Expression cassette for reducing lentivirus integration risk and application and product thereof

Abstract

The invention provides an expression cassette for reducing lentivirus integration risk and application and a product thereof, and belongs to the technical field of gene engineering. According to the invention, further optimization is carried out based on the prior art, reverse assembly is carried out on an expression cassette, and optimization selection of a polyA sequence finds that the vector disclosed by the invention can improve the gene expression quantity of a single copy and realize the same expression effect with a relatively low lentivirus integration copy number, so that the risk caused by virus integration is further reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to an expression cassette for reducing the risk of lentivirus integration, and an application and product thereof. Background Art

[0002] β-thalassemia is one of the most common monogenic autosomal recessive inherited diseases in the world. It is caused by defects in the β-globin gene or its regulatory sequence, which leads to reduced or impaired synthesis of β-globin chains, and then causes an imbalance in the α / β-globin ratio and an inability to produce enough hemoglobin. The clinical manifestations of β-thalassemia are closely related to the degree of α / β-globin chain imbalance. Most patients with the β0 / β0 genotype and some patients with the βE / β0 genotype produce almost no β chains, often showing transfusion-dependent β-thalassemia, with particularly severe clinical symptoms, requiring >100 mL / kg of concentrated RBCs (red blood cells) or ≥8 transfusions per year. Moderate and severe β-thalassemia manifests as severe hypochromic microcytic anemia in infancy, with total Hb (hemoglobin) ≤7g / dL.

[0003] A Chinese patent with publication number CN114457119A discloses a β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA is designed so that its gene expression cassette is oriented in the same direction as the transcription direction of the viral packaging main plasmid promoter. Its cell transduction is superior to that of conventional reverse expression cassettes, and the overall gene expression level is comparable. Therefore, the protein expression level provided by a single viral copy may be further improved.

[0004] On this basis, there is still a lot of room for development to develop new expression vector forms to increase the expression level of β-globin in the expression vector, and even to form an expression vector technology platform to promote the expression of other target proteins. Summary of the invention

[0005] The purpose of the present invention is to further increase the protein expression of a single vector copy, thereby reducing the number of vector integration copies and reducing the potential risks brought by vector integration; continuously optimize the gene expression cassette to maintain the gene expression level unchanged when the vector copy number is reduced, thereby improving safety. It plays an important role in viral vectors in particular.

[0006] In summary, the present invention firstly describes a β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β described in the patent publication with publication number CN114457119A. A-T87Q -RbPA based on LCR2.7K-pHBB-CI-β A-T87QA reverse assembly was performed, and different ployA sequences were screened based on the reverse sequence to increase mRNA stability and promote β A-T87Q Express.

[0007] Furthermore, the optimization direction of the present invention lies in the connection order of each element, and unexpected technical effects are achieved through reverse assembly. Those skilled in the art can easily think of applying it to the expression of other proteins.

[0008] The present invention specifically provides the following technical solutions: (1) The present invention provides a reverse assembly vector and provides the choice of different polyA sequences. After constructing the clone, the virus is packaged and the effects of different polyA sequences on gene integration and expression levels are verified in hematopoietic stem cells.

[0009] (2) The present invention provides a method for reducing the copy number of viral vectors and increasing the expression level of genes with a single copy number, thereby reducing the vector integration copy number while maintaining similar gene expression levels, thereby reducing the risk of vector integration.

[0010] The carrier design principle of the present invention: Use pCCL-SIN-cPPT-MCS-RbPA backbone as vector (containing Figure 1 The expression cassette elements were sequentially adjusted to polyA-CDS β in the 5'-3' direction. AT87Q -chimeric intron-βglobin promoter-LCR2.7, replace the polyA sequence with different sequences (design scheme see Figure 2 The completed plasmid vectors were named pHBB-508B-bGHPA-RE, pHBB-508B-LTR-RE, pHBB-508B-βPA-RE, pHBB-508B-αPA-RE, pHBB-508B-SPA-RE, and pHBB-508B-W3SL-RE. The lentiviral vectors (LVV) were named 508B-RE-bGH-PA, 508B-RE-LTR, 508B-RE-βPA, 508B-RE-αPA, 508B-RE-SPA, and 508B-RE-W3SL.

[0011] In the present invention, "sequentially" and "order" have the same meaning, indicating that there is a sequential relationship between the connections of the elements on the carrier.

[0012] Based on the general understanding in the art, "β-globin", "β-globin", "β-globin", and "βglobin" have the same meaning; "α-globin", "α-globin", "α-globin", and "αglobin" have the same meaning.

[0013] Specifically, the present invention provides an expression vector containing a target protein expression cassette, characterized in that the expression vector includes gene fragment 1-gene fragment 2-gene fragment 3-gene fragment 4-gene fragment 5 in the order of 5'-3' direction; The gene fragment 1 is a polyA element pA; The gene fragment 2 is a CDS element for expressing the target protein; The gene segment 3 is a chimeric intron element E; The gene fragment 4 is the promoter element P of the target protein; The gene fragment 5 is the LCR2.7 regulatory sequence; the gene fragment 5 corresponds to Figure 2 For the specific selection of the HS4-HS3-HS2, those skilled in the art can replace it by conventional means.

[0014] Preferably, the target protein is selected from α-globin, β-globin CDS β AT87Q , any one of pyruvate kinases, and the amino acid sequences are SEQ ID NOs: 1-3 respectively.

