rich sequence that can effectively block enhancer activity. With further research, scientists have found that the insulator sequence can block the activity of enhancers and protect downstream sequences from abnormal activation.

[0011] However, the function of the insulator has a polarity feature, that is, the function of the insulator is directional, and only the upstream promoter / enhancer or silencer of the insulator sequence can be inhibited, and the downstream sequence cannot be regulated. That is, when a functional insulator is placed in reverse, it has no function.

[0012] The prior art shows that the insulator works by blocking the interaction between the adjacent regulatory elements and the promoter they regulate. The work of Cai and Levine et al. (1995, Nature, 376:533-536) proves that the insulator inhibits the function of the enhancer with a polarity feature, that is, only the upstream enhancer of the insulator can be inhibited, and the downstream chromosomal domain enhancer has no effect. That is, the function of the insulator is directional, and only the upstream promoter / enhancer of the insulator sequence can be inhibited, and the downstream sequence cannot be regulated, and can only function in one direction, and cannot simultaneously regulate gene expression and protect genes from external regulation.

[0013] Most of the insulator sequences that can prevent or regulate gene expression are more than 100bp in length, and the capacity and modification degree of the vector used for gene therapy are very limited. The modification and addition of too long an insulator sequence to the vector will affect the packaging and production of the viral vector, and cannot be widely used in the field of gene therapy. The work of Naoya Uchida et al. (2011, Molecular Therapy, 19, 133-139) proves that the placement of 1200bp, 500bp and 250bp length insulators in viral vectors has different degrees of influence on the packaging of viral vectors, and greatly reduces the titer of viral vectors. The work of Olivier Negre et al. (2016, Human Gene Therapy, 27, 2) also proves that the insulator significantly reduces the titer of viral vectors in the clinical stage, resulting in the inability of the insulator to be widely used. Due to the difficulty in screening and constructing the insulator, the currently available insulator elements have a single function and cannot moderately regulate gene expression. The function of the insulator is directional, and only the upstream promoter / enhancer of the insulator sequence can be inhibited, and the downstream sequence cannot be regulated. SUMMARY

[0014] Therefore, in order to solve the above technical problems, the present application provides an element capable of regulating gene expression and application.

[0015] The technical solution provided by the present application is as follows:

[0016] <the first aspect>

[0017] A regulatory element capable of regulating gene expression, the sequence of the regulatory element being any one of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6.

[0018] The length of the regulatory element is not more than 100 bp, and the regulatory element is centrally symmetric (the regulatory sequence is centrally symmetric on the DNA double-stranded structure).

[0019] The regulatory element has the effect of gradient regulating gene expression, regulatory element 1 (SEQ ID NO. 1) can prevent more than 90% of gene expression, regulatory element 2 (SEQ ID NO. 2) can prevent 70-80% of gene expression, regulatory element 3 (SEQ ID NO. 3) can prevent 60-70% of gene expression, and regulatory element 4 (SEQ ID NO. 4) can prevent 40-60% of gene expression.

[0020] The regulatory element can affect / hinder the function of the promoter to realize the gene expression.

[0021] As an embodiment of the present application, the present application provides a regulatory element capable of gradient regulating gene expression, the sequence of the regulatory element being any one of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6.

[0022] The regulatory element can bidirectionally inhibit enhancer / promoter activity, and the inhibition efficiency is adjustable. Among them, regulatory element 1 (SEQ ID NO. 1) can prevent more than 90% of gene expression, regulatory element 2 (SEQ ID NO. 2) can prevent 70-80% of gene expression, regulatory element 3 (SEQ ID NO. 3) can prevent 60-70% of gene expression, and regulatory element 4 (SEQ ID NO. 4) can prevent 40-60% of gene expression.

[0023] <the second aspect>

[0024] A recombinant plasmid comprising the above-mentioned regulatory element.

[0025] The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a viral vector; or, is obtained by integrating the regulatory element into a non-viral vector transposon vector or plasmid delivery vector.

[0026] The viral vector includes one or several of a lentiviral vector, a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0027] As an embodiment of the present application, the recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a lentiviral vector, and the packaging master plasmid of the lentiviral vector includes one of pCCL, pRRL, pRLL, pCLL, pLKO.1, pLVX, pCDH, pSIN, pLenti, pWPI, pFUGW, pLL3.7, pGIPZ, and pSicoR series plasmid vectors; or,

[0028] The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a retroviral vector, and the packaging master plasmid of the retroviral vector includes one of pBABE, pMX, and pMSCV; or,

[0029] The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adenoviral vector, and the packaging master plasmid of the adenoviral vector includes one of pAdEasy, pShuttle, pAdTrack-CMV, pAdBam, pAdΔE1, pAd / CMV / V5-DEST, pAd5F35, and pAdlox series plasmid vectors; or,

[0030] The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adeno-associated viral vector, and the packaging master plasmid of the adeno-associated viral vector includes pAAV series vectors; or,

[0031] The recombinant plasmid is obtained by integrating the regulatory element into a transposon vector plasmid, and the transposon vector plasmid includes pT2 series (Sleeping Beauty (SB) transposon system), pSB series (Sleeping Beauty (SB) transposon system), pPB series (PiggyBac (PB) transposon system), pPB-CAG series (PiggyBac (PB) transposon system), pTol2 series (Tol2 transposon system), pMi series (Minos transposon system), and pMar series (Tc1 / mariner transposon system) plasmid vectors; or,

[0032] The recombinant plasmid is obtained by integrating the regulatory element into a plasmid delivery vector, and the plasmid delivery vector is a delivery vector using a plasmid as a medium for expressing a target gene.

