An expression cassette for reducing the integration risk of lentivirus, and its applications and products

Abstract

The present invention provides an expression cassette for reducing the risk of lentivirus integration, as well as its applications and products, belonging to the technical field of genetic engineering. The present invention is further optimized based on the prior art. The expression cassette is reversely assembled, and through the optimized selection of the polyA sequence, it is found that the vector of the present invention can improve the gene expression level of a single copy, achieve the same expression effect with a lower lentivirus integration copy number, thereby further reducing the risk brought by virus integration.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an expression cassette for reducing the integration risk of lentivirus, its applications and products. Background Art

[0002] β-thalassemia is one of the most common monogenic autosomal recessive hereditary diseases in the world. It is caused by defects in the β-globin gene or its regulatory sequences, resulting in reduced or impaired synthesis of β-globin chains, and further leading to an imbalance in the α / β-globin ratio and an inability to produce sufficient 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 hardly produce β chains and often present with transfusion-dependent β-thalassemia, with particularly severe clinical symptoms, requiring >100 mL / kg of concentrated RBC (red blood cells) or ≥8 transfusions per year. Moderate and severe β-thalassemia presents as severe hypochromic microcytic anemia in infancy, with a total Hb (hemoglobin) ≤7 g / dL.

[0003] Chinese Patent with Publication No. CN114457119A discloses the design of a β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA. Since the direction of its gene expression cassette is consistent with the transcription direction of the promoter of the viral packaging main plasmid, its cell transduction is superior to that of conventional reverse expression cassettes, and the overall gene expression level is comparable. Therefore, there is still room for further improvement in the protein expression amount provided by a single viral copy.

[0004] On this basis, there is still a large room for development in developing new expression vector forms to increase the expression amount 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 amount of a single vector copy, thereby reducing the vector integration copy number and the potential risks brought by vector integration; continuously optimize the gene expression cassette to maintain the gene expression amount unchanged when the vector copy number is reduced, thereby improving safety. It plays an important role especially in viral vectors.

[0006] Generally, the present invention is first based on the β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA described in the patent disclosure text with Publication No. CN114457119A, and on the basis of LCR2.7K-pHBB-CI-β A-T87QReverse assembly was performed, and different polyA sequences were screened on the basis of reverse to increase the stability of mRNA and promote β A-T87Q expression.

[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:

[0009] (1) The present invention provides a reverse assembly vector, provides different polyA selections, packages the virus after constructing the clone, and verifies the effects of different polyA sequences on gene integration and expression level in hematopoietic stem cells.

[0010] (2) The present invention provides a method for reducing the copy number of the viral vector and increasing the expression level of a single-copy gene. While maintaining a similar gene expression level, the vector integration copy number is reduced, thereby reducing the vector integration risk.

[0011] The vector design principle of the present invention:

[0012] Use the pCCL-SIN-cPPT-MCS-RbPA backbone as the vector (containing the 508B structure as shown in Figure 1 ), and sequentially adjust the expression cassette elements in the 5'-3' direction to polyA-CDS β AT87Q -chimeric intron-βglobin promoter-LCR2.7. Replace the polyA sequence with different sequences (the design scheme is shown in the 508B-RE-pA structure as shown in Figure 2 ). After completion, the plasmid vectors are respectively named: pHBB-508B-bGHPA-RE, pHBB-508B-LTR-RE, pHBB-508B-βPA-RE, pHBB-508B-αPA-RE, pHBB-508B-SPA-RE, pHBB-508B-W3SL-RE. The lentiviral vectors (LVV) are respectively named: 508B-RE-bGH-PA, 508B-RE-LTR, 508B-RE-βPA, 508B-RE-αPA, 508B-RE-SPA, 508B-RE-W3SL.

[0013] In the present invention, "sequentially" and "order" have the same meaning, indicating that there is an order relationship in the connection of the elements on the vector.

[0014] Based on the general knowledge in the art, "β-globin", "β - globin", "β-globin", "βglobin" have the same meaning; "α-globin", "α - globin", "α-globin", "αglobin" have the same meaning.

[0015] Specifically, the present invention provides an expression vector containing an expression cassette of a target protein, characterized in that the expression vector sequentially includes gene fragment 1 - gene fragment 2 - gene fragment 3 - gene fragment 4 - gene fragment 5 in the 5'-3' direction;

[0016] The gene fragment 1 is the polyA element pA;

[0017] The gene fragment 2 is the CDS element for expressing the target protein;

[0018] The gene fragment 3 is the chimeric intron element E;

[0019] The gene fragment 4 is the promoter element P of the target protein;

[0020] The gene fragment 5 is the LCR2.7 regulatory sequence; gene fragment 5 corresponds to Figure 2 HS4 - HS3 - HS2 in it. For the selection of specific fragments, those skilled in the art can replace them by conventional means.

[0021] Preferably, the target protein is selected from any one of α-globin, β-globin CDS β AT87Q , pyruvate kinase, and the amino acid sequences are SEQ ID NO: 1 - 3 respectively.

[0022] Preferably, the gene fragment 2 has any one of the nucleotide sequences shown in SEQ ID NO: 4 - 9, where 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 wild-type sequence for expressing β-globin CDS β AT87Q ; SEQ ID NO: 7 is the codon-optimized sequence for expressing β-globin CDS β AT87Q ; SEQ ID NO: 8 is the wild-type sequence for expressing pyruvate kinase; SEQ ID NO: 9 is the codon-optimized sequence for expressing pyruvate kinase.

[0023] 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 has the nucleotide sequences shown in SEQ ID NO: 10-12, respectively.