[0015] Preferably, the gene fragment 2 has any one of the nucleotide sequences shown in SEQ ID NOs: 4-9, wherein SEQ ID NO: 4 is the wild-type sequence of the α-globin expression gene; SEQ ID NO: 5 is the codon-optimized sequence for expressing α-globin; SEQ ID NO: 6 is the codon-optimized sequence for expressing β-globin CDS β AT87Q SEQ ID NO: 7 is used to express β-globin CDS β AT87Q SEQ ID NO: 8 is the wild-type sequence expressing pyruvate kinase; SEQ ID NO: 9 is the codon-optimized sequence expressing pyruvate kinase.

[0016] Preferably, the gene fragment 4 is selected from any one of β-globin promoter S (β-globin promoter S), β-globin promoter L (β-globin promoter L), and α-globin promoter (α-globin promoter), and each has a nucleotide sequence as shown in SEQ ID NO: 10-12.

[0017] Preferably, the polyA element is selected from any one of bGH polyA, LTR polyA, β-globin polyA, α-globin polyA, Synthetic polyA, and Sv40 polyA. As an example, it is selected from any one of the following fragments: bGH polyA: 26-250 bp of SEQ ID NO.21; LTR polyA: 35bp-186bp of SEQ ID NO.23; β-globin polyA: 29 bp to 422 bp of SEQ ID NO. 25; α-globin polyA: 27bp-228bp of SEQ ID NO.38; Synthetic polyA: SEQ ID NO.39, 30 bp-166 bp; Sv40 polyA: 27bp-281bp of SEQ ID NO.31.

[0018] Preferably, the gene fragment 5 is selected from: LCR2.7k, LCR5.5k, LCR6.5k, and has a nucleotide sequence as shown in SEQ ID NO: 13-15.

[0019] Preferably, the gene fragment 3 is selected from any one of: a chimeric intron, MVM, HS2, IVS1, IVS1-2TF, IgG, and AID intron. As an example: the chimeric intron has a nucleotide sequence as shown in SEQ ID NO: 16; the MVM has a nucleotide sequence as shown in SEQ ID NO: 17; the HS2 has a nucleotide sequence as shown in SEQ ID NO: 18; the IVS1 has a nucleotide sequence as shown in SEQ ID NO: 19; and the IVS1-2TF has a nucleotide sequence as shown in SEQ ID NO: 20.

[0020] Preferably, the expression vector is a plasmid vector or a lentiviral vector.

[0021] More preferably, the expression vector is a lentiviral vector.

[0022] The present invention also provides a method for constructing the expression vector, which comprises inserting gene fragments 1-5 into the expression vector.

[0023] The present invention does not limit the specific preparation method or source of gene fragments 1-5. Therefore, whether the gene fragments are obtained by direct synthesis or amplification, or the gene fragments are obtained by other means, the method for constructing the vector described in the present invention is within the scope of protection of the present invention.

[0024] In the present invention, the "insertion" refers to the process of connecting the corresponding gene to the vector, which can be in the form of sequential insertion or non-sequential insertion, or technicians in this field can also choose to replace elements based on the existing plasmid backbone.

[0025] The carrier of the present invention may also include other basic elements not modified in the present invention, as known to those skilled in the art, such as Figure 2 Other elements except gene fragment 1 to gene fragment 5.

[0026] In some specific embodiments, the vector construction of the present invention uses pCCL-SIN-cPPT-MCS-RbPA as a plasmid backbone for reverse assembly and regulation including but not limited to polyA elements.

[0027] Accordingly, the present invention provides genetic engineering products related to the aforementioned expression vectors, including but not limited to: (1) a cell comprising the expression vector; (2) A lentivirus prepared by transfecting cells with the expression vector; etc.

[0028] The present invention also includes the use of the aforementioned expression vector or construction method or cell or lentivirus in the preparation of a drug for α / β-globin chain imbalance diseases; the α / β-globin chain imbalance may be a β-globin chain expression deficiency.

[0029] In some application scenarios, the drug may be a β-thalassemia drug.

[0030] Based on the above, the present invention also protects the drug itself.

[0031] At least the active ingredient of the drug is prepared by the expression vector, the construction method, the cell or the lentivirus.

[0032] The medicine may also include pharmaceutical excipients, which are also referred to as pharmaceutically acceptable excipients in the art.

[0033] Preferably, the drug can be an injection. In the preparation of the corresponding injection, a person skilled in the art can confirm suitable injection excipients by routine selection, and these excipients can be disclosed or undisclosed in the prior art. As an example, the excipients that can be selected by a person skilled in the art include, but are not limited to, any one or more of buffers, stabilizers, anticoagulants, and cryoprotectants (DMSO) of different concentrations.

[0034] The buffer includes, but is not limited to: any one or more of phosphate buffer, Tris buffer, and sodium chloride buffer; The stabilizer includes but is not limited to: any one or more of human albumin, sucrose, and magnesium chloride; The anticoagulants include but are not limited to bloxamer; The cryoprotectant includes but is not limited to DMSO.

[0035] Beneficial effects of the present invention: 1. The present invention optimizes the insertion method of each gene element and increases the expression amount of a single copy vector through a reversely assembled expression cassette.

[0036] 2. The present invention also reduces the average vector copy number of the lentivirus inserted into the genome, while reducing the risk of the lentivirus inserting into the genome.

