T cell preference promoter as well as biological material and application thereof

Abstract

The invention discloses a T cell preference promoter as well as a biological material and application thereof. The invention aims to provide a T cell preference type promoter which is used for realizing high expression of CD3 + T cells in PBMC (peripheral blood mononuclear cells) and low expression of packaging cell lines HEK293 and non-targeted cells (such as B cells and hepatocytes) in in-vivo lentiviral vector application, blocking transgene leakage in a production period and reducing cytotoxicity and immunogenicity. By means of single intravenous administration, the carrier is silenced in blood circulation, after entering T cells, CAR or gene editing protein expression is efficiently started, in-vivo CAR-T is accurately constructed or T is knocked out, the process is simplified, the cost is reduced, off-target integration and antibody generation are reduced, and a foundation is laid for safe and repeated administration.

Description

A T-cell preferred promoter, its biomaterials and applications Technical Field

[0001] This patent belongs to the field of synthetic biology and in vivo gene therapy delivery technology, specifically involving a T-cell-preferred promoter, its biomaterials, and applications. This promoter exhibits high-level transcriptional activity in CD3+ T cells within peripheral blood mononuclear cells (PBMCs), while its activity is significantly reduced in HEK293 production cells and B cells, achieving a targeted expression mode of "low leakage during production and high expression in vivo." Background Technology

[0002] Existing in vivo lentiviral vectors commonly employ strong viruses such as CMV, CAG, and EF1α, or housekeeping promoters, to drive the expression of CARs, gene editing enzymes, or cytokines. These sequences are also highly active in HEK293 production cells, leading to continuous and massive leakage of transgenes during the viral packaging stage. This results in cytotoxicity, depletion of cellular energy and cell membrane resources, and a significant reduction in viral titer and batch stability. More seriously, these panphilic promoters maintain strong activity in non-target cells that highly express transcription factors, such as hepatocytes, splenic macrophages, and B cells, causing widespread integration of the vector genome and the associated risk of insertional mutations. Although some have attempted to reduce non-T expression using the miR-142-3p target sequence or chromatin insulators, the effects have been limited, and the genome needs to be lengthened, reducing viral load. In addition, the inducible Tet-On system requires continuous administration of the inducer, resulting in complex in vivo pharmacokinetics and leakage. Therefore, the field currently lacks a promoter with a short sequence, low leakage during production, high expression on T cells and low expression on B cells and liver and kidney tissues in vivo. This promoter cannot simultaneously meet the multiple requirements of safety, yield, targeting, and simplified procedures, thus hindering the widespread clinical application of lentiviral gene therapy in vivo. Summary of the Invention

[0003] To address the aforementioned shortcomings in existing technologies, this invention provides a T-cell-preferred promoter, its biomaterials, and its applications. This promoter exhibits high-level transcriptional activity in CD3+ T cells within peripheral blood mononuclear cells (PBMCs), while its activity is significantly reduced in HEK293 production cells and B cells, achieving a targeted expression mode of "low leakage during production and high expression in vivo."

[0004] To address the aforementioned technical problems, in a first aspect, the present invention provides a nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0005] On the other hand, the present invention provides a nucleic acid construct comprising a promoter and a heteropolynucleotide sequence as shown in SEQ ID NO: 1, wherein the promoter and the heteropolynucleotide sequence are operatively linked.

[0006] On the other hand, the present invention provides an expression cassette comprising the above-described nucleic acid molecule or the above-described nucleic acid construct.

[0007] On the other hand, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct, or the above-mentioned expression cassette.

[0008] On the other hand, the present invention provides a vector plasmid, wherein the expression vector comprises the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct, or the above-mentioned expression cassette.

[0009] On the other hand, the present invention provides a set of expression vectors, which include the above-mentioned vector plasmids, envelope plasmids and / or packaging plasmids.

[0010] On the other hand, the present invention provides a host cell comprising the above-described nucleic acid molecule, the above-described nucleic acid construct, the above-described expression cassette, the above-described expression vector, the above-described vector plasmid, or the above-described expression vector group.

[0011] On the other hand, the present invention provides viral particles, which comprise the above-mentioned nucleic acid molecules, nucleic acid constructs, expression cassettes, expression vectors, vector plasmids, or groups of expression vectors.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned viral particles, the method comprising the steps of preparing the viral particles from the above-mentioned nucleic acid molecules, the above-mentioned nucleic acid constructs, the above-mentioned expression cassettes, the above-mentioned expression vectors, the above-mentioned vector plasmids, or the above-mentioned expression vector groups.

[0013] On the other hand, the present invention provides the use of the above-mentioned nucleic acid molecules, the above-mentioned nucleic acid constructs, the above-mentioned expression cassettes, the above-mentioned expression vectors, the above-mentioned vector plasmids, the above-mentioned expression vector sets, the above-mentioned host cells or the above-mentioned viral particles in any of the following: (1) the use of the biomaterials in T-cell-specific expression of heterologous polynucleotides; (2) the use of the biomaterials in the preparation of T-cell-specific expression systems for heterologous polynucleotides.