[0024] 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:

[0025] bGH polyA: bp 26-250 of SEQ ID NO.21;

[0026] LTR polyA: bp 35-186 of SEQ ID NO.23;

[0027] β-globin polyA: bp 29-422 of SEQ ID NO.25;

[0028] α-globin polyA: bp 27-228 of SEQ ID NO.38;

[0029] Synthetic polyA: bp 30-166 of SEQ ID NO.39;

[0030] Sv40 polyA: bp 27-281 of SEQ ID NO.31.

[0031] Preferably, the gene fragment 5 is selected from: LCR2.7k, LCR5.5k, LCR6.5k, and has the nucleotide sequences shown in SEQ ID NO: 13-15.

[0032] Preferably, the gene fragment 3 is selected from any one of: chimeric intron, MVM, HS2, IVS1, IVS1-2TF, IgG, and AID intron. As an example: the chimeric intron has the nucleotide sequence shown in SEQ ID NO: 16; the MVM has the nucleotide sequence shown in SEQ ID NO: 17; the HS2 has the nucleotide sequence shown in SEQ ID NO: 18; the IVS1 has the nucleotide sequence shown in SEQ ID NO: 19; the IVS1-2TF has the nucleotide sequence shown in SEQ ID NO: 20.

[0033] Preferably, the expression vector is a plasmid vector or a lentiviral vector.

[0034] Further preferably, the expression vector is an LVV lentiviral vector.

[0035] The present invention also provides a method for constructing the expression vector, which includes inserting gene fragments 1-5 into the expression vector.

[0036] 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, amplification, or other means, as long as they are used to construct the vector of the present invention, they are within the protection scope of the present invention.

[0037] In the present invention, the "insertion" refers to the process of ligating the corresponding gene to the vector, which can be in the form of sequential insertion, non-sequential insertion, or in the form of element substitution on the basis of the existing plasmid backbone by those skilled in the art.

[0038] As is well known to those skilled in the art, the vector of the present invention may also include other basic elements that are not adjusted in the present invention, such as Figure 2 other elements except gene fragment 1 - gene fragment 5.

[0039] In some specific embodiments, the vector construction in the present invention uses pCCL-SIN-cPPT-MCS-RbPA as the plasmid backbone, and performs reverse assembly and regulation including but not limited to the polyA element, etc.

[0040] Correspondingly, the present invention provides gene engineering products related to the foregoing expression vector, including but not limited to:

[0041] (1) cells including the expression vector;

[0042] (2) lentivirus prepared by transfecting cells with the expression vector; etc.

[0043] The present invention also includes the application of the foregoing expression vector, construction method, cell, or lentivirus in the preparation of drugs for α / β-globin chain imbalance diseases; the α / β-globin chain imbalance may be the absence of β-globin chain expression.

[0044] In some application scenarios, the drug may be a β-thalassemia drug.

[0045] Based on the above, the present invention also protects the drug itself.

[0046] At least the active ingredient of the drug is prepared by the expression vector, the construction method, the cell, or the lentivirus.

[0047] The described drug may also include pharmaceutical excipients, also known as pharmaceutically acceptable excipients in the art.

[0048] Preferably, the drug may be an injection. Those skilled in the art can confirm suitable injection excipients through routine selection during the preparation of the corresponding injection. These excipients may be those already disclosed or not disclosed in the prior art. As an example, the excipients that those skilled in the art can select include, but are not limited to: any one or more of buffers, stabilizers, anticoagulants, and cryoprotectants (DMSO) at different concentrations.

[0049] The buffers include, but are not limited to: any one or more of phosphate buffer, Tris buffer, and sodium chloride buffer;

[0050] The stabilizers include, but are not limited to: any one or more of human albumin, sucrose, and magnesium chloride;

[0051] The anticoagulants include, but are not limited to: Pluronic F68;

[0052] The cryoprotectants include, but are not limited to: DMSO.

[0053] Advantages of the present invention:

[0054] 1. The present invention optimizes the insertion method of each gene element, and improves the expression level of the single-copy vector through the reverse-assembled expression cassette.

[0055] 2. The present invention also reduces the average vector copy number of the lentivirus inserted into the genome, and at the same time reduces the risk of the lentivirus inserted into the genome.

[0056] Based on the above effects, the safety in the application of viral vectors in the art has been greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a schematic diagram of the 508B structure.

[0058] Figure 2 It is a schematic diagram of the 508B-RE-pA structure.

[0059] Figure 3 It is a structural diagram of the plasmid pHBB-508B-bGHPA-RE in Example 1.

[0060] Figure 4 It is a structural diagram of the plasmid pHBB-508B-LTR-RE in Example 1.

[0061] Figure 5 It is a structural diagram of the plasmid pHBB-508B-βPA-RE in Example 1.

[0062] Figure 6 It is the structural diagram of plasmid pHBB-508B-αPA-RE in Example 1.

[0063] Figure 7 It is the structural diagram of plasmid pHBB-508B-SPA-RE in Example 1.

[0064] Figure 8 It is the structural diagram of plasmid pHBB-508B-W3SL-RE in Example 1.

[0065] Figure 9 It is the detection result graph of lentiviral transduction titer in Example 1.

[0066] Figure 10 It is the detection result graph of VCN vector copy number in Example 1.

[0067] Figure 11A - Figure 11G It is the detection result graph of RP-HPLC after erythroid differentiation of hematopoietic stem cells infected with lentiviral vector in vitro in Example 1. Figure 11A - Figure 11G It is an automatically output picture of the detection system. Partial overlapping content does not affect the effect determination, and the relevant content has been analyzed and explained in Example 1.