[0037] Based on the above effects, the safety of viral vector applications in this field has been greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the 508B structure.

[0039] Figure 2 Schematic diagram of the structure of 508B-RE-pA.

[0040] Figure 3 This is the structure diagram of the plasmid pHBB-508B-bGHPA-RE in Example 1.

[0041] Figure 4 This is the structure diagram of the plasmid pHBB-508B-LTR-RE in Example 1.

[0042] Figure 5 This is the structure diagram of the plasmid pHBB-508B-βPA-RE in Example 1.

[0043] Figure 6 This is the structure diagram of the plasmid pHBB-508B-αPA-RE in Example 1.

[0044] Figure 7 This is the structure diagram of the plasmid pHBB-508B-SPA-RE in Example 1.

[0045] Figure 8 This is the structure diagram of the plasmid pHBB-508B-W3SL-RE in Example 1.

[0046] Fig. 9 This is a graph showing the results of lentiviral transduction titer detection in Example 1.

[0047] Fig.10 This is a graph showing the copy number detection results of the VCN vector in Example 1.

[0048] Figures 11A-11G This is a graph showing the RP-HPLC detection results of hematopoietic stem cells infected with the lentiviral vector after in vitro erythroid differentiation in Example 1. Figures 11A-11G For the detection system to automatically output pictures, some overlapping content does not affect the effect recognition, and the relevant content has been analyzed and explained in Example 1.

[0049] Fig.12 is Hbβ in Example 1 A-T87Q RP-HPLC statistical chart of percentage of HBA.

[0050] Fig.13 Provide Hbβ for the average number of viral vector copies in Example 1 A-T87Q RP-HPLC statistical chart of percentage of HBA.

[0051] Fig.14 This is a statistical chart comparing the results of positive and negative assembly virus detection after hematopoietic stem cell transduction in Example 1; this chart is used to compare 508B-RE-bGH-PA and 508B control, where A is the transduction titer comparison; B is the VCN vector copy number comparison; C is the Hbβ A-T87Q Comparison of percentage of HBA; D is the average number of copies of each viral vector providing Hbβ A-T87Q Comparison of percentage of HBA. DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0053] The information of some experimental materials used in the examples is shown in Table 1: Table 1 Experimental materials

[0054] Those skilled in the art know that the selection of specific experimental materials can be conventionally replaced according to the disclosure of the prior art, so the implementation of the technical solution of the present invention is not limited to the above types.

[0055] In the present invention, "plasmid vector", "plasmid" and "vector" have the same or similar meanings.

[0056] In the present invention, "Core Insulator" refers to a core insulator, which is used to prevent adjacent control elements from interfering with each other.

[0057] In the present invention, "promoter" refers to a promoter that initiates the transcription of a DNA sequence.

[0058] In the present invention, "Enhancer" refers to an enhancer that enhances the transcription of a DNA sequence.

[0059] In the present invention, "SV40 poly(A) signal" refers to the simian virus 40 polyadenylation signal, which increases the stability of mRNA.

[0060] In the present invention, "AmpR" refers to the ampicillin resistance gene, which is used for the selection of plasmids in Escherichia coli.

[0061] In the present invention, "KanR" refers to the kanamycin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0062] In the present invention, "ori" refers to the E. coli replication origin, which regulates the rolling circle replication of the entire plasmid in E. coli.

[0063] In the present invention, "f1 ori" refers to the f1 phage replication initiation site, which controls the replication of single-stranded DNA.

[0064] In the present invention, "LB" refers to Luria-Bertani medium.

[0065] In the present invention, "DMEM" refers to Dulbecco's Modified Eagle Medium.

[0066] In the present invention, "FBS" refers to fetal bovine serum.

[0067] In the present invention, "DPBS" refers to phosphate buffered saline.

[0068] In the present invention, "LVV" refers to a lentiviral vector.

[0069] In the present invention, "VCN" refers to the vector copy number In the present invention, XhoI enzyme, KpnI enzyme, EcoRI enzyme, sometimes abbreviated as XhoI, KpnI, EcoRI, those skilled in the art can understand that they represent the corresponding enzymes or restriction sites.

[0070] In the present invention, "RP-HPLC" refers to reverse phase high performance liquid chromatography.

[0071] In the present invention, "CDS" refers to a coding sequence.

[0072] In the present invention, “β A-T87Q ” refers to the mutation of codon 87 of β-globin from threonine to glutamine.

[0073] In the present invention, "overlap PCR" refers to overlapping extension polymerase chain reaction. As an example, the conditions of overlap PCR used in the embodiments of the present invention are: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 s, annealing at 58°C for 10 s, extension at 68°C for 40 s, and extension at 68°C for 2 min.

[0074] In the present invention, "qPCR" refers to real-time fluorescent quantitative polymerase chain reaction.

[0075] Unless otherwise specified, the gel recovery in the present invention is performed using the gel recovery kit in Table 1. Unless otherwise specified, the seamless cloning in the present invention is performed using the seamless cloning kit in Table 1.

[0076] Some plasmid maps in the present invention are produced by software, and the output format is retained. Some of the retained English contents are all contents that can be understood by those skilled in the art in combination with the content of the invention and the definitions of related terms.

