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

[0001] This application claims a divisional application of Chinese patent application No. 2025102669850, filed on March 7, 2025, entitled “An expression cassette for reducing the risk of lentiviral integration, its application and product”. The entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of genetic engineering technology, and specifically relates to an expression cassette for reducing the risk of lentivirus integration, and its application and product. Background Art

[0003] β-thalassemia is one of the most common monogenic autosomal recessive inherited disorders worldwide. It is caused by defects in the β-globin gene or its regulatory sequences, resulting in reduced or impaired β-globin chain synthesis, which in turn leads to an imbalance in the α / β-globin ratio and insufficient hemoglobin production. 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 with the βE / β0 genotype produce virtually no β chains, often presenting with transfusion-dependent β-thalassemia. Clinical symptoms are particularly severe, requiring >100 mL / kg of packed red blood cells (RBCs) or ≥8 transfusions annually. Moderate to severe β-thalassemia manifests in infancy as severe hypochromic microcytic anemia, with total hemoglobin (Hb) ≤7 g / dL.

[0004] The Chinese patent publication number CN114457119A discloses a β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA is designed. Because the direction of its gene expression cassette is consistent with the transcription direction of the viral packaging main plasmid promoter, its cell transduction is better than that of conventional reverse expression cassettes, and the overall gene expression level is comparable. Therefore, the protein expression amount provided by a single viral copy has the potential to continue to increase.

[0005] 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

[0006] The present invention aims to further increase protein expression from a single vector copy, thereby reducing the number of vector integration copies and mitigating potential risks associated with vector integration. This approach also aims to optimize gene expression cassettes to maintain gene expression levels even when the vector copy number is reduced, thereby improving safety. This approach is particularly important in viral vectors.

[0007] In summary, the present invention firstly develops a β-globin recombinant lentiviral vector pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β described in the patent publication No. CN114457119A. A-T87Q -RbPA, based on LCR2.7K-pHBB-CI-β A-T87Q A reverse assembly was performed, and different ployA sequences were screened based on the reverse to increase mRNA stability and promote β A-T87Q Express.

[0008] Furthermore, the optimization direction of the present invention lies in the connection order of each element. Unexpected technical effects can be achieved through reverse assembly. Those skilled in the art can easily think of applying it to the expression of other proteins.

[0009] 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.

[0010] (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. While maintaining similar gene expression levels, the vector integration copy number is reduced, thereby reducing the risk of vector integration.

[0011] 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 (Figure 5B). 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.

[0012] In the present invention, "sequentially" and "order" have the same meaning, indicating that there is a sequential relationship in the connection of elements on the carrier.

[0013] 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.

[0014] Specifically, the present invention provides an expression vector containing a target protein expression cassette, characterized in that the expression vector comprises gene fragment 1-gene fragment 2-gene fragment 3-gene fragment 4-gene fragment 5 in the 5'-3' direction order; The gene segment 1 is a polyA element pA; The gene fragment 2 is the CDS element for expressing the target protein; The gene segment 3 is a chimeric intron element E; The gene segment 4 is the promoter element P of the target protein; The gene segment 5 is the LCR2.7 regulatory sequence; the gene segment 5 corresponds to Figure 2 For the selection of specific fragments, those skilled in the art can replace them by conventional means.

[0015] Preferably, the target protein is selected from α-globin, β-globin CDS β AT87Q , any one of pyruvate kinases, whose amino acid sequences are SEQ ID NOs: 1-3 respectively.

[0016] 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 The wild-type sequence of 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.

[0017] Preferably, the gene fragment 4 is selected from any one of the following: β-globin promoter S (β-globin promoter S), β-globin promoter L (β-globin promoter L), α-globin promoter (α-globin promoter), each having a nucleotide sequence as shown in SEQ ID NO: 10-12.

[0018] Preferably, the polyA element is selected from any one of the following: 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: SEQ ID NO. 21, 26-250 bp; LTR polyA: SEQ ID NO. 23, 35 bp-186 bp; β-globin polyA: SEQ ID NO. 25, 29 bp-422 bp; α-globin polyA: SEQ ID NO. 38, 27 bp-228 bp; synthetic polyA: SEQ ID NO. 39, 30 bp-166 bp; Sv40 polyA: SEQ ID NO. 31, 27 bp-281 bp.

[0019] Preferably, the gene fragment 5 is selected from any one of the following: LCR2.7k, LCR5.5k, and LCR6.5k, each having a nucleotide sequence as shown in SEQ ID NO: 13-15.

[0020] Preferably, the gene fragment 3 is selected from any one of the following: 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.

[0021] Preferably, the expression vector is a plasmid vector or a lentivirus vector.