[0068] Figure 12 It is Hbβ in Example 1 A-T87Q RP-HPLC statistical graph of the percentage of HBA.

[0069] Figure 13 It is the RP-HPLC statistical graph of the percentage of Hbβ provided by the average number of virus vector copies in Example 1 A-T87Q of HBA.

[0070] Figure 14 It is the comparative statistical graph of the results of detecting forward and reverse assembled viruses after transduction of patients' hematopoietic stem cells in Example 1; this graph is used to compare 508B-RE-bGH-PA with the 508B control. Among them, A is the comparison of transduction titers; B is the comparison of VCN vector copy numbers; C is the comparison of the percentage of Hbβ A-T87Q in HBA; D is the comparison of the percentage of Hbβ provided by the average number of virus vector copies A-T87Q in HBA. Specific implementation mode

[0071] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are, unless otherwise specified, the experimental methods without specific conditions in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0072] The information of some experimental materials used in the examples is as shown in Table 1 below:

[0073] Table 1 Experimental Materials

[0074]

[0075] Those skilled in the art know that for the selection of specific experimental materials, conventional substitutions can be made according to the disclosure of the prior art. Therefore, the implementation of the technical solution of the present invention is not limited to the above types.

[0076] In the present invention, "plasmid vector", "plasmid", and "vector" have the same or similar meanings.

[0077] In the present invention, "Core Insulator" refers to the core insulator, which is used to block the mutual interference of adjacent regulatory elements.

[0078] In the present invention, "promoter" refers to the promoter, which initiates the transcription of the DNA sequence.

[0079] In the present invention, "Enhancer" refers to the enhancer, which enhances the transcription of the DNA sequence.

[0080] In the present invention, "SV40 poly(A) signal" refers to the Simian vacuolating virus 40 polyadenylation signal, which increases the stability of mRNA.

[0081] In the present invention, "AmpR" refers to the ampicillin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0082] In the present invention, "KanR" refers to the kanamycin resistance gene, which is used for the screening of plasmids in Escherichia coli.

[0083] In the present invention, "ori" refers to the Escherichia coli replication origin, which regulates the rolling circle replication of the entire plasmid in Escherichia coli.

[0084] In the present invention, "f1 ori" refers to the f1 phage replication origin, which controls the replication of single-stranded DNA.

[0085] In the present invention, "LB" refers to Luria-Bertani medium.

[0086] In the present invention, "DMEM" refers to Dulbecco's Modified Eagle Medium.

[0087] In the present invention, "FBS" refers to fetal bovine serum.

[0088] In the present invention, "DPBS" refers to phosphate buffered saline.

[0089] In the present invention, "LVV" refers to a lentiviral vector.

[0090] In the present invention, "VCN" refers to the vector copy number

[0091] In the present invention, the XhoI enzyme, KpnI enzyme, and EcoRI enzyme, sometimes abbreviated as XhoI, KpnI, and EcoRI, can be understood by those skilled in the art to represent the corresponding enzymes or restriction sites.

[0092] In the present invention, "RP-HPLC" refers to reverse-phase high-performance liquid chromatography.

[0093] In the present invention, "CDS" refers to the coding sequence.

[0094] In the present invention, "β A-T87Q " refers to the mutation of codon 87 of β-globin from threonine to glutamine.

[0095] In the present invention, "overlap PCR" refers to overlap extension polymerase chain reaction. As an example, the conditions of overlap PCR used in the example part 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 further extension at 68°C for 2 min.

[0096] In the present invention, "qPCR" refers to real-time fluorescence quantitative polymerase chain reaction.

[0097] Unless otherwise specified, the gel extraction in the present invention is completed using the gel extraction kit in Table 1. Unless otherwise specified, the seamless cloning in the present invention is completed using the seamless cloning kit in Table 1.

[0098] Some plasmid maps in the present invention are made using software, and the output format is retained. Some of the retained English content can be understood by those skilled in the art in combination with the invention content and relevant vocabulary interpretations.

[0099] As is well known to those skilled in the art, operations such as restriction digestion, primer amplification, and ligation are involved in the process of constructing a vector. Therefore, in most cases, some fragments involved in the construction process may retain some non-functional fragments (including but not limited to fragments related to restriction sites, primer-related fragments, etc.). Therefore, some fragments in the examples are in an inclusion relationship with the target fragment, rather than being exactly the same as the target fragment, which can be understood by those skilled in the art.

[0100] Example 1

[0101] Specifically, it includes the following steps:

[0102] I. Construction of plasmid vector

[0103] The relevant information of the plasmid vector constructed in this example is shown in Table 2:

[0104] Table 2 Plasmid Vector Information

[0105]

[0106] The construction methods of each plasmid vector in Table 2 are as follows:

[0107] 1. Construction of plasmid pHBB-508B-bGHPA-RE (serial number 1):

[0108] 1.1 Double digest the pCCL-SIN-cPPT-MCS-RbPA plasmid (refer to pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA in the patent with application number 202210373730.0) with XhoI and KpnI and recover the gel to obtain a linear vector with a size of 5507 bp.

[0109] 1.2 Use the scAAV-CBA-EGFP-bGHpolyA plasmid with bGH polyA as a template for PCR amplification to obtain fragment 1-1, with the sequence of SEQ ID NO.21, and the 26th - 250th bp is bGH polyA.

[0110] 1.3 Use the gene-synthesized p508B plasmid as a template for PCR amplification to obtain fragment 1-2, with the sequence of SEQ IDNO.22.