[0033] The insertion site of the regulatory element in the recombinant plasmid is between the promoter delivered by the vector and the target gene.

[0034] The promoter includes one or more of PGK promoter, CAG promoter, and EF1a promoter.

[0035] As an embodiment of the present application, the insertion site of the regulatory element in the recombinant plasmid is upstream or / and downstream of the expression frame of the target gene delivered by the vector.

[0036] In the lentiviral vector or retroviral vector, the insertion site of the regulatory element can be in the LTR region.

[0037] <Third aspect>

[0038] A construction method of a recombinant plasmid, comprising the following steps:

[0039] Step 1, the non-viral vector transposon vector or plasmid delivery vector is subjected to restriction enzyme digestion to obtain a linearized vector fragment;

[0040] Step 2, the regulatory element of claim 1 is synthesized into an oligonucleotide chain with an enzyme digestion site, annealed into double-stranded DNA, and then subjected to enzyme digestion;

[0041] Step 3, the enzyme-digested regulatory element and the vector fragment are connected by a ligase;

[0042] Step 4, the ligation product is subjected to competent transformation and monoclonal sequencing verification; and the correct one is the recombinant plasmid.

[0043] Step 1 is specifically: the lentiviral vector packaging master plasmid pCCL-PGK-eGFP is subjected to restriction enzyme digestion (such as BamH I digestion) at the BamH I site to obtain a linearized vector fragment.

[0044] Step 2 is specifically: the single-stranded DNA of the regulatory element is annealed into a double-stranded sequence with a restriction enzyme digestion site and subjected to restriction enzyme digestion (such as BamH I digestion).

[0045] In step 3, the ligase includes T4 DNA ligase.

[0046] As an embodiment of the present application, the construction method of the recombinant plasmid comprises the following steps:

[0047] Step 1, the lentiviral vector packaging master plasmid pCCL-PGK-eGFP is subjected to restriction enzyme digestion (such as BamH I digestion) at the BamH I site to obtain a linearized vector fragment;

[0048] Step 2, annealing the single-stranded DNA (containing complementary strands) of the regulatory element as described above to form double-stranded DNA with a BamH I enzyme cutting site sequence and cutting using BamH I enzyme;

[0049] Step 3, connecting the regulatory element into the linearized lentivirus packaging master plasmid pCCL-PGK-eGFP through T4 DNA ligase;

[0050] Step 4, verifying the ligation product by using competent transformation and selecting single clones for sequencing;

[0051] Step 5, extracting the plasmid of the correct clone to obtain the recombinant plasmid.

[0052] The construction method of the recombinant plasmid can also use the commonly used cloning construction method in the art, including but not limited to the method of gene synthesis, restriction enzyme cutting and ligation, and homologous recombination connection.

[0053] <Fourth aspect>

[0054] The application of the regulatory element in the preparation of drugs for treating alpha-thalassemia, beta-thalassemia, macula, Parkinson also belongs to the protection scope of the present application.

[0055] Compared with the prior art, the present application has the following beneficial effects:

[0056] 1. The present application has the beneficial effects of preventing the enhancer / promoter carried by the vector from affecting the inserted host gene and preventing the enhancer / promoter of the inserted host gene from producing uncontrolled and unintended expression, thereby increasing the safety of the vector application.

[0057] 2、The application found in the research process, and not any bidirectional symmetry of regulatory elements can meet the requirements, bidirectional design of regulatory elements belong to the generalized palindrome structure, palindrome structure may terminate transcription, thereby causing false positive results, even in the virus packaging process to terminate the reverse transcription of viral RNA and thus lead to normal packaging. In order to solve these problems, the present application in the test of different length (<100bp) of bidirectional regulatory elements, first, these different length of regulatory elements do not affect the normal packaging of viral vectors; second, further research found that the regulatory strength of short regulatory elements in (<100bp) range and its specific length is not related, thereby proving that the bidirectional design of insulator is a generalized palindrome sequence, but it has a unique function, and is not achieved by forming a hairpin structure; further research found that not any (<100bp) of short regulatory elements can well play the purpose of bidirectional regulation of gene expression, by comparing research found that the regulatory elements (such as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6) have good effect. And regulatory elements 1, 2, 3 and 4 (corresponding to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4) have a gradient effect of regulatory effect. The effect of gradient regulation of gene expression is that regulatory element 1 (SEQ ID NO. 1) can prevent more than 90% of gene expression, regulatory element 2 (SEQ ID NO. 2) can prevent 70-80% of gene expression, regulatory element 3 (SEQ ID NO. 3) can prevent 60-70% of gene expression, and regulatory element 4 (SEQ ID NO. 4) can prevent 40-60% of gene expression.