[0022] Further preferably, the expression vector is an LVV lentivirus vector.

[0023] The present invention also provides a method for constructing the expression vector, which comprises inserting gene fragments 1-5 into the expression vector.

[0024] 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.

[0025] 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 those skilled in the art can also choose to replace elements based on the existing plasmid backbone.

[0026] The carrier of the present invention may further 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 segment 1 to gene segment 5.

[0027] In some specific embodiments, the vector of the present invention is constructed using pCCL-SIN-cPPT-MCS-RbPA as a plasmid backbone for reverse assembly and regulation including but not limited to polyA elements.

[0028] 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) Lentivirus prepared by transfecting cells with the expression vector; etc.

[0029] 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 can be a lack of β-globin chain expression.

[0030] In some application scenarios, the drug may be a β-thalassemia drug.

[0031] Based on the above, the present invention also protects the drug itself.

[0032] At least the active ingredient of the drug is prepared by the expression vector, the construction method, the cell or the lentivirus.

[0033] The medicine may also include pharmaceutical excipients, which are also referred to as pharmaceutically acceptable excipients in the art.

[0034] Preferably, the drug may be an injectable formulation. Those skilled in the art can routinely select and identify suitable injectable excipients in the preparation of the corresponding injectable formulation. These excipients may be disclosed or undisclosed in the prior art. For example, excipients that can be selected by those skilled in the art include, but are not limited to, any one or more of buffers, stabilizers, anticoagulants, and cryoprotectants (DMSO) at varying concentrations.

[0035] 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.

[0036] 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 reverse assembly of the expression cassette.

[0037] 2. The present invention also reduces the average vector copy number of the lentivirus inserted into the genome, while reducing the risk of lentivirus insertion into the genome.

[0038] Based on the above effects, the safety of viral vector applications in this field has been greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the 508B structure.

[0040] Figure 2 Schematic diagram of the 508B-RE-pA structure.

[0041] Figure 3 This is a structural diagram of the plasmid pHBB-508B-bGHPA-RE in Example 1.

[0042] Figure 4 This is a structural diagram of the plasmid pHBB-508B-LTR-RE in Example 1.

[0043] Figure 5 This is a structural diagram of the plasmid pHBB-508B-βPA-RE in Example 1.

[0044] Figure 6 This is a structural diagram of the plasmid pHBB-508B-αPA-RE in Example 1.

[0045] Figure 7 This is a structural diagram of the plasmid pHBB-508B-SPA-RE in Example 1.

[0046] Figure 8 The structure diagram of the plasmid pHBB-508B-W3SL-RE in Example 1 is shown.

[0047] Figure 9 This is a graph showing the results of lentiviral transduction titer detection in Example 1.

[0048] Figure 10 This is a graph showing the copy number detection results of the VCN vector in Example 1.

[0049] Figures 11A-11G This is a graph showing the RP-HPLC test results after in vitro erythroid differentiation of hematopoietic stem cells infected with the lentiviral vector in Example 1. Figures 11A-11G The detection system automatically outputs pictures, and some overlapping content does not affect the effect determination. The relevant content has been analyzed and explained in Example 1.

[0050] Figure 12 Hbβ in Example 1 A-T87Q RP-HPLC statistical chart of the percentage of HBA.

[0051] Figure 13 Provides Hbβ for the average number of viral vector copies in Example 1 A-T87Q RP-HPLC statistical chart of the percentage of HBA.

[0052] Figure 14 This is a statistical chart comparing the results of the forward and reverse assembled virus detection after the patient's hematopoietic stem cell transduction in Example 1; this figure 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 Hbβ copies per viral vector A-T87Q Comparison of percentage of HBA. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0054] Some experimental material information used in the examples is shown in Table 1: Table 1 Experimental materials

[0055] The person skilled in the art knows that the selection of specific experimental materials can be routinely replaced according to the disclosure of the prior art, and therefore the implementation of the technical solutions of the present application is not limited to the above types.

[0056] In the present application, "plasmid vector", "plasmid" and "vector" have the same or similar meanings.

[0057] In the present application, "Core Insulator" refers to a core insulator for blocking the mutual interference of adjacent regulatory elements.

[0058] In the present application, "promoter" refers to a promoter that initiates the transcription of a DNA sequence.

[0059] In the present application, "Enhancer" refers to an enhancer that enhances the transcription of a DNA sequence.

[0060] In the present application, "SV40 poly(A) signal" refers to a simian vacuolating virus 40 polyadenylation signal that increases the stability of mRNA.

[0061] In the present application, "AmpR" refers to an ampicillin resistance gene used for plasmid screening in E. coli.