[0111] 1.4 Then perform Overlap PCR on the 2 fragments, obtain the final fragment and recover the gel to obtain an amplification product with a length of 4004 bp, which is fragment 1.

[0112] 1.5 Connect the recovered linear vector and fragment 1 using a seamless cloning kit, in a 10 μL system, react at 50 °C for 15 min.

[0113] 1.6 Transform the ligation product into Escherichia coli competent TransStbl3, gently mix; incubate on ice for 20 min, heat shock at 42 °C for 45 s, immediately incubate on ice for 2 min; add antibiotic-free liquid LB, shake culture at 37 °C for 60 min; discard part of the supernatant and use a sterile spreading rod to evenly spread the bacterial solution onto an LB agar plate containing ampicillin; incubate upside down at 37 °C for 16 h. Pick a single colony and inoculate it into liquid LB containing ampicillin, shake culture at 37 °C for 16 h.

[0114] 1.7 Extract the plasmid using a plasmid miniprep kit (TIANGEN rapid plasmid miniprep kit), which is the pHBB-508B-bGHPA-RE plasmid. After double digestion identification with XhoI and KpnI, then perform sequencing identification and it is correct.

[0115] Subsequent plasmid vector construction was carried out using pHBB-508B-bGHPA-RE (Serial No. 1) as a template, replacing the polyA sequence with LTR polyA, β-globin polyA, α-globin polyA, synthetic polyA or Sv40 polyA.

[0116] 2. Construction of plasmid pHBB-508B-LTR-RE (Serial No. 2):

[0117] 2.1 The pHBB-508B-bGHPA-RE plasmid was digested with XhoI and EcoRI, and the gel was recovered to obtain a linear vector with a size of 5507 bp.

[0118] 2.2 PCR amplification was performed using the pHBB-508B-bGHPA-RE plasmid as a template to obtain fragment 2-1, with the sequence of SEQ ID NO.23; the 35th bp - 186th bp is LTR polyA.

[0119] 2.3 The sequence of fragment 2-2 is SEQ ID NO.24.

[0120] 2.4 Then, the two fragments were subjected to Overlap PCR to obtain the final fragment, and the fragment with a length of 741 bp was recovered by gel.

[0121] 2.5 The recovered vector and the fragment were ligated using a seamless cloning kit, in a 10 μL system, and reacted at 50 °C for 15 min.

[0122] 2.6 The ligation product was transformed into Escherichia coli competent TransStbl3, gently mixed; ice-bathed for 20 min, heat-shocked at 42 °C for 45 s, and immediately ice-bathed for 2 min; added liquid LB without antibiotics, cultured with shaking at 37 °C for 60 min; after discarding part of the supernatant, the bacterial solution was evenly spread on an LB agar plate containing ampicillin using a sterile spreading rod; cultured upside down at 37 °C for 16 h.

[0123] 2.7 Single colonies were picked and inoculated into liquid LB containing ampicillin, cultured with shaking at 37 °C for 16 h; the plasmid pHBB-508B-LTR-RE was extracted using a plasmid miniprep kit (TIANGEN rapid plasmid miniprep kit). The plasmid was digested with XhoI and EcoRI and then sequenced for identification.

[0124] 3. Construction of pHBB-508B-βPA-RE (Serial No. 3) plasmid:

[0125] 3.1 The plasmid pHBB-508B-bGHPA-RE (Serial No. 1) was digested with XhoI and EcoRI, and the gel was recovered to obtain a linear vector with a size of 5507 bp.

[0126] 3.2 Using the pHBB-508B-bGHPA-RE plasmid as a template, PCR amplification was performed to obtain fragment 3-1, with the sequence being SEQ ID NO.25; the 29th bp - 422nd bp is the β-globin polyA.

[0127] 3.3 The sequence of fragment 3-2 is SEQ ID NO.26.

[0128] 3.4 Then, Overlap PCR was performed on the 2 fragments to obtain the final fragment, and a fragment with a length of 906 bp was recovered by gel extraction.

[0129] 3.5 The recovered vector and the fragment were ligated using a seamless cloning kit, in a 10 μL system, and reacted at 50 °C for 15 min.

[0130] 3.6 The ligation product was transformed into Escherichia coli competent TransStbl3, gently mixed; incubated on ice for 20 min, heat shocked at 42 °C for 45 s, and immediately incubated on ice for 2 min; added liquid LB without antibiotics, cultured with shaking at 37 °C for 60 min; after discarding part of the supernatant, the bacterial solution was evenly spread onto an LB agar plate containing ampicillin using a sterile spreading rod; cultured inverted at 37 °C for 16 h.

[0131] 3.7 Single colonies were picked and inoculated into liquid LB containing ampicillin, cultured with shaking at 37 °C for 16 h; the plasmid was extracted using a plasmid mini-prep kit (TIANGEN rapid plasmid mini-prep kit). After double digestion identification with XhoI and EcoRI, sequencing identification was performed to obtain the pHBB-508B-βPA-RE plasmid.

[0132] 4. Construction of plasmid pHBB-508B-αPA-RE (serial number 4):

[0133] 4.1 The pHBB-508B-bGHPA-RE plasmid (serial number 1) was digested with XhoI and EcoRI, and the gel was recovered to obtain a linear vector with a size of 5507 bp.

[0134] 4.2 Without a template, directly use 3UTR-119F (SEQ ID NO.33) and 3UTR-88R (SEQ ID NO.34) for PCR amplification and gel recovery to obtain fragment 4-1, with the sequence being SEQ ID NO.27.

[0135] 4.3 Using the pHBB-508B-bGHPA-RE plasmid as a template, PCR was performed to obtain fragment 4-2, with the sequence being SEQ ID NO.28.