[0077] As is well known to those skilled in the art, the process of constructing a vector involves operations such as enzyme cleavage, primer amplification, and enzyme coupling. Therefore, some of the fragments involved in the construction process may retain some non-functional fragments (including but not limited to fragments related to enzyme cleavage sites, primer related fragments, etc.) in most cases. Therefore, in the embodiments, some of the fragments are included in the target fragment, rather than being completely consistent with the target fragment, which can be understood by those skilled in the art.

[0078] Example 1 The specific steps include: 1. Plasmid vector construction The relevant information of the plasmid vector constructed in this example is shown in Table 2: Table 2 Plasmid vector information

[0079] The construction methods of each plasmid vector in Table 2 are as follows: 1. Construction of plasmid pHBB-508B-bGHPA-RE (sequence number 1): 1.1 Plasmid pCCL-SIN-cPPT-MCS-RbPA (refer to pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β in patent application number 202210373730.0) A-T87Q -RbPA) was double-digested with XhoI and KpnI and recovered on gel to obtain a linear vector with a size of 5507 bp.

[0080] 1.2 PCR amplification was performed using the scAAV-CBA-EGFP-bGHpolyA plasmid carrying bGH polyA as a template to obtain fragment 1-1, the sequence of which is SEQ ID NO.21, and the 26th to 250th bp are bGH polyA.

[0081] 1.3 Using the gene-synthesized p508B plasmid as a template, PCR amplification was performed to obtain fragment 1-2, the sequence of which is SEQ ID NO.22.

[0082] 1.4 The two fragments were then subjected to Overlap PCR to obtain the final fragment and the amplified product with a length of 4004 bp was recovered from the gel, which was fragment 1.

[0083] 1.5 The recovered linear vector was connected with fragment 1 using a seamless cloning kit, 10 μL system, and reacted at 50°C for 15 min.

[0084] 1.6 Transform the ligation product into competent E. coli TransStbl3, mix gently; place on ice for 20 minutes, heat shock at 42℃ for 45 seconds, and immediately place on ice for 2 minutes; add liquid LB without antibiotics, and culture at 37℃ with shaking for 60 minutes; discard part of the supernatant and evenly spread the bacterial solution on the LB agar plate containing ampicillin with a sterile spreader; culture at 37℃ with inversion for 16 hours. Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture at 37℃ with shaking for 16 hours.

[0085] 1.7 Use a plasmid extraction kit (TIANGEN Rapid Plasmid Extraction Kit) to extract the plasmid, pHBB-508B-bGHPA-RE plasmid. Perform double restriction digestion with XhoI and KpnI to identify the plasmid, and then perform sequencing to identify the plasmid.

[0086] Subsequent plasmid vector constructions were all constructed using pHBB-508B-bGHPA-RE (sequence number 1) as a template, replacing the polyA sequence with LTR polyA, β-globin polyA, α-globin polyA, synthetic polyA, or Sv40 polyA.

[0087] 2. Construction of plasmid pHBB-508B-LTR-RE (sequence number 2): 2.1 The pHBB-508B-bGHPA-RE plasmid was double-digested with XhoI and EcoRI, and gel-recovered to obtain a linear vector with a size of 5507 bp.

[0088] 2.2 PCR amplification was performed using pHBB-508B-bGHPA-RE plasmid as a template to obtain fragment 2-1, the sequence of which is SEQ ID NO.23; the 35 bp-186 bp is LTR polyA.

[0089] 2.3 The sequence of fragment 2-2 is SEQ ID NO.24.

[0090] 2.4 The two fragments were subjected to Overlap PCR to obtain the final fragment and the fragment of 741 bp in length was recovered by gel extraction.

[0091] 2.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0092] 2.6 Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0093] 2.7 Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16 hours; use a plasmid mini-extraction kit (TIANGEN rapid plasmid mini-extraction kit) to extract the plasmid pHBB-508B-LTR-RE. After double restriction digestion with XhoI and EcoRI, the plasmid was sequenced and identified.

[0094] 3. Construction of pHBB-508B-βPA-RE (sequence number 3) plasmid: 3.1 The plasmid pHBB-508B-bGHPA-RE (sequence number 1) was double-digested with XhoI and EcoRI, and gel-recovered to obtain a linear vector with a size of 5507 bp.

[0095] 3.2 PCR amplification was performed using pHBB-508B-bGHPA-RE plasmid as a template to obtain fragment 3-1, the sequence of which is SEQ ID NO.25; the 29 bp-422 bp is β-globin polyA.

[0096] 3.3 The sequence of fragment 3-2 is SEQ ID NO.26.

[0097] 3.4 The two fragments were subjected to Overlap PCR to obtain the final fragment and a fragment with a length of 906 bp was recovered from the gel.

[0098] 3.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0099] 3.6 Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0100] 3.7 Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16h; use a plasmid mini-extraction kit (TIANGEN rapid plasmid mini-extraction kit) to extract the plasmid. After double restriction digestion with XhoI and EcoRI, sequencing was performed to obtain pHBB-508B-βPA-RE plasmid.

[0101] 4. Construction of plasmid pHBB-508B-αPA-RE (sequence number 4): 4.1 The pHBB-508B-bGHPA-RE plasmid (sequence number 1) was double-digested with XhoI and EcoRI, and gel-recovered to obtain a linear vector with a size of 5507 bp.

[0102] 4.2 No template is required. 3UTR-119F (SEQ ID NO.33) and 3UTR-88R (SEQ ID NO.34) are directly used for PCR amplification and gel recovery to obtain fragment 4-1 with the sequence of SEQ ID NO.27.