[0014] Beneficial effects: This application provides a T-cell-preferred promoter. This promoter is packaged in HEK293 using the target vectors pHiT-BCMA and pHiTE-BCMA, respectively, and the titer is comparable to that of EF1a-BCMA. After the virus packaged with the plasmid vector containing this promoter (pHiT-BCMA) is transduced into human PBMCs, the CAR expression level of CD3⁺ T cells is comparable to the expression level of the target gene by the plasmid vector containing the EF1a promoter, while the expression in B cells and hepatocytes is <1% (MOI=1), showing strict T-cell preference. This demonstrates that the T-cell-preferred promoter of this gene achieves high expression of CD3+ T cells in PBMCs and low expression in packaging cell line HEK293 and non-target cells (such as B cells and hepatocytes) in in vivo lentiviral vector applications, blocking transgene leakage during production and reducing cytotoxicity and immunogenicity. Furthermore, a single intravenous administration silences the vector in the bloodstream, allowing it to efficiently initiate CAR or gene-edited protein expression after entering T cells, precisely constructing in vivo CAR-T or knockout T cells, simplifying the process, reducing costs, and minimizing off-target integration and antibody production, thus laying the foundation for safe and repeated dosing. Attached Figure Description

[0015] Figure 1 is a flowchart of the packaging process for Group 1 virus, Group 2 virus and Group 3 virus in Example 1.

[0016] Figure 2 is a flowchart of the transfer of group 1 virus, group 2 virus and group 3 virus to PMBC in Example 1.

[0017] Figure 3 is a flowchart of the transduction of Group 1 virus, Group 2 virus and Group 3 virus into B cells and hepatocytes in Example 1. Detailed Implementation

[0018] The specific embodiments of this application will be further described in detail below with reference to the accompanying drawings. These embodiments are only for illustrating this application and are not intended to limit the scope of the invention.

[0019] In a first aspect, the present invention provides a nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0020] In a specific embodiment, the nucleic acid molecule can promote the transcription or expression of operable heteropolynucleotides in T cells.

[0021] In a specific embodiment, the T cells are selected from mice, rats, hamsters, monkeys, or humans.

[0022] In a specific embodiment, the nodular nucleic acid molecule further includes having at least 85%, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1, and being capable of specifically initiating or promoting the expression of operablely linked heteropolynucleotides in T cells, and / or not being capable of initiating or promoting the expression of operablely linked heteropolynucleotides in B cells or hepatocytes.

[0023] On the other hand, the present invention provides a nucleic acid construct comprising a promoter and a heteropolynucleotide sequence as shown in SEQ ID NO: 1, wherein the promoter and the heteropolynucleotide sequence are operatively linked.

[0024] In a specific embodiment, the nucleic acid construct further includes polyA.

[0025] In a specific embodiment, the polyA is operatively linked to a heterologous polynucleotide sequence.

[0026] In a specific embodiment, the heteropolynucleotide encodes a transmembrane protein. In a specific embodiment, the transmembrane protein is a type I transmembrane protein.

[0027] In a specific embodiment, the nodular nucleic acid molecule further includes having at least 85%, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2, and being able to enhance the expression of the target gene specifically by the promoter shown in SEQ ID NO:1 in T cells, but not being able to specifically initiate the expression of the target gene in B cells or epithelial cells or not being able to specifically initiate the expression of the target gene in B cells or epithelial cells with the promoter shown in SEQ ID NO:1.

[0028] In a specific embodiment, the nucleic acid construct further includes an enhancer with a nucleotide sequence as shown in SEQ ID NO: 2, the enhancer being operatively linked to the heterologous nucleic acid sequence.

[0029] In a specific embodiment, the nucleic acid construct sequentially includes a promoter, a heteropolynucleotide, and an enhancer; in a specific embodiment, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO: 6.

[0030] On the other hand, the present invention provides an expression cassette comprising the above-described nucleic acid molecule or the above-described nucleic acid construct.

[0031] On the other hand, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct, or the above-mentioned expression cassette.

[0032] On the other hand, the present invention provides a vector plasmid, wherein the expression vector comprises the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct, or the above-mentioned expression cassette.

[0033] In a specific embodiment, the vector plasmid is selected from adeno-associated virus vectors, lentiviral vectors, adenovirus vectors, retroviral vectors, herpes simplex virus vectors, vaccinia virus vectors, or baculovirus vectors.

[0034] In a specific embodiment, the lentiviral vector further includes a 5' long terminal repeat sequence, a packaging signal sequence, a Rev response element, a central polypurine region / central termination sequence, and / or a 3' self-inactivating long terminal repeat sequence.

[0035] In specific embodiments, the backbone vector of the vector plasmid includes pLVX-IRES-Puro, pLVX-PuropLVX-IRES-ZsGreen1, pLVX-EF1α-IRES-Puro, pLVX-CMV-MCS-IRES-RFP, pLenti-CMV-GFP-Puro, pLenti-CMV-MCS-Puro, pLenti-EF1α-MCS-P2A-Puro, and pCDH-CMV-MCS-E. F1-Puro, pCDH-CMV-MCS-EF1-copGFP, pCDH-MSCV-MCS-EF1-GFP, pWPI, pWPXL, pLV-EGFP, pLV-mCherry, SICO-E F1α-GFP, pSICO-CMV-mCherry, pLVCT-tTR-KRAB, pLEX-MCS-IRES-Puro, pLJM1-Flag-GFP, pLV-Fluc-P2A-Puro.