[0062] In the present application, "KanR" refers to a kanamycin resistance gene used for plasmid screening in E. coli.

[0063] In the present application, "ori" refers to an E. coli replication initiation site that regulates the rolling circle replication of the entire plasmid in E. coli.

[0064] In the present application, "f1 ori" refers to an f1 bacteriophage replication initiation site that controls the replication of single-stranded DNA.

[0065] In the present application, "LB" refers to Luria-Bertani medium.

[0066] In the present application, "DMEM" refers to Dulbecco's Modified Eagle Medium.

[0067] In the present application, "FBS" refers to fetal bovine serum.

[0068] In the present application, "DPBS" refers to phosphate buffered saline.

[0069] In the present application, "LVV" refers to a lentiviral vector.

[0070] In the present application, "VCN" refers to the copy number of a vector In the present invention, XhoI enzyme, KpnI enzyme, EcoRI enzyme are sometimes abbreviated as XhoI, KpnI, EcoRI, and those skilled in the art will understand that they represent corresponding enzymes or enzyme cleavage sites.

[0071] In the present invention, "RP-HPLC" refers to reverse phase high performance liquid chromatography.

[0072] In the present invention, "CDS" refers to a coding sequence.

[0073] In the present invention, "β A-T87Q ” refers to the mutation of codon 87 of β-globin from threonine to glutamine.

[0074] In the present invention, "overlap PCR" refers to overlap extension polymerase chain reaction. As an example, the overlap PCR conditions used in the Examples of the present invention are: pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 10 seconds, annealing at 58°C for 10 seconds, extension at 68°C for 40 seconds, and further extension at 68°C for 2 minutes.

[0075] In the present invention, "qPCR" refers to real-time fluorescent quantitative polymerase chain reaction.

[0076] Unless otherwise specified, gel recovery in the present invention was performed using the gel recovery kit in Table 1. Unless otherwise specified, seamless cloning in the present invention was performed using the seamless cloning kit in Table 1.

[0077] Some of the plasmid maps in the present invention were produced using software, and the output format was retained. Some of the retained English content is content that can be understood by those skilled in the art in combination with the content of the invention and the definitions of related terms.

[0078] 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 linkage. Therefore, some fragments involved in the construction process may in most cases retain some non-functional fragments (including but not limited to fragments related to enzyme cleavage sites, primer-related fragments, etc.). Therefore, in the embodiments, some fragments are included in the target fragment, rather than being completely consistent with the target fragment. This is understandable to those skilled in the art.

[0079] 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

[0080] The construction methods of each plasmid vector in Table 2 are as follows: 1. Construction of plasmid pHBB-508B-bGHPA-RE (SEQ ID NO. 1): 1.1 The pCCL-SIN-cPPT-MCS-RbPA plasmid (pCCL-SIN-cPPT-LCR2.7K-pHBB-CI-β A-T87Q -RbPA in the patent with application number 202210373730.0) was double digested with XhoI and KpnI and gel recovered to obtain a linear vector with a size of 5507 bp.

[0081] 1.2 PCR amplification was performed using the scAAV-CBA-EGFP-bGHpolyA plasmid with bGH polyA as the template to obtain fragment 1-1, the sequence of which is SEQ ID NO. 21, and the 26-250 bp is bGH polyA.

[0082] 1.3 PCR amplification was performed using the p508B plasmid synthesized by gene synthesis as the template to obtain fragment 1-2, the sequence of which is SEQ ID NO. 22.

[0083] 1.4 Overlap PCR was then performed on the two fragments to obtain the final fragment and gel recover an amplification product with a length of 4004 bp, which is fragment 1.

[0084] 1.5 The recovered linear vector and fragment 1 were connected using a seamless cloning kit in a 10 μL system at 50°C for 15 min.

[0085] 1.6 The ligation product was transformed into E. coli competent TransStbl3, gently mixed; ice bath for 20 min, 42°C heat shock for 45 s, immediately ice bath for 2 min; add liquid LB without antibiotics, 37°C shaking culture for 60 min; discard part of the supernatant and evenly spread the bacterial solution on LB agar plates containing ampicillin using a sterile spreader; 37°C inverted culture for 16 h. Single colony was picked and inoculated in liquid LB containing ampicillin, and 37°C shaking culture for 16 h.

[0086] 1.7 The plasmid was extracted using a plasmid extraction kit (TIANGEN rapid plasmid extraction kit) to obtain the pHBB-508B-bGHPA-RE plasmid. After XhoI and KpnI double digestion identification, sequencing identification was performed again and no errors were found.

[0087] Subsequent plasmid vector construction used pHBB-508B-bGHPA-RE (SEQ ID NO. 1) as the template, and replaced the polyA sequence with LTR polyA, β globin polyA, α globin polyA, synthetic polyA, or Sv40 polyA.