[0058] 3、The length of the regulatory element of the present application is not more than 100bp, which solves the problem of long regulatory element length that cannot be used for vector modification, and can be effectively used for modification of viral vectors without affecting the normal therapeutic effect.

[0059] 4、The present application obtains regulatory elements of different intensities by screening, and different regulatory elements have different inhibitory activities on transcription, which solves the problem of single function of existing insulators and realizes different degrees of regulation of transcription intensity, which can effectively control the expression intensity of genes, so that the expression degree of the required protein is maintained. BRIEF DESCRIPTION OF DRAWINGS

[0060] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments with reference to the following drawings:

[0061] Figure 1 is a bidirectional design of the regulatory element of the present application;

[0062] Figure 2 is a comparison chart of green fluorescence expression after 293T cells are transiently transfected with plasmids carrying control regulatory elements;

[0063] Figure 3 is a comparison chart of green fluorescence expression after 293T cells are transiently transfected with plasmids carrying different lengths of unidirectional regulatory elements;

[0064] Figure 4 is a comparison chart of green fluorescence expression after 293T cells are transiently transfected with plasmids carrying different lengths of bidirectional regulatory elements;

[0065] Figure 5 is a comparison chart of green fluorescence expression after 293T cells are transiently transfected with plasmids carrying different lengths of bidirectional regulatory elements after further design;

[0066] Figure 6 is the titer detection result after plasmids carrying different regulatory elements are packaged into lentiviral vectors;

[0067] Figure 7 is the regulatory expression effect of different regulatory elements on green fluorescent protein eGFP; wherein A is the proportion of GFP positive cells; and B is the relative fluorescence intensity;

[0068] Figure 8 is the application of the regulatory element in the lentiviral vector when the regulatory element is placed in the forward direction;

[0069] Figure 9 is the application of the regulatory element in the lentiviral vector when the regulatory element is placed in the reverse direction;

[0070] Figure 10 is the application of the regulatory element in the lentiviral vector without directionality;

[0071] Figure 11 is a regulatory effect chart of the regulatory element of Example 4 under the control of different promoters;

[0072] Figure 12 is a regulatory effect chart of the regulatory element of Example 5 under the control of different promoters after the regulatory element is applied to the viral vector. DETAILED DESCRIPTION

[0073] The present application will be described in detail below with reference to examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0074] The present application constructs multiple regulatory elements with bidirectional function through innovative bidirectional symmetric design, and clones the regulatory elements to the vector. After gene transduction to cells, the function is verified by the signal strength of the downstream gene green fluorescent protein, and multiple regulatory elements with bidirectional expression regulation are screened.

[0075] In the following examples and comparative examples:

[0076] Table 1

[0077] Example 1 Construction of plasmid vector containing regulatory element

[0078] 1. Regulatory element design:

[0079] Since DNA sequence has 5' to 3' directionality, in order to solve the limitation that insulator can only play a one-way role in the prior art, a bidirectional symmetric scheme is designed for the regulatory element, as shown in Figure 1 (where 1 is a one-way regulatory element (traditional insulator) effect verification model; 2 is a one-way regulatory element reverse complementary sequence effect verification model; 3 is a bidirectional regulatory element of the present application), the second half of the sequence of the regulatory element is the reverse complementary sequence of the first half, so that the sequence of the two directions (sense strand, antisense strand, 5'-3') of the regulatory element is completely identical, which can play a bidirectional regulation effect.

[0080] Construction of plasmid vector containing regulatory element and control regulatory element plasmid vector.

[0081] The design of the regulatory element sequence is shown in Table 2, which contains single and bidirectional sequences, and the following single-stranded DNA of the regulatory element is synthesized according to the sequence shown in Table 2. The sequence of the 36bp insulator regulatory element for control is 5'-AAGGGAGACATCTAGTGATATAAGTGTGAACTACAC-3'(forward one-way, SEQ ID No. 17) and its reverse complementary sequence 5'-GTGTAGTTCACACTTATATCACTAGATGTCTCCCTT-3'(reverse one-way, SEQ ID No. 18). When synthesizing, 4bp protection base and 6bp BamHI enzyme cutting site sequence (5'-TAGCggatcc-3') are added at 5'-end of each sequence, and 6bp BamHI enzyme cutting site and 4bp protection base sequence (5'-ggatccGCTA-3') are added at 3'-end of the sequence. When synthesizing the oligonucleotide chain, the regulatory element of the single direction needs to synthesize the antisense oligonucleotide chain at the same time for complementary pairing into double-stranded DNA for plasmid construction.