[0136] 4.4 Then perform Overlap PCR on the 2 fragments to obtain the final fragment and recover the 712 bp fragment by gel extraction. The sequence is SEQ ID NO.38 (the 27th bp - 228th bp is the α-globin polyA).

[0137] 4.5 Ligate the recovered vector and the fragment using a seamless cloning kit in a 10 μL system and react at 50 °C for 15 min.

[0138] 4.6 Transform the ligation product into Escherichia coli competent TransStbl3, gently mix; incubate on ice for 20 min, heat shock at 42 °C for 45 s, and immediately incubate on ice for 2 min; add antibiotic-free liquid LB and culture with shaking at 37 °C for 60 min; discard part of the supernatant and evenly spread the bacterial solution on an LB agar plate containing ampicillin using a sterile spreading rod; incubate upside down at 37 °C for 16 h.

[0139] 4.7 Pick a single colony and inoculate it into liquid LB containing ampicillin, culture with shaking at 37 °C for 16 h; extract the plasmid using a plasmid mini-prep kit (TIANGEN rapid plasmid mini-prep kit). After double digestion with XhoI and EcoRI for identification and then sequencing identification, obtain the plasmid pHBB-508B-αPA-RE.

[0140] 5. Construction of plasmid pHBB-508B-SPA-RE (No. 5):

[0141] 5.1 Perform double digestion of the plasmid pHBB-508B-bGHPA-RE (No. 1) with XhoI and EcoRI and recover the gel to obtain a linear vector with a size of 5507 bp.

[0142] 5.2 Without a template, use 3UTR-SPA-F (SEQ ID NO.35) and 3UTR-88R (SEQ ID NO.34) for PCR amplification and recover the gel to obtain fragment 5-1 with the sequence SEQ ID NO.29.

[0143] 5.3 Use the plasmid pHBB-508B-bGHPA-RE as a template for PCR to obtain fragment 5-2 with the sequence SEQ ID NO.30.

[0144] 5.4 Then perform Overlap PCR on the 2 fragments to obtain the final fragment and recover the 650 bp fragment by gel extraction. The sequence is SEQ ID NO.39, and the 30th bp - 166th bp is the synthetic polyA.

[0145] 5.5 Ligate the recovered vector and the fragment using a seamless cloning kit in a 10 μL system and react at 50 °C for 15 min.

[0146] 5.6 Transform the ligation product into competent Escherichia coli TransStbl3, mix gently; incubate on ice for 20 min, heat shock at 42 °C for 45 s, and immediately incubate on ice for 2 min; add antibiotic-free liquid LB, shake culture at 37 °C for 60 min; discard part of the supernatant and spread the bacterial solution evenly onto an LB agar plate containing ampicillin using a sterile spreading rod; incubate inverted at 37 °C for 16 h.

[0147] 5.7 Pick a single colony and inoculate it into liquid LB containing ampicillin, shake culture at 37 °C for 16 h; extract the plasmid using a plasmid mini-prep kit (TIANGEN Fast Plasmid Mini-Prep Kit). After double digestion with NotI and EcoRI for identification, then perform sequencing identification to obtain the plasmid pHBB-508B-SPA-RE.

[0148] 6. Construction of plasmid pHBB-508B-W3SL-RE (No. 6):

[0149] 6.1 Perform double digestion of plasmid pHBB-508B-bGHPA-RE (No. 1) with XhoI and EcoRI, and recover the gel to obtain a linear vector with a size of 5507 bp.

[0150] 6.2 Use SV40-XhoI-F (SEQ ID NO.36) and β-WPRE-R (SEQ ID NO.37) for PCR amplification and recover the gel to obtain fragment 6-1 with the sequence of SEQ ID NO.31 (the 27th bp - 281st bp is Sv40 polyA).

[0151] 6.3 Use pHBB-508B-bGHPA-RE (No. 1) as a template to perform PCR to obtain fragment 6-2 with the sequence of SEQ ID NO.32.

[0152] 6.4 Then perform Overlap PCR on the two fragments to obtain the final fragment and recover the 937 bp fragment from the gel;

[0153] 6.5 Ligate the recovered vector and fragment using a seamless cloning kit, 10 μL system, react at 50 °C for 15 min.

[0154] 6.6 Transform the ligation product into competent Escherichia coli TransStbl3, mix gently; incubate on ice for 20 min, heat shock at 42 °C for 45 s, and immediately incubate on ice for 2 min; add antibiotic-free liquid LB, shake culture at 37 °C for 60 min; discard part of the supernatant and spread the bacterial solution evenly onto an LB agar plate containing ampicillin using a sterile spreading rod; incubate inverted at 37 °C for 16 h.

[0155] 6.7 Pick a monoclonal colony and inoculate it into liquid LB containing ampicillin, and culture it with shaking at 37°C for 16 h; use a plasmid miniprep kit to extract plasmid pHBB-508B-W3SL-RE, and perform double digestion with NotI and EcoRI followed by sequencing identification.

[0156] II. Lentivirus packaging

[0157] (1) Preheat the Wayne293TM transient transfection medium in a water bath for 30 - 60 minutes. Irradiate the biosafety cabinet with ultraviolet light for 30 min.

[0158] (2) Take a cell sample for counting and measure the glucose concentration. When the viable cell density is 2.2 - 2.7E+06 cells / mL and the viability is ≥90%, supplement glucose to a final concentration of 6 g / L and prepare for transfection.

[0159] (3) Take out the relevant plasmids. The total amount of plasmid 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 plasmid amount (mass, μg) = 2:1; the amount of medium used in the transfection system is 10% of the volume of the cell suspension.