[0088] 2. Plasmid pHBB-508B-LTR-RE (SEQ ID NO. 2) construction: 2.1 The pHBB-508B-bGHPA-RE plasmid was double digested with XhoI and EcoRI, and the linear vector with a size of 5507 bp was recovered by gel.

[0089] 2.2 The fragment 2-1 was obtained by PCR amplification using the pHBB-508B-bGHPA-RE plasmid as a template, and the sequence was SEQ ID NO. 23; the 35th-186th bp was LTR polyA.

[0090] 2.3 The sequence of fragment 2-2 was SEQ ID NO. 24.

[0091] 2.4 The two fragments were subjected to overlap PCR to obtain the final fragment and the 741 bp length fragment was recovered by gel.

[0092] 2.5 The recovered vector and the fragment were connected using a seamless cloning kit, and the 10 μL system was reacted at 50°C for 15 min.

[0093] 2.6 The ligation product was transformed into E. coli competent TransStbl3, and gently mixed; ice bath for 20 min, 42°C heat shock for 45 s, immediately ice bath for 2 min; add liquid LB without antibiotics, 37°C shaking culture for 60 min; after discarding part of the supernatant, the bacterial solution was evenly coated on the LB agar plate containing ampicillin by sterile coating rod; 37°C inverted culture for 16 h.

[0094] 2.7 A single colony was picked and inoculated in liquid LB containing ampicillin, and cultured at 37°C for 16 h; the plasmid pHBB-508B-LTR-RE was extracted by plasmid extraction kit (TIANGEN rapid plasmid extraction kit). The plasmid was double digested with XhoI and EcoRI, and then sequenced.

[0095] 3. Plasmid pHBB-508B-βPA-RE (SEQ ID NO. 3) construction: 3.1 The plasmid pHBB-508B-bGHPA-RE (SEQ ID NO. 1) was double digested with XhoI and EcoRI, and the linear vector with a size of 5507 bp was recovered by gel.

[0096] 3.2 The fragment 3-1 was obtained by PCR amplification using the pHBB-508B-bGHPA-RE plasmid as a template, and the sequence was SEQ ID NO. 25; the 29th-422nd bp was β globin polyA.

[0097] 3.3 The sequence of fragment 3-2 was SEQ ID NO. 26.

[0098] 3.4 The two fragments were subjected to Overlap PCR to obtain the final fragment and a 906 bp fragment was recovered by gel.

[0099] 3.5 The recovered vector and the fragment were connected using a seamless cloning kit in a 10 μL system at 50°C for 15 min.

[0100] 3.6 The connection product was transformed into E. coli competent TransStbl3, mixed gently; ice bath for 20 min, 42°C heat shock for 45 s, immediately ice bath for 2 min; add liquid LB without antibiotics, 37°C shaking culture for 60 min; after discarding part of the supernatant, the bacterial solution was evenly coated on LB agar plates containing ampicillin; 37°C inverted culture for 16 h.

[0101] 3.7 A single colony was picked and inoculated in liquid LB containing ampicillin, and 37°C shaking culture was performed for 16 h; the plasmid was extracted using a plasmid extraction kit (TIANGEN rapid plasmid extraction kit). After XhoI and EcoRI double enzyme digestion identification, sequencing identification was performed, and the pHBB-508B-βPA-RE plasmid was obtained.

[0102] 4. Construction of plasmid pHBB-508B-αPA-RE (SEQ ID NO. 4): 4.1 The pHBB-508B-bGHPA-RE plasmid (SEQ ID NO. 1) was subjected to XhoI and EcoRI double enzyme digestion, and a linear vector with a size of 5507 bp was recovered.

[0103] 4.2 Without template, 3UTR-119F (SEQ ID NO. 33) and 3UTR-88R (SEQ ID NO. 34) were directly used for PCR amplification and gel recovery to obtain fragment 4-1 with the sequence of SEQ ID NO. 27.

[0104] 4.3 The pHBB-508B-bGHPA-RE plasmid was used as a template for PCR to obtain fragment 4-2 with the sequence of SEQ ID NO. 28.

[0105] 4.4 The two fragments were subjected to Overlap PCR to obtain the final fragment and a 712 bp fragment was recovered by gel, with the sequence of SEQ ID NO. 38 (the 27th-228th bp is α globin polyA).

[0106] 4.5 The recovered vector and the fragment were connected using a seamless cloning kit in a 10 μL system at 50°C for 15 min.