[0082] Table 2

[0083] 3. Single-stranded DNA annealing

[0084] After dissolving each single-stranded DNA sequence of the regulatory element to a concentration of 100 μM with ultrapure water, each single-stranded DNA sequence is annealed under the following conditions to form a double-stranded DNA fragment:

[0085] Table 3

[0086] The substances and amounts in Table 3 were added into a 1.5 mL centrifuge tube, mixed well, and placed in a water bath at 95°C for 5 minutes. Then the water bath was turned off and the tube was allowed to cool naturally. The annealing was completed.

[0087] The double-stranded DNA fragments of each regulatory element formed by annealing were subjected to agarose gel electrophoresis. The target bands were cut and recovered using a DNA product purification kit (Tiangen; DP204-3).

[0088] 4. Vector and annealing product digestion

[0089] 4.1 Vector digestion

[0090] The green fluorescent protein-expressible lentiviral vector packaging master plasmid pCCL-PGK-eGFP was treated with restriction enzymes under the following conditions.

[0091] Table 4

[0092] The substances and amounts in Table 4 were added into a 1.5 mL EP tube, mixed well, and subjected to enzyme digestion at 37°C for 2 hours. The digested vector was subjected to agarose gel electrophoresis. The target bands were cut and recovered using a DNA product purification kit (Tiangen; DP204-3).

[0093] 4.2 Annealing product digestion

[0094] The double-stranded DNA fragments of each regulatory element after annealing were treated with restriction enzymes under the conditions in Table 2.

[0095] Table 5

[0096] The substances and amounts in Table 5 were added into a 1.5 mL EP tube, mixed well, and subjected to enzyme digestion at 37°C for 2 hours. The digested double-stranded DNA fragments were subjected to agarose gel electrophoresis. The target bands were cut and recovered using a DNA product purification kit (Tiangen; DP204-3).

[0097] 5. Connection of plasmid vector and regulatory element DNA fragments

[0098] The double-stranded DNA fragments of each regulatory element after enzyme digestion and purification in step 4.2 were connected to the vector after enzyme digestion in step 4.2 using T4 DNA ligase under the conditions in Table 6.

[0099] Table 6

[0100] The substances and amounts in Table 4 were added to a 1.5 mL centrifuge tube, mixed and left at room temperature overnight to obtain recombinant vectors carrying different regulatory elements as shown in Table 7:

[0101] Table 7

[0102] 6. Monoclonal screening

[0103] Each recombinant plasmid was added to 100 μL competent cells (Dali Life Technology; DL1030), and after 30 minutes of ice bath, 42°C water bath heat shock for 45 seconds, quickly placed on ice for 2 minutes, then added to non-resistant LB medium, 37°C, 200 rpm recovery for 60 minutes, spread on plates and cultured overnight. After 14-18 hours, single colonies were picked, amplified and cultured for sequencing verification. Sequencing primer: 5'-actagtctcgtgcagatgga-3'.

[0104] Example 2 Verification of the effect of plasmid vectors containing regulatory elements on expression and screening of vectors

[0105] The plasmid vectors containing regulatory elements constructed in Example 1 were transfected into 293T cell lines.

[0106] Using plasmids carrying different regulatory element sequences: pCCL-PGK-IN(M28F)-eGFP, pCCL-PGK-IN(L30F)-eGFP, pCCL-PGK-IN(R30F)-eGFP, pCCL-PGK-IN(L32F)-eGFP, pCCL-PGK-IN(R32F)-eGFP, pCCL-PGK-IN(L36)-eGFP, pCCL-PGK-IN(M36)-eGFP, pCCL-PGK-IN(R36)-eGFP, pCCL-PGK-IN(L56)-eGFP, pCCL-PGK-IN(M56)-eGFP, pCCL-PGK-IN(R56)-eGFP, pCCL-PGK-IN(L60)-eGFP, pCCL-PGK-IN(M60)-eGFP, pCCL-PGK-IN(R60)-eGFP, pCCL-PGK-IN(L64)-eGFP, pCCL-PGK-IN(R64)-eGFP, plasmids of control vectors pCCL-PGK-IN(36F)-eGFP (positive control of regulatory function, containing a forward insulator regulatory element), pCCL-PGK-IN(36R)-eGFP (reverse complementary sequence of the positive control regulatory element), and pCCL-PGK-eGFP (positive control of GFP expression) carrying no regulatory element were respectively transfected into 293T cells (4E5 cells / well were plated into 6-well plates 24 h before transfection of 1 μg of plasmid per well), and green fluorescence expression in the cells after transient transfection was observed 48 h later to characterize the effect of the insulator. The untransfected blank cells served as a no-fluorescence control (Blank).The results are shown in Figure 2. In the control group of regulatory elements, the regulatory element with a length of 36 bp has a strong effect on preventing gene expression (pCCL-PGK-(36F)-eGFP). When the reverse complementary sequence is inserted into the promoter and GFP gene, it is difficult to hinder the expression of the GFP gene, indicating that the reverse insulation effect of the insulator (pCCL-PGK-(36R)-eGFP) is weak, i.e., it cannot function as a bidirectional insulator. In the unidirectional regulatory element group (Figure 3), the plasmids containing shorter regulatory elements (≤32 bp) have a weak effect on inhibiting the expression of the GFP gene. Only pCCL-PGK-IN(R32F)-eGFP shows a slightly obvious inhibitory effect, and the effects of other regulatory elements are weak or none. In the regulatory elements with a central symmetric structure composed of short regulatory elements and their reverse complementary sequences (Figure 4), a stronger effect on inhibiting gene expression is shown. Among them, the regulatory elements in pCCL-PGK-IN(M56)-eGFP and pCCL-PGK-IN(R64)-eGFP have better effects, the regulatory element in pCCL-PGK-IN(L60)-eGFP has a second-best effect, and the effects of the other two regulatory elements are weak. The reverse complementary sequence of the regulatory sequence with a central symmetric structure is the same as the forward sequence, so it has a bidirectional regulatory effect. Therefore, the regulatory element with a central symmetric structure can not only strengthen the effect of inhibiting gene expression, but also has a bidirectional inhibitory effect. Therefore, in the screening of regulatory elements with a central symmetric structure, further screening of elements with an inhibitory effect on gene expression is performed (Figure 5). Among them, the regulatory element in pCCL-PGK-IN(R56)-eGFP has the best effect, the regulatory element in pCCL-PGK-IN(L36)-eGFP has a better effect, and the effects of the other regulatory elements such as pCCL-PGK-IN(R36)-eGFP are weak.