[0160] (4) Use a pipette to take the medium with a volume of 5% of the cell suspension volume into 2 centrifuge tubes, and label them as tube A and tube B respectively.

[0161] (5) Use a pipette to add the above-calculated 4 plasmids to tube A. After tightening the lid, gently invert it 10 times evenly and then let it stand at room temperature.

[0162] (6) Use a pipette to add the calculated transfection reagent to tube B. After tightening the lid, gently invert it 10 times evenly and then start to let it stand at room temperature for 5 min.

[0163] (7) Use a pipette to add the solution in tube B to tube A, then tighten the lid, gently invert it 10 times evenly and then start to let it stand at room temperature for 15 min.

[0164] (8) Use a pipette to evenly add the solution in tube A to the cell suspension to be transfected. After evenly mixing the cell suspension, put the shaking flask back on the shaker and culture it at 37°C.

[0165] (9) 24 ± 4 hours after transfection, add 33 μL of anti-clumping agent to the cell suspension according to 0.1% volume of the total volume of the cell suspension, supplement glucose to a final concentration of 6 g / L. Supplement the universal nuclease to a final concentration of 25 U / mL, and at the same time add MgCl2 to a final concentration of 2 mM.

[0166] (10) 48 ± 4 hours after transfection, count the cells and harvest the virus.

[0167] III. Lentivirus Purification

[0168] Using a tangential flow filtration - chromatography system, and adopting the core700 chromatography and Q ImpRes chromatography purification processes, the lentivirus was purified. The specific purification process is as follows:

[0169] (1) Nuclease digestion: Treat with 25 U / mL nuclease at 37 °C for 1 hour to remove contaminants such as plasmid DNA remaining from the transfection process and genomic DNA released by lysed cells;

[0170] (2) MF (clarification): The virus harvest fluid was filtered (0.45 μm) to remove insoluble particles such as HEK293T cells and cell debris, improving the clarity of the solution for subsequent chromatography purification;

[0171] (3) UF / DF (concentration and diafiltration): The clarified digested sample was concentrated and diafiltered using a UFP - 750 - E - 3X2MA hollow fiber with a molecular weight cut - off of 750 kDa, effectively removing small - molecule impurities to achieve the purpose of purification;

[0172] (4) SEC (size - exclusion chromatography): The sample after UF / DF was further purified through the equilibrated Capto Core700 packing. Particles larger than 700 kDa, such as viruses, were discharged and collected through the void volume, while impurities with smaller molecular weights entered the pores of the packing and were adsorbed;

[0173] (5) IEX (ion - exchange chromatography): The sample after size - exclusion chromatography was refined through the Capto Q impres packing. The virus sample was loaded onto the equilibrated Capto Q impres packing. At this time, impurities were not adsorbed by the packing and were discharged, and then the virus was eluted through a 1M NaCl step - gradient;

[0174] (6) Formulation: The virus solution after IEX was concentrated and salt - exchanged through a hollow fiber with a molecular weight cut - off of 750 kDa UFP - 750 - E - 2U, and the virus was exchanged into PBS containing 2% HSA.

[0175] (7) Preservation: The virus was filtered through a 0.2 μm membrane, aliquoted, and stored at - 80 °C.

[0176] IV. Detection of Lentivirus Transduction Titer

[0177] 1. Plating and infecting 293T cells:

[0178] (1) Experiment preparation: DMEM medium and FBS were pre - heated in a water bath at 37 °C for 20 min in advance; mix well for standby.

[0179] (2) Cell digestion and termination: Add 10 mL of DMEM medium (containing 10% FBS), place it flat to cover the bottom surface completely, and completely terminate the digestion with 0.25% Trypsin-EDTA (1×). Gently pipette the bottom surface of the flask to detach the cells and break up the cell clusters simultaneously.

[0180] (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. Record the cell concentration and viability; calculate the required number of cell wells N based on the number of virus samples to be infected.

[0181] (4) Cell medium change and lentivirus infection: Take out the polybrene and melt it at room temperature. Mix it thoroughly with the required DMEM 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 well and then place it in a carbon dioxide incubator for 20 h; take out the 12-well plate, aspirate the supernatant, add 2 mL of DMEM medium (containing 10% FBS), and continue to culture it in the carbon dioxide incubator for 52 h.

[0182] 2. Cell genomic DNA extraction

[0183] (1) Select a magnetic bead-based tissue extraction kit. Add 200 μL of GL and 180 μL of GTL buffer to each cell sample, mix well by shaking, and then centrifuge briefly for 3 - 15 s. Then add them to a 96DW deep well plate according to Table 3 below:

[0184] Table 3

[0185]

[0186] (2) After extraction, transfer the elution products in columns 6 and 12 of the deep well plate to a 200 μL eight-strip centrifuge tube, and dilute to 40 ng / μL using a DNA sample concentration detector.

[0187] (3) qPCR system amplification: Take 7 1.5 mL centrifuge tubes, label them as ST1, ST2, ST3, ST4, ST5, ST6, and ST7 respectively. Add 90 μL of sterile and enzyme-free water to each tube. Dilute the standard STD (2 ng / μL) by 10-fold in 7 gradients. The sample addition system is as shown in Table 4 below:

[0188] Table 4

[0189]

[0190] (4) After sealing the 96-well plate with an optical film, centrifuge it at 2500 rpm for 30 s and load it onto a fluorescence quantitative PCR instrument (ABI, 7500). Set the program parameters as shown in Table 5 below:

[0191] Table 5

[0192]

[0193] 3. Result Calculation

[0194] (1) The copy number of the integrated lentiviral vector in the measured DNA sample is calibrated with the number of genomes to obtain the number of viral copies integrated per genome. The calculation formula is as follows: TU / ml = (C × N × D × 1000) / V.