[0107] 4.6 Transform the ligation product into competent E. coli TransStbl3 and mix gently; incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately incubate on ice for 2 minutes; add antibiotic-free liquid LB and incubate at 37°C with shaking for 60 minutes; discard part of the supernatant and evenly spread the bacterial solution onto an LB agar plate containing ampicillin using a sterile spreader; incubate inverted at 37°C for 16 hours.

[0108] 4.7 Pick a single colony and inoculate it in liquid LB containing ampicillin. Cultivate with shaking at 37°C for 16 hours. Extract the plasmid using a plasmid extraction kit (TIANGEN Rapid Plasmid Extraction Kit). After double digestion with XhoI and EcoRI, confirm the identity of the plasmid by sequencing. The pHBB-508B-αPA-RE plasmid was obtained.

[0109] 5. Construction of plasmid pHBB-508B-SPA-RE (sequence number 5): 5.1 The pHBB-508B-bGHPA-RE plasmid (SEQ ID NO. 1) was double-digested with XhoI and EcoRI, and the resulting vector was recovered on gel to obtain a linear vector of 5507 bp.

[0110] 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.

[0111] 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.

[0112] 5.4 Overlap PCR was performed on the two fragments to obtain the final fragment and the 650 bp fragment was recovered by gel recovery. The sequence was SEQ ID NO. 39, and the 30 bp to 166 bp fragment was synthetic poly A.

[0113] 5.5 The recovered vector and fragment were connected using a seamless cloning kit, 10 μL system, 50°C for 15 minutes.

[0114] 5.6 Transform the ligated product into competent E. coli TransStbl3 and mix gently; incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately incubate on ice for 2 minutes; add antibiotic-free liquid LB and incubate at 37°C with shaking for 60 minutes; discard part of the supernatant and evenly spread the bacterial solution onto an LB agar plate containing ampicillin using a sterile spreader; incubate inverted at 37°C for 16 hours.

[0115] 5.7 Pick a single colony and inoculate it in liquid LB containing ampicillin. Incubate with shaking at 37°C for 16 hours. Extract the plasmid using a plasmid extraction kit (TIANGEN Rapid Plasmid Extraction Kit). After double digestion with NotI and EcoRI, confirm the identity of the plasmid by sequencing to obtain the pHBB-508B-SPA-RE plasmid.

[0116] 6. Construction of plasmid pHBB-508B-W3SL-RE (sequence number 6): 6.1 Double-digest the pHBB-508B-bGHPA-RE plasmid (SEQ ID NO. 1) with XhoI and EcoRI, and recover the fragment on gel to obtain a linear vector of 5507 bp.

[0117] 6.2 PCR amplification and gel recovery were performed using SV40-XhoI-F (SEQ ID NO. 36) and β-WPRE-R (SEQ ID NO. 37) to obtain fragment 6-1, the sequence of which is SEQ ID NO. 31 (bp 27-281 is Sv40 polyA).

[0118] 6.3 PCR was performed using pHBB-508B-bGHPA-RE (SEQ ID NO. 1) as a template to obtain fragment 6-2, the sequence of which is SEQ ID NO. 32.

[0119] 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 ligated using a seamless cloning kit, 10 μL system, 50°C for 15 min.

[0120] 6.6 Transform the ligated product into competent E. coli TransStbl3 and mix gently; incubate on ice for 20 minutes, heat shock at 42°C for 45 seconds, and immediately incubate on ice for 2 minutes; add antibiotic-free liquid LB and incubate at 37°C with shaking for 60 minutes; discard some of the supernatant and evenly spread the bacterial solution onto an LB agar plate containing ampicillin using a sterile spreader; incubate inverted at 37°C for 16 hours.

[0121] 6.7 Pick a single colony and inoculate it into liquid LB containing ampicillin. Cultivate with shaking at 37°C for 16 h. Use a plasmid extraction kit to extract the plasmid pHBB-508B-W3SL-RE. Digest it with NotI and EcoRI and then sequence it.

[0122] 2. Lentiviral Packaging (1) Preheat Wayne 293™ Transient Transfection Medium in a water bath for 30-60 minutes. Irradiate the medium with UV light in a biosafety cabinet for 30 minutes.

[0123] (2) Cell sampling, counting and glucose concentration measurement. When the viable cell density is 2.2-2.7E+06 / mL and the viability is ≥90%, add glucose to a final concentration of 6 g / L, and prepare for transfection.

[0124] (3) Take out the relevant plasmids. The total amount of plasmids used is 0.5 μg per million cells. The molar ratio of the four plasmids is GOI plasmid: pMDLg / pRRE Kan: pRSV-Rev Kan: pMD2.g Kan = 4:2:1:1. The transfection reagent (volume, μL): total amount of plasmids (mass, μg) = 2:1. The amount of transfection system medium used is 10% of the volume of the cell suspension.