[0107] Example 3 verifies the application effect of the vector containing the regulatory element in the lentiviral vector

[0108] Using 3 helper plasmids pMD2.G: 9 ng, pMDLg-RRE: 7 ng, pRSV-REV: 15 ng, and the green fluorescent expression vector recombinant vector pCCL-PGK-IN(R56)-eGFP, pCCL-PGK-IN(R64)-eGFP, pCCL-PGK-IN(M56)-eGFP, and pCCL-PGK-IN(R36)-eGFP carrying different regulatory element sequences screened above, and the control group vector plasmids pCCL-PGK-IN(36F)-eGFP (positive control of regulatory function), pCCL-PGK-IN(36R)-eGFP (reverse sequence of the positive control regulatory element), and the vector plasmid pCCL-PGK-eGFP (GFP expression positive control) without carrying a regulatory element, 15 ng each, were used to transfect 293T cells (293T cells were plated in 15 cm dishes, 1.3E7 cells / dish, and the plasmids were transfected after 24 h) to package the lentiviral vector, and the cell supernatant was collected at 48 h and 72 h after transduction, and the lentiviral vector was collected using ultracentrifugation (25000 rpm, 2 h, 4℃). The lentiviral vectors obtained were LV-IN(R36)-eGFP, LV-IN(M56)-eGFP, LV-IN(R56)-eGFP, LV-IN(R64)-eGFP, LV-IN(36F)-eGFP, LV-IN(36R)-eGFP, and LV-eGFP (wherein each lentiviral vector contains the promoter PGK, which is located after the central poly-puromycin region (cPPT) of the lentiviral vector and before the regulatory element and green fluorescent protein gene; for example, the lentiviral vector is LV-PGK-IN(R36)-eGFP). The titer was detected (as shown in Table 8 and FIG. 6). The corresponding lentiviral vectors were used to infect 293T cells (293T cells were plated in 24-well plates, 1E5 cells / well), and cells not transduced with any lentiviral vector were used as a blank control (Blank), MOI = 2, and the expression proportion of green fluorescence and the relative fluorescence intensity were detected by flow cytometry at 48 h after transduction to characterize the effects of different regulatory elements. The lower the proportion of green fluorescent expression and the lower the relative fluorescence intensity, the stronger the effect of the regulatory element on preventing gene expression. The GFP positive cell ratio is shown in FIG. 7A and Table 9, and the relative fluorescence intensity is shown in FIG. 7B and Table 10. According to the data analysis in Tables 9 and 10, the four regulatory elements verified in this experiment have the effect of gradient regulation of gene expression. Regulatory element 1 (SEQ ID NO. 1) can prevent more than 90% of gene expression, regulatory element 2 (SEQ ID NO. 2) can prevent 70-80% of gene expression, regulatory element 3 (SEQ ID NO. 3) can prevent 60-70% of gene expression, and regulatory element 4 (SEQ ID NO. 4) can prevent 40-60% of gene expression.

[0109] Table 8

[0110] Table 9

[0111] Table 10

[0112] The traditional regulatory element effect verification model shown as 1 in FIG. 1 inserts the regulatory element between the PGK promoter and the target gene (eGFP), and the regulatory element can theoretically exert a regulatory effect and reduce the expression of the target gene to different degrees. Since the complementary sequence of the traditional regulatory element is not consistent with itself (2 in FIG. 1), it can only be insulated in one direction, while the regulatory element of the present application is centrosymmetric and has no directionality, and can exert an insulation effect in both directions (3 in FIG. 1).