[0103] 4.3 PCR was performed using pHBB-508B-bGHPA-RE plasmid as a template to obtain fragment 4-2, the sequence of which is SEQ ID NO.28.

[0104] 4.4 Overlap PCR was performed on the two fragments to obtain the final fragment and a fragment of 712 bp in length was recovered by gel electrophoresis, the sequence of which was SEQ ID NO.38 (the 27 bp-228 bp were α-globin polyA).

[0105] 4.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0106] 4.6 Transform the ligation product into competent E. coli TransStbl3 and mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0107] 4.7 Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16h; use a plasmid mini-extraction kit (TIANGEN rapid plasmid mini-extraction kit) to extract the plasmid. After double restriction digestion with XhoI and EcoRI, sequencing was performed to obtain pHBB-508B-αPA-RE plasmid.

[0108] 5. Construction of plasmid pHBB-508B-SPA-RE (sequence number 5): 5.1 The pHBB-508B-bGHPA-RE plasmid (sequence number 1) was double-digested with XhoI and EcoRI, and gel-recovered to obtain a linear vector with a size of 5507 bp.

[0109] 5.2 PCR amplification was performed using 3UTR-SPA-F (SEQ ID NO.35) and 3UTR-88R (SEQ ID NO.34) without template and gel recovery was performed to obtain fragment 5-1, the sequence of which is SEQ ID NO.29.

[0110] 5.3 PCR was performed using pHBB-508B-bGHPA-RE plasmid as a template to obtain fragment 5-2, the sequence of which is SEQ ID NO.30.

[0111] 5.4 Overlap PCR was performed on the two fragments to obtain the final fragment and a 650 bp fragment was recovered by gel electrophoresis. The sequence was SEQ ID NO.39, and the 30 bp to 166 bp fragment was synthetic poly A.

[0112] 5.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0113] 5.6 Transform the ligation product into competent E. coli TransStbl3, mix gently; place on ice for 20 min, heat shock at 42°C for 45 s, and immediately place on ice for 2 min; add liquid LB without antibiotics and culture at 37°C with shaking for 60 min; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 h.

[0114] 5.7 Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37℃ with shaking for 16h; use a plasmid mini-extraction kit (TIANGEN rapid plasmid mini-extraction kit) to extract the plasmid. After double restriction digestion with NotI and EcoRI, sequencing identification was performed to obtain pHBB-508B-SPA-RE plasmid.

[0115] 6. Construction of plasmid pHBB-508B-W3SL-RE (sequence number 6): 6.1 The pHBB-508B-bGHPA-RE plasmid (sequence number 1) was double-digested with XhoI and EcoRI, and gel-recovered to obtain a linear vector with a size of 5507 bp.

[0116] 6.2 SV40-XhoI-F (SEQ ID NO.36) and β-WPRE-R (SEQ ID NO.37) were used for PCR amplification and gel recovery to obtain fragment 6-1, the sequence of which was SEQ ID NO.31 (bp 27-281 was Sv40 polyA).

[0117] 6.3 PCR was performed using pHBB-508B-bGHPA-RE (sequence number 1) as a template to obtain fragment 6-2, the sequence of which is SEQ ID NO.32.

[0118] 6.4 Overlap PCR was performed on the two fragments to obtain the final fragment and the 937 bp fragment was recovered by gel extraction; 6.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0119] 6.6 Transform the ligation product into competent E. coli TransStbl3, mix gently; place on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately place on ice for 2 minutes; add liquid LB without antibiotics and culture at 37°C with shaking for 60 minutes; discard part of the supernatant and use a sterile spreading stick to evenly spread the bacterial solution on an LB agar plate containing ampicillin; and culture inverted at 37°C for 16 hours.

[0120] 6.7 Pick a single clone colony and inoculate it in liquid LB containing ampicillin, and culture it at 37°C with shaking for 16 hours; use a plasmid extraction kit to extract the plasmid pHBB-508B-W3SL-RE, and then sequence and identify it after double digestion with NotI and EcoRI.

[0121] 2. Lentivirus Packaging (1) Preheat Wayne293TM transient culture medium in a water bath for 30-60 minutes. Irradiate the biosafety cabinet with UV light for 30 minutes.

[0122] (2) Take a sample of cells, count them and measure the glucose concentration. When the viable cell density is between 2.2-2.7E+06 cells / mL and the viability is ≥90%, add glucose to a final concentration of 6 g / L and prepare for transfection.

[0123] (3) Take out the relevant plasmids. The total amount of plasmids used is: 0.5 μg / million cells. The molar ratio of the four plasmids is: GOI plasmid: pMDLg / pRREKan: pRSV-RevKan: pMD2.gKan = 4:2:1:1. The transfection reagent (volume, μL): total amount of plasmid (mass, μg) = 2:1. The amount of transfection system culture medium used is 10% of the volume of the cell suspension.

[0124] (4) Use a pipette to transfer 5% of the volume of the cell suspension culture medium into two centrifuge tubes and mark them as Tube A and Tube B respectively.

[0125] (5) Use a pipette to add the four plasmids calculated above to tube A. After tightening the lid, invert it 10 times at a constant speed and leave it at room temperature.

[0126] (6) Use a pipette to add the calculated transfection reagent to tube B. After tightening the lid, invert it 10 times at a constant speed and let it stand at room temperature for 5 minutes.