[0125] (4) Use a pipette to take 5% of the volume of the cell suspension medium into two centrifuge tubes, respectively, and label them as tube A and tube B.

[0126] (5) Use a pipette to add the above calculated four plasmids to tube A. After tightening the cap, rotate and invert at a uniform speed for 10 times, and then place it at room temperature.

[0127] (6) Use a pipette to add the calculated transfection reagent to tube B. After tightening the cap, rotate and invert at a uniform speed for 10 times, and then start room temperature standing for 5 min.

[0128] (7) After adding the solution in tube B to tube A with a pipette, tighten the cap, rotate and invert at a uniform speed for 10 times, and then start room temperature standing for 15 min.

[0129] (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 shake flask back on the shaker and cultivate at 37°C.

[0130] (9) 24±4 hours after transfection, add 33 μL of anti-clumping agent to the cell suspension at a volume of 0.1% of the total volume of the cell suspension, and add glucose to a final concentration of 6 g / L. Add nuclease to a final concentration of 25 U / mL, and add MgCl2 to a final concentration of 2 mM.

[0131] (10) 48±4 hours after transfection, count the cells and harvest the virus.

[0132] III. Lentivirus purification Use tangential flow filtration-chromatography system, use core700 chromatography, Q ImpRes chromatography purification process, purify lentivirus, the specific purification process is as follows: (1) Nuclease digestion: treat with 25 U / mL of nuclease at 37°C for 1 hour to remove residual plasmid DNA and contaminants such as genome released by lysed cells during transfection; (2) MF (clarification): The virus harvest solution is filtered (0.45 μm) to remove insoluble particles such as HEK293T cells and debris, thereby improving the clarity of the solution for subsequent chromatographic 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 Core 700 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 packing. The virus sample is loaded into the equilibrated Capto Q impres packing. At this time, the impurities will not be adsorbed by the packing and will be discharged. The virus is then eluted by a 1 M 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.

[0133] (7) Storage: Filter the virus using a 0.2 μm membrane, aliquot, and store at -80°C.

[0134] 4. Lentiviral transduction titer detection 1. Plating and infection of 293T cells: (1) Experimental preparation: Preheat DMEM culture medium and FBS in a water bath at 37°C for 20 minutes; mix well and set aside.

[0135] (2) Cell digestion and termination: Add 10 mL of DMEM medium (containing 10% FBS) and lay the bottle flat so that it spreads evenly on the bottom surface. Completely terminate the 0.25% trypsin-EDTA (1×) digestion. Gently use a pipette to blow the bottom surface of the bottle to blow the cells down and disperse the cell clusters.

[0136] (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.

[0137] (4) Cell culture 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, aspirate and discard the supernatant, add 2 mL of DMEM culture medium (containing 10% FBS), and place it in a carbon dioxide incubator for further 52 hours.

[0138] 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 below: Table 3

[0139] (2) After the extraction is completed, the eluted products in columns 6 and 12 of the deep-well plate are transferred to 200 μL eight-row centrifuge tubes and diluted to 40 ng / μL using the test DNA sample concentration.

[0140] (3) qPCR system amplification: Take 7 1.5 mL centrifuge tubes, mark 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 in 7 gradients. The sample addition system is as shown in Table 4: Table 4

[0141] (4) After sealing the 96-well plate with 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

[0142] 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 viral copies integrated per genome. The calculation formula is as follows: TU / ml = (C × N × D × 1000) / V.

[0143] Where: C = average number of virus copies integrated per genome (Gag copies / RNase P copies); N = number of cells at the time of infection; D = dilution factor of viral vector; V = volume of diluted virus added.

[0144] (2) Detection and analysis results such as Figure 9 shown.

[0145] V. Lentivirus transduction and cell differentiation 1. Virus transduction: (1) CD34+ cells were taken out from liquid nitrogen, and the appropriate culture density was selected. After overnight culture, transduction was performed.

[0146] (2) Add transduction-promoting reagent: directly add LentiBoost and protamine sulfate to the cells and mix gently.

[0147] (3) Virus thawing: thaw lentivirus at 4°C.

[0148] (4) Add lentivirus for transduction: select appropriate MOI, MOI is 50, calculate the required amount of lentivirus, add to the cell culture medium, and mix gently.

[0149] (5) Normal culture conditions overnight culture of cells.

[0150] (6) Cell differentiation: change to differentiation medium after 24h transduction, the change is marked as D0, and in vitro liquid red blood cell differentiation is performed, a total of 21 days.