[0113] This embodiment uses three lentivirus helper plasmids pMD2.G, pMDLg-RRE, and pRSV-REV, and a main plasmid carrying a regulatory element to package a lentivirus vector carrying a regulatory element: a main plasmid carrying a target gene expression frame is synthesized by whole gene synthesis, and the regulatory element is designed in the U3 region of the 3' end long terminal repeat sequence of the plasmid vector. The required helper plasmid and the above-mentioned main plasmid are co-transfected into HEK293T cells using a lentivirus vector to package the lentivirus vector. After the lentivirus vector is transduced into HEK293T cells, it is integrated after reverse transcription, at which time it is converted into a provirus. During this process, the 3' end long terminal repeat sequence will be replicated to the 5' end long terminal repeat sequence of the viral vector through its unique "jumps" feature, so that the target gene expression frame delivered in the provirus has regulatory element sequences at both ends.

[0114] After the viral vector carrying a one-way regulatory element is integrated into the host cell genome, the regulatory elements carried at both ends can only function in a single direction, and can only prevent abnormal activation of downstream non-target genes or resist chromatin heterogeneity. After the viral vector carrying a bidirectional regulatory element is integrated into the host cell genome, it can function in both directions, and can prevent abnormal activation of upstream and downstream non-target genes, and can also prevent the upstream and downstream promoter / enhancer signals from activating the inserted genes to produce unintended expression.

[0115] As shown in Figure 8, the disclosed 36bp insulator regulatory sequence with directionality (5'-AAGGGAGACATCTAGTGATATAAGTGTGAACTACAC-3', SEQ ID No. 17) is placed in the 3' end LTR-U3 region of the lentiviral vector production master plasmid. After the lentiviral vector is transduced into a provirus, the regulatory element at the 3' end can only prevent the abnormal activation of the downstream gene by the promoter carried by the vector itself, and cannot resist the influence of the downstream promoter on the genes carried by the vector. The 5' end regulatory element can resist the influence of the upstream promoter on the genes carried by the vector, and cannot prevent the abnormal activation of the upstream gene by the promoter carried by the vector itself.

[0116] As shown in Figure 9, when the disclosed 36bp regulatory sequence with directional insulator is reversed (5'-GTGTAGTTCACACTTATATCACTAGATGTCTCCCTT-3', SEQ ID No. 18) and placed in the LTR-U3 region, the direction of the regulatory element at the 3' end of the provirus is reversed (opposite to the direction shown in Figure 8). At this time, the regulatory element at the 3' end can only resist the influence of the downstream promoter on the genes carried by the vector, and cannot prevent the abnormal activation of the downstream gene by the promoter carried by the vector itself. The 5' end regulatory element can prevent the abnormal activation of the upstream gene by the promoter carried by the vector itself, and cannot resist the influence of the upstream promoter on the genes carried by the vector.

[0117] As shown in Figure 10, when the regulatory element with central symmetry structure without directionality is placed in the 3' end LTR-U3 region of the lentiviral vector production master plasmid, the lentiviral vector is transduced into a provirus. At this time, the regulatory elements at both ends of the provirus can exert the function in both directions, i.e. the regulatory element at the 3' end can prevent the abnormal activation of the downstream gene by the promoter carried by the vector itself, and can also resist the influence of the downstream promoter on the genes carried by the vector. The 5' end regulatory element can prevent the abnormal activation of the upstream gene by the promoter carried by the vector itself, and can also resist the influence of the upstream promoter on the genes carried by the vector.

[0118] Example 4 Verification of the regulatory function of the regulatory element on different promoters

[0119] By using two enzyme digestion sites of PshAI and BamHI, the PGK promoter of the three plasmids pCCL-PGK-IN(R56)-eGFP, pCCL-PGK-IN(M56)-eGFP, pCCL-PGK-IN(36R)-eGFP containing regulatory element 1, regulatory element 3 and regulatory function positive control is replaced by CAG promoter and EF1a promoter, respectively, to form new plasmids as shown in Table 11 (the construction method is referred to Example 1).

[0120] Table 11 Different promoter plasmid tables

[0121] Wherein:

[0122] The sequence of the PGK promoter is: see CN 114672515 A

[0123] The sequence of the CAG promoter is: SEQ ID No. 19

[0124] The sequence of the EF1a promoter is: SEQ ID No. 20

[0125] The different promoter plasmid vectors containing regulatory elements constructed in Table 11 were transfected into 293T cell lines (Reference Example 2).