[0195] Where: C = the number of viral copies integrated per genome on average (Gag copy number / RNase P copy number); N = the number of cells at the time of infection; D = the dilution factor of the viral vector; V = the volume of the diluted virus added.

[0196] (2) The detection and analysis results are as Figure 9 shown.

[0197] V. Lentiviral Transduction and Cell Differentiation

[0198] 1. Viral Transduction:

[0199] (1) CD34+ cells are taken out of liquid nitrogen for resuscitation, and an appropriate culture density is selected. After culturing overnight, transduction is carried out.

[0200] (2) Add transduction-promoting reagents: directly add LentiBoost and protamine sulfate to the cells and gently mix.

[0201] (3) Viral thawing: Thaw the lentivirus at 4°C.

[0202] (4) Add lentivirus for transduction: Select an appropriate MOI, the MOI is selected as 50, calculate the required amount of lentivirus, add it to the cell culture medium, and gently mix.

[0203] (5) Culture the cells overnight under normal culture conditions.

[0204] (6) Cell differentiation: After 24 hours of transduction, change to the differentiation medium. The medium change is marked as D0, and in vitro liquid erythroid differentiation is carried out for a total of 21 days.

[0205] (7) Cell resuscitation (D0): Transfer the cells transduced for 24 hours to a 15 mL centrifuge tube, add 3 times the volume of DPBS, centrifuge at 350g for 10 minutes at room temperature, discard the supernatant, and resuspend the cells with 1 mL of the first-stage medium (basic medium IMDM supplemented with SCF, IL-3, and EPO). Take 20 μL of the cells for trypan blue staining, mix well, and perform cell counting, and record the data;

[0206] (8) Adjust the cell culture density to 1E5 cells / mL, select an appropriate culture container, and supplement the first-stage medium for culture;

[0207] (9) Observe the cells every day from D5 to D6;

[0208] 2. Complete medium change for cells (D4)

[0209] (1) Aspirate all the liquid in the culture vessel into a centrifuge tube. Wash the bottom of the culture vessel with 1 - 2 mL of pre-warmed DPBS and add it to the centrifuge tube containing the cells. Centrifuge at 350g for 10 min at room temperature. After discarding the supernatant, resuspend the cells with 2 mL of the first-stage medium. Take 20 μL of the cells for trypan blue staining, mix well and perform cell counting, and record the data;

[0210] (2) Adjust the cell culture density to 1×10⁵ cells / mL, select a suitable culture vessel, and supplement with the first-stage medium for culture;

[0211] 3. Complete medium change for cells (D7)

[0212] (1) Aspirate all the liquid in the culture vessel into a centrifuge tube. Wash the bottom of the culture vessel with 1 - 2 mL of pre-warmed DPBS and add it to the centrifuge tube containing the cells. Centrifuge at 350g for 10 min at room temperature. After discarding the supernatant, resuspend the cells with 2 mL of the second-stage medium (basic medium IMDM supplemented with SCF and EPO). Take 20 μL of the cells for trypan blue staining, mix well and perform cell counting, and record the data;

[0213] (2) Send 2×10⁵ cells for VCN testing;

[0214] (3) Adjust the density of the remaining cells to 1×10⁵ cells / mL, select a suitable culture vessel, and supplement with the second-stage medium for culture;

[0215] (4) Observe the cells every day from D8 to D10, and supplement the second-stage medium or expand the culture according to the cell growth status;

[0216] 4. Complete medium change for cells (D11)

[0217] (1) Aspirate all the liquid in the culture vessel into a centrifuge tube. Wash the bottom of the culture vessel with 1 - 2 mL of pre-warmed DPBS and add it to the centrifuge tube containing the cells. Centrifuge at 350g for 10 min at room temperature. After discarding the supernatant, resuspend the cells with 2 mL of the third-stage medium (basic medium IMDM supplemented with EPO). Take 20 μL of the cells for trypan blue staining, mix well and perform cell counting, and record the data;

[0218] (2) Adjust the density of the remaining cells to 1×10⁶ cells / mL, select a suitable culture vessel, and supplement with the third-stage medium for culture;

[0219] (3) Observe the cells every day from D12 to D14, and supplement the third-stage medium or expand the culture according to the cell growth status;

[0220] 5. Complete cell medium change (D15)

[0221] (1) Aspirate all the liquid in the culture vessel into a centrifuge tube. Wash the bottom of the culture vessel with 1 - 2 mL of pre-warmed DPBS and add it to the centrifuge tube containing the cells. Centrifuge at 350 g for 10 min at room temperature. After discarding the supernatant, resuspend the cells with 2 mL of the fourth-stage medium. Take 20 μL of the cells for trypan blue staining, mix well and perform cell counting, and record the data;

[0222] (2) Adjust the culture density of the remaining cells to 5×10⁶ cells / mL. Select a suitable culture vessel and supplement with the fourth-stage medium for culture;

[0223] (3) Observe the cells daily from D15 to D17. Depending on the cell status, supplement with the fourth-stage medium or expand the culture;

[0224] 6. Cell harvesting (D18)

[0225] (1) Aspirate all the liquid in the culture vessel into a centrifuge tube. Wash the bottom of the culture vessel with 1 - 2 mL of pre-warmed DPBS and add it to the centrifuge tube containing the cells. Centrifuge at 350 g for 10 min at room temperature. After discarding the supernatant, resuspend the cells with 2 mL of DPBS. Take 20 μL of the cells for trypan blue staining, mix well and perform cell counting, and record the data;

[0226] (2) Centrifuge again at 350 g for 10 min at room temperature. After discarding the supernatant, send the cell pellet for β A-T87Q .