[0127] (7) Pipette the solution in tube B into tube A, tighten the lid, invert it 10 times at a constant speed, and let it stand at room temperature for 15 minutes.

[0128] (8) Use a pipette to uniformly add the solution in tube A to the cell suspension to be transfected. After uniformly mixing the cell suspension, place the flask back on the shaker and incubate at 37°C.

[0129] (9) 24 ± 4 hours after transfection, add 33 μL of anti-agglomerating agent to the cell suspension at 0.1% of the total volume of the cell suspension, supplement glucose to a final concentration of 6 g / L, supplement omnipotent nuclease to a final concentration of 25 U / mL, and add MgCl2 to a final concentration of 2 mM.

[0130] (10) 48±4 hours after transfection, count the cells and harvest the virus.

[0131] 3. Lentivirus Purification The lentivirus was purified by using a tangential flow filtration-chromatography system, core700 chromatography, and Q ImpRes chromatography purification process. The specific purification process is as follows: (1) Nuclease digestion: Treat with 25 U / mL nuclease at 37°C for 1 hour to remove contaminants such as residual plasmid DNA from the transfection process and genomes released from lysed cells; (2) MF (clarification): The virus harvest solution is filtered (0.45 μm) to remove insoluble particles such as HEK293T cells and debris, and to improve the clarity of the solution for subsequent chromatography purification; (3) UF / DF (concentration and diafiltration): After clarification and digestion, the sample is concentrated and diafiltered using a UFP-750-E-3X2MA hollow fiber with a molecular weight cutoff of 750 kDa. Small molecular impurities can be effectively removed, thus achieving the purpose of purification; (4) SEC (size exclusion chromatography): The sample after UF / DF is further purified by equilibrated Capto Core700 filler. Viruses and other particles larger than 700 kDa are discharged and collected through the external water volume, while impurities with smaller molecular weight enter the filler pores and are adsorbed there. (5) IEX (anion exchange chromatography): The sample after size exclusion chromatography is purified by Capto Q impres filler, and the virus sample is loaded into the equilibrated Capto Q impres filler. At this time, the impurities will not be adsorbed by the filler and discharged, and then the virus is eluted by a 1M NaCl step gradient; (6) Preparation: The virus solution after IEX was concentrated and salt exchanged through a hollow fiber with a molecular weight cutoff of 750 kDa UFP-750-E-2U, and the virus was replaced in PBS containing 2% HSA.

[0132] (7) Storage: Filter the virus using a 0.2 μm membrane, aliquot, and store at -80°C.

[0133] IV. Lentiviral transduction titer detection 1. Plating and infection of 293T cells: (1) Experimental preparation: Preheat DMEM medium and FBS in a water bath at 37°C for 20 min; mix well and set aside.

[0134] (2) Cell digestion and termination: Add 10 mL of DMEM medium (containing 10% FBS) and lay the bottle flat so that it covers the bottom surface. Completely terminate the 0.25% trypsin-EDTA (1×) digestion. Gently use a pipette to blow the bottom of the bottle to blow the cells off and disperse the cell clusters.

[0135] (3) Cell counting and plating: Take an appropriate amount of cells (20 μL) and add the same volume of trypan blue solution for cell counting, and record the cell concentration and viability; calculate the required number of cell wells N based on the number of virus samples to be infected.

[0136] (4) Cell medium replacement and lentiviral infection: Take out polybrene and melt it at room temperature, and mix it thoroughly with the required DMEM culture medium (containing 10% FBS) in a 50 mL centrifuge tube; mix the virus to be infected, take 5 μL and add it to the well, mix it and place it in a carbon dioxide incubator for 20 hours; take out the 12-well plate, discard the supernatant, add 2 mL of DMEM culture medium (containing 10% FBS), and continue to culture it in a carbon dioxide incubator for 52 hours.

[0137] 2. Cell genome extraction (1) Select the magnetic bead tissue extraction kit, add 200 μL GL and 180 μL GTL buffer to each cell sample, shake and mix, and centrifuge for 3-15 seconds. Then add to the 96DW deep well plate according to Table 3: Table 3

[0138] (2) After extraction, transfer the eluted products in columns 6 and 12 of the deep-well plate to 200 μL eight-row centrifuge tubes and dilute them to 40 ng / μL using the test DNA sample concentration.

[0139] (3) qPCR system amplification: Take 7 1.5 mL centrifuge tubes and label them as ST1, ST2, ST3, ST4, ST5, ST6, and ST7. Add 90 μL of sterile enzyme-free water to each tube and dilute the standard STD (2 ng / μL) by 10 times into 7 gradients. The sample addition system is as shown in Table 4: Table 4

[0140] (4) After sealing the 96-well plate with an optical film, centrifuge at 2500 rpm for 30 seconds and load the plate with a fluorescence quantitative PCR instrument (ABI, 7500). The program parameters are set as shown in Table 5: Table 5

[0141] 3. Result calculation (1) The measured number of lentiviral vector copies integrated in the DNA sample was calibrated with the number of genomes to obtain the number of integrated viral copies per genome. The calculation formula is as follows: TU / ml = (C×N×D×1000) / V.

[0142] Where: C = average number of virus copies integrated per genome (Gag copy number / RNase P copy number); N = number of cells at the time of infection; D = dilution factor of viral vector; V = volume of diluted virus added.