[0126] Using plasmids carrying different regulatory element sequences: pCCL-PGK-IN(R56)-eGFP, pCCL-CAG-IN(R56)-eGFP, pCCL-EF1a-IN(R56)-eGFP, pCCL-PGK-IN(M56)-eGFP, pCCL-CAG-IN(M56)-eGFP, pCCL-EF1a-IN(M56)-eGFP, and control vector plasmids: pCCL-PGK-IN(36R)-eGFP, pCCL-CAG-IN(36R)-eGFP, pCCL-EF1a-IN(36R)-eGFP (positive control for regulatory function, containing a positive insulator regulatory element), and different promoter vector plasmids without carrying regulatory elements: pCCL-PGK-eGFP, pCCL-CAG-eGFP, pCCL-EF1a-eGFP (positive control for GFP expression) were transfected into 293T cells (4E5 cells / well were plated into a 6-well plate 24h before transfection of 1ug plasmid per well), and the green fluorescence expression in the cells after transient transfection was observed 48h later to characterize the effect of the regulatory element under different promoters. The lower the green fluorescence expression, the stronger the effect of the regulatory element on gene expression. Untransfected blank cells served as a no fluorescence control (Blank). The results are shown in Figure 11. The control regulatory element has a strong regulatory effect under the PGK promoter, with essentially no green fluorescence expression, but has essentially no regulatory effect under the CAG promoter and the EF1a promoter. Regulatory element 1 has a strong regulatory effect under the PGK promoter, the CAG promoter, and the EF1a promoter. Regulatory element 3 has a weak regulatory effect under the PGK promoter and the EF1a promoter, and essentially no regulatory effect under the CAG promoter.

[0127] Example 5 Verification of the function of the regulatory element under the control of different promoters after application to viral vectors

[0128] Using 3 helper plasmids pMD2.G: 9 ng, pMDLg-RRE: 7 ng, pRSV-REV: 15 ng, and the plasmids containing regulatory elements and different promoters constructed in Example 4: pCCL-PGK-IN(R56)-eGFP, pCCL-CAG-IN(R56)-eGFP, pCCL-EF1α-IN(R56)-eGFP, pCCL-PGK-IN(M56)-eGFP, pCCL-CAG-IN(M56)-eGFP, pCCL-EF1α-IN(M56)-eGFP, and control vector plasmids: pCCL-PGK-IN(36R)-eGFP, pCCL-CAG-IN(36R)-eGFP, pCCL-EF1α-IN(36R)-eGFP (regulatory function positive control, containing a positive insulator regulatory element), and different promoter vector plasmids pCCL-PGK-eGFP, pCCL-CAG-eGFP, pCCL-EF1α-eGFP (GFP expression positive control) were used at 15 ng, respectively, to co-transfect 293T cells (293T cells were plated in 15 cm dishes at 1.3E7 cells / dish, and the plasmids were transfected after 24 h). The supernatant was collected at 48 h and 72 h after transduction, and the lentiviral vectors were collected using ultracentrifugation (25000 rpm, 2 h, 4℃). Lentiviral vectors: LV-PGK-eGFP, LV-PGK-IN(R56)-eGFP, LV-PGK-IN(M56)-eGFP, LV-PGK-IN(36R)-eGFP, LV-CAG-eGFP, LV-CAG-IN(R56)-eGFP, LV-CAG-IN(M56)-eGFP, LV-CAG-IN(36R)-eGFP, LV-EF1α-eGFP, LV-EF1α-IN(R56)-eGFP, LV-EF1α-IN(M56)-eGFP, LV-EF1α-IN(36R)-eGFP were obtained. The titers were detected (as shown in Table 12)

[0129] Table 12

[0130] The 293T cells were infected with corresponding lentiviral vectors (the 293T cells were spread in 24-well plates, 1E5 cells per well), and the cells not transduced with any lentiviral vector were used as blank control (Blank), MOI=2, and the expression proportion of green fluorescence was detected by flow cytometry 48 hours after transduction to characterize the effects of different regulatory elements. The lower the expression proportion of green fluorescence, the stronger the effect of the regulatory element on preventing gene expression. The GFP positive cell ratio is shown in Figure 12. After the regulatory elements were applied to the viral vectors, for the PGK promoter, both the regulatory element 1 and the control regulatory element had good regulatory effects, and could inhibit more than 95% of the expression of the green fluorescent gene, and the regulatory element 3 had a weaker regulatory effect; for the CAG promoter, the regulatory element 1 had a good regulatory effect, and could inhibit more than 95% of the expression of the green fluorescent gene, and the control regulatory element only had a weak regulatory effect, and the regulatory element 3 had no regulatory effect; for the EF1a promoter, the regulatory element 1 had a good regulatory effect, and could inhibit more than 95% of the expression of the green fluorescent gene, and the control regulatory element had a weak regulatory effect, and the regulatory element 3 was better than the control regulatory element, and had a moderate regulatory effect.

[0131] The specific embodiments of the present application are described above. It needs to be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which does not affect the essential content of the present application.

Claims

1. A regulatory element capable of modulating gene expression, characterized in that, The sequence of the regulatory element is one or more of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.

6.