[0227] VI. Detection of VCN vector copy number

[0228] 1. Cell genome extraction

[0229] (1) Select the Magbead Tissue DNA Kit. Add 200 μL of GL and 180 μL of GTL buffer to each cell sample respectively. After shaking and mixing well, centrifuge briefly for 3 - 15 s. Then add them to the 96DW deep well plate according to Table 6 below:

[0230] Table 6

[0231]

[0232] (2) After the extraction is completed, transfer the elution products in columns 6 and 12 of the deep well plate to a 200 μL eight-strip centrifuge tube and dilute the detected DNA sample concentration to 40 ng / μL.

[0233] 2. qPCR system amplification

[0234] (1) Take 7 1.5 mL centrifuge tubes, labeled as ST1, ST2, ST3, ST4, ST5, ST6, and ST7 respectively. Add 90 μL of sterile and enzyme-free water to each tube. Dilute the standard product STD (2 ng / μL) in 7 gradients at a 10-fold dilution. The sample addition system is as shown in Table 7 below:

[0235] Table 7

[0236]

[0237] (2) After sealing the 96-well plate with an optical film, centrifuge it at 2500 rpm for 30 s and load it onto the fluorescence quantitative PCR instrument (ABI, 7500). The program parameters are set as shown in Table 8 below:

[0238] Table 8

[0239]

[0240] 3. Result calculation

[0241] (1) VCN (lentiviral vector copy number) = (copy number of GAG / copy number of RNase P) × 2;

[0242] (2) The detection and analysis results are as Figure 10 shown.

[0243] VII. RP-HPLC quantification of Hbβ A-T87Q expression.

[0244] 1. Sample treatment

[0245] (1) Take 1.0 × 10 6 quantity of cells, centrifuge at 300 g for 10 min, and discard the supernatant;

[0246] (2) Resuspend with 40 μL of water, freeze at -80 °C for 10 min, rapidly thaw at 37 °C, and mix by oscillation;

[0247] (3) Repeat the freeze-thaw cycle three times to fully lyse the cells;

[0248] (4) Centrifuge at 9000 g at 4 °C for 10 min, collect the supernatant for detection.

[0249] 2. Buffer preparation

[0250] (1) Buffer A: Aqueous solution containing 1.2% TFA, pH 3.0;

[0251] (2) Buffer B: Acetonitrile solution containing 0.08% TFA.

[0252] (3) The detection procedure is shown in Table 9 below:

[0253] Table 9

[0254]

[0255] (4)The detection and analysis results are as Figure 11A - Figure 11G and Figure 12 shown.

[0256] For the reverse expression cassette designed in the present invention combined with different polyA combinations, the β A-T87Q / α / VCN ratio increases. Among them: the ratio of the 508B control is 1.80% ( Figure 11A ), the ratio of 508B-RE-bGH-PA is 2.64% ( Figure 11B ), the 508B-RE-LTR is not detected ( Figure 11C ), the ratio of 508B-RE-βPA is 3.26% ( Figure 11D ), the ratio of 508B-RE-αPA is 2.38% ( Figure 11E ), the ratio of 508B-RE-SPA is 1.87% ( Figure 11F ), the ratio of 508B-RE-W3SL is 0.48% ( Figure 11G ).

[0257] The analysis results are as Figure 13 and Figure 14 shown. The results show that the ratios of bGHpolyA, β-globin polyA, and α-globin polyA are all higher than those of the forward 508B. Among them, the β-globin polyA is the highest. Therefore, while overexpressing β A-T87Q , the corresponding vector copy number decreases, reducing the carcinogenic risk caused by lentivirus insertion into the genome.

[0258] The above embodiments are only used to illustrate the present invention and are not used 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 a target protein expression cassette, and the target protein expression cassette is composed of gene fragments 1-5, and in the 5'-3' direction sequence is gene fragment 1 - gene fragment 2 - gene fragment 3 - gene fragment 4 - gene fragment 5; the expression vector is an LVV lentiviral vector; The gene fragment 1 is a polyA element pA, and the nucleotide sequence is as shown in bp 26-250 of SEQ ID NO. 21; The gene fragment 2 described above is the CDS of the element expressing the target protein; the target protein is β-globin CDS β AT87Q , and the amino acid sequence of the target protein is shown in SEQ ID NO.2; The gene fragment 3 is a chimeric intron element E, and the nucleotide sequence is as shown in SEQ ID NO: 16; The gene fragment 4 is a promoter element P of the target protein, and the nucleotide sequence is as shown in SEQ ID NO.10; The gene fragment 5 is an LCR2.7 regulatory sequence.

2. The method for constructing the expression vector according to claim 1, characterized in that It includes inserting the target protein expression cassette described in claim 1 into an expression vector.

3. A cell comprising the expression vector described in claim 1.

4. A lentivirus prepared by transfecting cells with the expression vector described in claim 1.

5. Use of the expression vector described in claim 1 or the construction method described in claim 2 or the cell described in claim 3 or the lentivirus described in claim 4 in the preparation of drugs for β-thalassemia.

6. A drug prepared by the expression vector described in claim 1 or the construction method described in claim 2 or the cell described in claim 3 or the lentivirus described in claim 4.

7. The drug according to claim 6, characterized in that, The drug also includes pharmaceutical excipients.

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