[0143] (2) The test and analysis results are as follows: Fig. 9 shown.

[0144] 5. Lentiviral transduction and cell differentiation 1. Viral transduction: (1) CD34+ cells were taken out of liquid nitrogen and revived. The appropriate culture density was selected and transduction was performed after overnight culture.

[0145] (2) Add transduction-enhancing reagents: Add LentiBoost and protamine sulfate directly to the cells and mix gently.

[0146] (3) Virus reconstitution: Reconstitute the lentivirus at 4°C.

[0147] (4) Add lentivirus for transduction: Select an appropriate MOI, set MOI to 50, calculate the required amount of lentivirus, add it to the cell culture medium, and mix gently.

[0148] (5) Culture cells overnight under normal culture conditions.

[0149] (6) Cell differentiation: 24 hours after transduction, the medium was changed to differentiation medium, marked as D0, and in vitro liquid erythroid differentiation was performed for a total of 21 days.

[0150] (7) Cell recovery (D0): Transfer the transduced cells for 24 h to a 15 mL centrifuge tube, add 3 times the volume of DPBS, centrifuge at 350 g for 10 min at room temperature, discard the supernatant and resuspend the cells in 1 mL of the first phase culture medium (basal culture medium IMDM, supplemented with SCF, IL-3 and EPO), take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (8) Adjust the cell culture density to 1E5 cells / mL, select a suitable culture container, and add the first phase culture medium for culture; (9) Observe cells every day from D5 to D6; 2. Replace all cell medium (D4) (1) Pipette all the liquid in the culture container into a centrifuge tube, wash the bottom of the culture container with 1-2 mL of warm DPBS and add it to the centrifuge tube containing cells, centrifuge at 350g for 10 min at room temperature, discard the supernatant and resuspend with 2 mL of the first phase culture medium, take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (2) Adjust the cell culture density to 1E5 cells / mL, select a suitable culture container, and add the first phase culture medium for culture; 3. Replace all cell medium (D7) (1) Pipette all the liquid in the culture container into a centrifuge tube, wash the bottom of the culture container with 1-2 mL of warm DPBS and add it to the centrifuge tube containing cells, centrifuge at 350g for 10 min at room temperature, discard the supernatant and resuspend with 2 mL of the second phase culture medium (basal culture medium IMDM, supplemented with SCF and EPO), take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (2) Take 2E5 cells and send them to VCN for testing; (3) Adjust the culture density of the remaining cells to 1E5 cells / mL, select a suitable culture container, and add the second phase culture medium for culture; (4) Observe the cells every day from D8 to D10, and add the second phase culture medium or expand the culture depending on the cell growth status; 4. Replace all cell medium (D11) (1) Pipette all the liquid in the culture container into a centrifuge tube, wash the bottom of the culture container with 1-2 mL of warm DPBS and add it to the centrifuge tube containing cells, centrifuge at 350g for 10 min at room temperature, discard the supernatant and resuspend with 2 mL of the third phase culture medium (basal culture medium IMDM, with EPO added), take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (2) Adjust the culture density of the remaining cells to 1E6 cells / mL, select a suitable culture container, and add the third phase culture medium for culture; (3) Observe the cells every day from D12 to D14, and add the third-phase culture medium or expand the culture depending on the cell growth status; 5. Replace all cell medium (D15) (1) Pipette all the liquid in the culture container into a centrifuge tube, wash the bottom of the culture container with 1-2 mL of warm DPBS and add it to the centrifuge tube containing cells, centrifuge at room temperature at 350g for 10 min, discard the supernatant and resuspend with 2 mL of Phase IV culture medium, take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (2) Adjust the culture density of the remaining cells to 5E6 cells / mL, select a suitable culture container, and add the fourth phase culture medium for culture; (3) Observe the cells every day from D15 to D17, and add the fourth-stage culture medium or expand the culture depending on the cell status; 6. Cell harvest (D18) (1) Pipette all the liquid in the culture container into a centrifuge tube, wash the bottom of the culture container with 1-2 mL of warm DPBS and add it to the centrifuge tube containing cells, centrifuge at room temperature at 350g for 10 min, discard the supernatant and resuspend with 2 mL of DPBS, take 20 μL of cells for trypan blue staining, mix well, count the cells, and record the data; (2) Centrifuge again at 350g for 10 min at room temperature, discard the supernatant and send the cell pellet for β A-T87Q .

[0151] 6. VCN vector copy number detection 1. Cell genome extraction (1) Select Magbead Tissue DNA Kit. Add 200μL GL and 180μL GTL buffer to each cell sample, shake and mix, and centrifuge for 3-15s. Then add to the 96DW deep-well plate according to Table 6: Table 6

[0152] (2) After extraction, transfer the eluted products in columns 6 and 12 of the deep-well plate to 200 μL eight-row centrifuge tubes and dilute them to 40 ng / μL using the test DNA sample concentration.

[0153] 2. qPCR system amplification (1) Take 7 1.5 mL centrifuge tubes and label them as ST1, ST2, ST3, ST4, ST5, ST6, and ST7. Add 90 μL of sterile enzyme-free water to each tube and dilute the standard STD (2 ng / μL) by 10 times into 7 gradients. The sample addition system is as shown in Table 7: Table 7

[0154] (2) After sealing the 96-well plate with an optical film, centrifuge at 2500 rpm for 30 seconds and load the plate with a fluorescence quantitative PCR instrument (ABI, 7500). The program parameters are set as shown in Table 8: Table 8

[0155] 3. Result calculation (1) VCN (lentiviral vector copy number) = (GAG copy number / RNase P copy number) × 2; (2) The test and analysis results are as follows: Fig.10 shown.