2. The regulatory element of claim 1, wherein, The regulatory element has the effect of gradient regulation of gene expression.

3. A recombinant plasmid comprising the regulatory element of claim 1 or 2.

4. The recombinant plasmid of claim 3, wherein, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a viral vector; or, integrating the regulatory element into a non-viral vector transposon vector; or, constructing a plasmid delivery vector.

5. The recombinant plasmid of claim 4, wherein, The viral vector comprises one or more of a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector.

6. The recombinant plasmid of claim 3, wherein, In the recombinant plasmid, the insertion site of the regulatory element is located between the promoter and the target gene delivered by the vector.

7. The recombinant plasmid of claim 3, wherein, In the recombinant plasmid, the insertion site of the regulatory element is located upstream or / and downstream of the expression frame of the target gene delivered by the vector.

8. The recombinant plasmid of claim 3, wherein, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a lentiviral vector or a retroviral vector; in the lentiviral vector or the retroviral vector, the insertion site of the regulatory element is located in the LTR region.

9. The recombinant plasmid of claim 3, wherein, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a lentiviral vector, and the packaging master plasmid of the lentiviral vector comprises one of pCCL, pRRL, pRLL, pCLL, pLKO.1, pLVX, pCDH, pSIN, pLenti, pWPI, pFUGW, pLL3.7, pGIPZ, pSicoR series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a retroviral vector; the packaging master plasmid of the retroviral vector comprises one of pBABE, pMX, pMSCV; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adenoviral vector; the packaging master plasmid of the adenoviral vector comprises one of pAdEasy, pShuttle, pAdTrack-CMV, pAdBam, pAdΔE1, pAd / CMV / V5-DEST, pAd5F35, pAdlox series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adeno-associated viral vector; the packaging master plasmid of the adeno-associated viral vector is pAAV series vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a transposon vector plasmid; the transposon vector plasmid comprises pT2 series, pSB series, pPB series, pPB-CAG series, pTol2 series, pMi series, pMar series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a plasmid delivery vector; the plasmid delivery vector is any plasmid used as a medium for delivery of the target gene.

10. A method of constructing a recombinant plasmid, characterized by, The method comprises the following steps: The sequence of the regulatory element is one or more of the sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.

6. The regulatory element has the effect of gradient regulation of gene expression.

3. A recombinant plasmid comprising the regulatory element of claim 1 or 2. The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a viral vector; or, integrating the regulatory element into a non-viral vector transposon vector; or, constructing a plasmid delivery vector. The viral vector comprises one or more of a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector. In the recombinant plasmid, the insertion site of the regulatory element is located between the promoter and the target gene delivered by the vector. In the recombinant plasmid, the insertion site of the regulatory element is located upstream or / and downstream of the expression frame of the target gene delivered by the vector. The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a lentiviral vector or a retroviral vector; in the lentiviral vector or the retroviral vector, the insertion site of the regulatory element is located in the LTR region. The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a lentiviral vector, and the packaging master plasmid of the lentiviral vector comprises one of pCCL, pRRL, pRLL, pCLL, pLKO.1, pLVX, pCDH, pSIN, pLenti, pWPI, pFUGW, pLL3.7, pGIPZ, pSicoR series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of a retroviral vector; the packaging master plasmid of the retroviral vector comprises one of pBABE, pMX, pMSCV; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adenoviral vector; the packaging master plasmid of the adenoviral vector comprises one of pAdEasy, pShuttle, pAdTrack-CMV, pAdBam, pAdΔE1, pAd / CMV / V5-DEST, pAd5F35, pAdlox series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a packaging master plasmid of an adeno-associated viral vector; the packaging master plasmid of the adeno-associated viral vector is pAAV series vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a transposon vector plasmid; the transposon vector plasmid comprises pT2 series, pSB series, pPB series, pPB-CAG series, pTol2 series, pMi series, pMar series plasmid vectors; or, The recombinant plasmid is obtained by integrating the regulatory element into a plasmid delivery vector; the plasmid delivery vector is any plasmid used as a medium for delivery of the target gene. The method comprises the following steps: Step 1, linearize the packaging master plasmid of the viral vector, or the transposon vector of the non-viral vector, or the plasmid delivery vector by restriction enzyme digestion; Step 2, synthesize the oligonucleotide chain added with the restriction enzyme site of the regulatory element of claim 1 or 2, anneal into double-stranded DNA and then digest; Step 3, ligate the digested regulatory element and the vector fragment; Step 4, use competent transformation for the ligation product and verify by single colony sequencing; the correct one is the recombinant plasmid.

11. Use of the regulatory element of claim 1, or the recombinant plasmid of any one of claims 3-9 in the preparation of a drug for treating alpha-thalassemia, beta-thalassemia, macula, Parkinson's disease, hemophilia, adrenoleukodystrophy, eczema, thrombocytopenia with immunodeficiency syndrome, chronic granulomatous disease, severe combined immunodeficiency, adenosine deaminase severe combined immunodeficiency or leukemia.

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