[0151] (7) Cell recovery (D0): transfer the cells transduced for 24h to a 15 mL centrifuge tube, add 3 times the volume of DPBS, centrifuge at 350g at room temperature for 10 min, discard the supernatant, and resuspend the cells with 1 mL of the first stage medium (IMDM base medium, supplemented with SCF, IL-3 and EPO). Take 20 μL of cells for trypan blue staining, mix well and perform cell counting, and record the data. (8) Adjust the cell culture density to 1E5 / mL, select the appropriate culture vessel, and add the first stage medium for culture. (9) Observe the cells every day from D5 to D6. 2. Cell full medium change (D4) (1) Absorb all the liquid in the culture vessel into a centrifuge tube, wash the bottom of the culture vessel with 1-2 mL of warm DPBS, then add it to the centrifuge tube containing the cells, centrifuge at 350g at room temperature for 10 min, discard the supernatant, and resuspend with 2 mL of the first stage medium. Take 20 μL of cells for trypan blue staining, mix well and perform cell counting, and record the data. (2) Adjust the cell culture density to 1E5 / mL, select the appropriate culture vessel, and add the first stage medium for culture. 3. Cell full medium change (D7) (1) Absorb all the liquid in the culture container into the centrifuge tube, wash the bottom of the culture container with 1-2 mL of DPBS warmed, then add it into the centrifuge tube containing the cells, centrifuge at 350 g for 10 min at room temperature, discard the supernatant, then resuspend with 2 mL of the second-stage culture medium (IMDM basic medium, added with SCF and EPO), take 20 μL of the cells for trypan blue staining, mix well, then perform cell counting, and record the data; (2) Take 2E5 cells for VCN inspection; (3) Adjust the culture density of the remaining cells to 1E5 cells / mL, select a suitable culture container, and supplement the second-stage culture medium for culture; (4) Observe the cells every day from D8 to D10, and supplement the second-stage culture medium or expand the culture according to the cell growth state; 4. Cell complete medium exchange (D11) (1) Absorb all the liquid in the culture container into the centrifuge tube, wash the bottom of the culture container with 1-2 mL of DPBS warmed, then add it into the centrifuge tube containing the cells, centrifuge at 350 g for 10 min at room temperature, discard the supernatant, then resuspend with 2 mL of the third-stage culture medium (IMDM basic medium, added with EPO), take 20 μL of the cells for trypan blue staining, mix well, then perform cell counting, and record the data; (2) Adjust the culture density of the remaining cells to 1E6 cells / mL, select a suitable culture container, and supplement the third-stage culture medium for culture; (3) Observe the cells every day from D12 to D14, and supplement the third-stage culture medium or expand the culture according to the cell growth state; 5. Cell complete medium exchange (D15) (1) Absorb all the liquid in the culture container into the centrifuge tube, wash the bottom of the culture container with 1-2 mL of DPBS warmed, then add it into the centrifuge tube containing the cells, centrifuge at 350 g for 10 min at room temperature, discard the supernatant, then resuspend with 2 mL of the fourth-stage culture medium, take 20 μL of the cells for trypan blue staining, mix well, then perform cell counting, and record the data; (2) Adjust the culture density of the remaining cells to 5E6 cells / mL, select a suitable culture container, and supplement the fourth-stage culture medium for culture; (3) Observe the cells every day from D15 to D17, and supplement the fourth-stage culture medium or expand the culture according to the cell state; 6. Cell harvesting (D18) (1) Absorb all the liquid in the culture container into the centrifuge tube, wash the bottom of the culture container with 1-2 mL of DPBS warmed, then add it into the centrifuge tube containing the cells, centrifuge at 350 g for 10 min at room temperature, discard the supernatant, then resuspend with 2 mL of DPBS, take 20 μL of the cells for trypan blue staining, mix well, then perform cell counting, 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 .

[0152] 6. VCN vector copy number detection 1. Cell genome extraction (1) Select the Magbead Tissue DNA Kit. Add 200 μL of GL and 180 μL of GTL buffer to each cell sample, shake and mix thoroughly, and centrifuge for 3-15 seconds. Then add the sample to a 96DW deep-well plate according to Table 6: Table 6

[0153] (2) After the extraction is completed, the eluted products in columns 6 and 12 of the deep-well plate are transferred to 200 μL eight-row centrifuge tubes and diluted to 40 ng / μL using the test DNA sample concentration.

[0154] 2. qPCR system amplification (1) Take seven 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 seven gradients. The sample addition system is as shown in Table 7: Table 7

[0155] (2) After sealing the 96-well plate with 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

[0156] 3. Result calculation (1) VCN (lentiviral vector copy number) = (GAG copy number / RNase P copy number) × 2; (2) Detection and analysis results such as Figure 10 As shown, the results showed that the lentiviral copy number of 508B was the highest. After the reverse expression cassette was added with different polyA combinations, the expression vectors 508B-RE-bGH-PA, 508B-RE-LTR, 508B-RE-βPA, 508B-RE-αPA, 508B-RE-SPA and 508B-RE-W3SL all had lower lentiviral copy numbers, among which 508B-RE-βPA had the lowest lentiviral copy number.