[0156] 7. RP-HPLC Quantification of Hbβ A-T87Q expression.

[0157] 1. Sample processing (1) Take 1.0×10 6 The cells were centrifuged at 300 g for 10 min and the supernatant was discarded; (2) Resuspend in 40 μL water, freeze at -80°C for 10 min, thaw quickly at 37°C, and oscillate to mix; (3) Repeat the freezing and thawing process three times to fully lyse the cells; (4) Centrifuge at 9000g, 4°C for 10 min and collect the supernatant for detection.

[0158] 2. Buffer preparation (1) Buffer A: aqueous solution containing 1.2% TFA, pH 3.0; (2) Buffer B: acetonitrile solution containing 0.08% TFA.

[0159] (3) The test procedure is shown in Table 9 below: Table 9

[0160] (4) Test and analysis results such as Figures 11A-11G and Fig.12 shown.

[0161] The reverse expression cassette designed by the present invention is added with different polyA combinations, β A-T87Q The / α / VCN ratio increased, among which: the 508B control ratio was 1.80% ( Fig.11A ), 508B-RE-bGH-PA is 2.64% ( Fig. 11B ), 508B-RE-LTR was not detected ( Fig. 11C ), 508B-RE-βPA is 3.26% ( Fig.11D ), 508B-RE-αPA is 2.38% ( Fig.11E ), 508B-RE-SPA is 1.87% ( Fig.11F ), 508B-RE-W3SL is 0.48% ( Fig.11G ).

[0162] The analysis results are as follows Fig.13 and Fig.14 As shown in the figure, the results show that the ratios of bGH polyA, β-globin polyA, and α-globin polyA are all higher than those of positive 508B, among which β-globin polyA is the highest. A-T87Q At the same time as the overexpression of the corresponding vector, the copy number of the corresponding vector is reduced, which reduces the risk of carcinogenesis caused by the insertion of the lentivirus into the genome.

[0163] The above embodiments are only used to illustrate the present invention, not to limit the present invention. Adjustments and improvements of conventional technical means made by those skilled in the art without departing from the technical concept of the present invention are all within the scope of protection of the present invention.

Claims

1. An expression vector containing a target protein expression cassette, characterized in that: The expression vector includes gene fragment 1-gene fragment 2-gene fragment 3-gene fragment 4-gene fragment 5 in the order of 5'-3' direction; The gene fragment 1 is a polyA element pA; The gene fragment 2 is a CDS element for expressing the target protein; The gene segment 3 is a chimeric intron element E; The gene fragment 4 is the promoter element P of the target protein; The gene fragment 5 is the LCR2.7 regulatory sequence.

2. The expression vector according to claim 1, characterized in that The target protein is selected from α-globin, β-globin CDS β AT87Q , any one of pyruvate kinases, and the amino acid sequences are SEQ ID NOs: 1-3 respectively.

3. The expression vector according to claim 2, characterized in that The gene fragment 2 has any one of the nucleotide sequences shown in SEQ ID NOs: 4-9.

4. The expression vector according to claim 2, characterized in that The gene fragment 4 is selected from any one of β-globin promoter S, β-globin promoter L, and α-globin promoter, and each has a nucleotide sequence as shown in SEQ ID NO: 10-12.

5. The expression vector according to claim 1, characterized in that In the gene fragment 1, the polyA element is selected from any one of: bGH polyA, LTR polyA, β-globin polyA, α-globin polyA, synthetic polyA, and Sv40 polyA.

6. The expression vector according to claim 1, characterized in that The gene fragment 3 is selected from any one of: chimeric intron, MVM, HS2, IVS1, IVS1-2TF, IgG, and AID intron.

7. The expression vector according to claim 1, characterized in that The gene fragment 5 is selected from any one of LCR2.7k, LCR5.5k, and LCR6.5k, and has a nucleotide sequence as shown in SEQ ID NO: 13-15.

8. The expression vector according to any one of claims 1 to 7, characterized in that The expression vector is a plasmid vector or a lentiviral vector.

9. The expression vector according to claim 8, characterized in that The expression vector is a lentiviral vector.

10. The method for constructing the expression vector according to any one of claims 1 to 9, characterized in that: The method comprises inserting gene fragments 1-5 into an expression vector.

11. A cell comprising the expression vector according to any one of claims 1 to 9.

12. A lentivirus prepared by transfecting cells with the expression vector according to any one of claims 1 to 9.

13. Use of the expression vector according to any one of claims 1 to 9, the construction method according to claim 10, the cell according to claim 11, or the lentivirus according to claim 12 in the preparation of a drug for treating α / β-globin chain imbalance diseases, characterized in that: The α / β-globin chain imbalance is the lack of β-globin chain expression.

14. The use according to claim 13, characterized in that The drug is a β-thalassemia drug.

15. A drug prepared by the expression vector according to any one of claims 1 to 9, the construction method according to claim 10, the cell according to claim 11, or the lentivirus according to claim 12.

16. The drug according to claim 15, characterized in that The medicine also includes pharmaceutical excipients.

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