[0157] 7. RP-HPLC Quantification of Hbβ A-T87Q expression.

[0158] 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 of water, freeze at -80°C for 10 min, thaw rapidly 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 and 4℃ for 10 min, and collect the supernatant for detection.

[0159] 2. Buffer preparation (1) Buffer A: 1.2% TFA in water, pH 3.0; (2) Buffer B: acetonitrile solution containing 0.08% TFA.

[0160] (3) The test procedure is shown in Table 9 below: Table 9

[0161] (4) Detection and analysis results such as Figures 11A-11G and Figure 12 shown.

[0162] according to Figure 12 Hbβ A-T87Q The RP-HPLC statistical chart of the percentage of HBA showed that the β-amino acids of the expression vectors 508B-RE-bGH-PA, 508B-RE-LTR, 508B-RE-βPA, 508B-RE-αPA, and 508B-RE-SPA were obtained by adding different polyA combinations to the reverse expression cassette. A-T87Q The / α value is above 4%, among which 508B and 508B-RE-bGH-PA have β A-T87Q / α value is the highest and can reach more than 8%.

[0163] Combined with the lentiviral copy number, it reflects the Hbβ per unit vector copy number A-T87Q As a percentage of HBA, see Figure 13 The ratio of 508B control was 1.80%, 508B-RE-bGH-PA was 2.64%, 508B-RE-LTR was not detected, 508B-RE-βPA was 3.26%, 508B-RE-αPA was 2.38%, 508B-RE-SPA was 1.87%, and 508B-RE-W3SL was 0.48%. Except for 508B-RE-W3SL, the reverse expression cassette designed by the present invention plus different poly A combinations showed that compared with the 508B control, β A-T87QThe ratios of Hbβ / α / VCN increased to varying degrees, among which 508B-RE-βPA was more excellent, which means that at the unit copy number of 508B-RE-βPA, Hbβ A-T87Q A higher percentage of HBAs.

[0164] Vector integration is essential for the expression of exogenous genes and the achievement of gene therapy effects. However, vector integration activity also carries the risk of unintended host genomic alterations, mutations, or carcinogenesis. J. Ma et al., in their study "An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products," documented that an increase in viral vector-integrated transgene copies (VCN) is associated with a higher risk of uncontrolled integration, which can lead to insertional mutagenesis by interfering with the transcription of adjacent host genes. Although secondary malignancies have been reported in patients receiving genetically engineered cell therapies, the risk is considered low and not significantly different from that of patients receiving standard therapy. However, products with higher VCNs may increase genotoxicity. Therefore, careful monitoring of integrated VCN is crucial to determine the VCN level that maximizes clinical efficacy while minimizing the genotoxic and carcinogenic potential of the viral vector.

[0165] The 508B-RE-βPA of the present invention successfully achieves "low-copy, high-expression" lentiviral vectors in β-thalassemia gene therapy. 508B-RE-βPA of the present invention requires fewer viral copies to integrate into the patient's cell genome. This directly reduces the potential risk of oncogene activation or tumor suppressor gene inactivation due to random viral integration, laying an important technical foundation for the development of safer and more effective β-thalassemia gene therapy drugs.

[0166] 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 carried out 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, which is composed of gene segments 1-5, and the order from 5' to 3' is gene segment 1-gene segment 2-gene segment 3-gene segment 4-gene segment 5; the expression vector is an LVV lentiviral vector; The gene fragment 1 is a polyA element pA, and the nucleotide sequence is shown in SEQ ID NO. 25, 29 bp to 422 bp; The gene fragment 2 is the element CDS for expressing the target protein; the target protein is β-globin CDS β AT87Q , the amino acid sequence of the target protein is shown in SEQ ID NO.2; The gene segment 3 is a chimeric intron element E, and the nucleotide sequence is shown in SEQ ID NO: 16; The gene fragment 4 is the promoter element P of the target protein, and the nucleotide sequence is shown in SEQ ID NO.10; The gene segment 5 is the LCR2.7 regulatory sequence.

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

3. A cell comprising the expression vector of claim 1.

4. A lentivirus prepared by transfecting cells with the expression vector according to claim 1.

5. Use of the expression vector according to claim 1, the construction method according to claim 2, the cell according to claim 3, or the lentivirus according to claim 4 in the preparation of a β-thalassemia drug.

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

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

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