[0036] In a specific embodiment, the vector plasmid is a vector obtained by inserting the above-mentioned nucleic acid construct into the above-mentioned backbone vector.

[0037] In a specific embodiment, the insertion site is between CMV and WPRE.

[0038] On the other hand, the present invention provides a set of expression vectors, which include the above-mentioned vector plasmids, envelope plasmids and / or packaging plasmids.

[0039] As described in this application, the term "packaging plasmid" encodes proteins essential for viral replication and the formation of the internal structure of the particle, but lacks the packaging signal Ψ, therefore its own RNA is not packaged into the viral particle. It typically includes: Gag: a structural protein of the virus that forms the capsid and matrix. Pol: viral enzymes, including reverse transcriptase, integrase, and proteases, responsible for converting the RNA genome into DNA and integrating it into the host genome.

[0040] Vector plasmids are plasmids that are encapsulated within viral particles and ultimately integrated into the host genome to express the target gene. They typically contain (1) 5′LTR (or Δ5′LTR) + 3′LTR; (2) ψ (packaging signal); (3) RRE; (4) cPPT (optional, to enhance transduction); (5) target gene; (6) promoter; (7) polyA; (8) WPRE (to enhance mRNA stability). In specific embodiments, the backbone plasmids include pLVX-Puro, pCDH-CMV-MCS-EF1α-copGFP, pLenti-CMV-GFP-P2A-Puro, or pLEX-MCS.

[0041] Packaging plasmids refer to transviral structures / enzyme proteins that are not encapsulated within viral particles. They typically contain (1) gag (matrix + capsid + nucleocapsid); (2) pol (reverse transcriptase + integrase + protease); (3) tat (still required in generation 1; removed in generation 3); and (4) REV response element (Rev gene). In specific embodiments, the backbone plasmid includes psPAX2 (Addgene#12260), pCMV-ΔR8.74, or pLP1 (Thermo triple plasmid kit). In specific embodiments, the packaging plasmid is a lentiviral packaging plasmid. The lentiviral packaging plasmid is a third-generation lentiviral packaging plasmid. The third-generation lentiviral plasmid includes pHi002 (Hillgene HG-pHi002-G) and pHi003 (Hillgene HG-pHi003-G).

[0042] Envelope plasmids are plasmids that provide heterologous coat glycoproteins and determine the host range (tropism) of viral particles. They typically contain only one outer membrane protein, ORF, located downstream of the CMV or RSV promoter. In specific embodiments, backbone plasmids include pMD2.G (VSV-G, Addgene #12259), pCMV-RD114, pCMV-BaEV-R / TR, pCMV-GALV-TR, or pCAG-EboV-GP. In specific embodiments, the envelope plasmid includes pHi001 (Hillgene HG-pHi001-G).

[0043] On the other hand, the present invention provides a host cell comprising the above-described nucleic acid molecule, the above-described nucleic acid construct, the above-described expression cassette, the above-described expression vector, the above-described vector plasmid, or the above-described expression vector group.

[0044] In a specific embodiment, the host cell is a microorganism, insect cell, plant cell, animal cell, or mammalian cell.

[0045] On the other hand, the present invention provides viral particles, which comprise the above-mentioned nucleic acid molecules, nucleic acid constructs, expression cassettes, expression vectors, vector plasmids, or groups of expression vectors.

[0046] In a specific embodiment, the virus particles are selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0047] In a specific embodiment, the virus particle comprises nucleic acid and a protein capsid.

[0048] In a specific embodiment, the nucleic acid includes the above-described nucleic acid molecule, the above-described nucleic acid construct, the above-described expression cassette, or the above-described expression vector.

[0049] In a specific embodiment, the protein capsid is selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0050] In a specific embodiment, the virus particle further includes a lipid envelope, which is selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0051] In a specific embodiment, the viral particle further includes a membrane protein selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0052] On the other hand, the present invention provides a method for preparing the above-mentioned viral particles, the method comprising the steps of preparing the viral particles from the above-mentioned nucleic acid molecules, the above-mentioned nucleic acid constructs, the above-mentioned expression cassettes, the above-mentioned expression vectors, the above-mentioned vector plasmids, or the above-mentioned expression vector groups.

[0053] In a specific embodiment, the method includes the step of co-infecting host cells with the nucleic acid molecule of claim 1, the nucleic acid construct of claim 2 or 3, the expression cassette of claim 4, the expression vector of claim 5, or the vector plasmid of claim 6, along with the envelope plasmid and the packaging plasmid.

[0054] In specific embodiments, the host cells include the HEK-293 series (such as HEK-293, HEK-293T, HEK-293A, HEK-293SF / 293F / 293S, VirusExpress®293T / 293AAV), HK-2, Vero, BSC-1 / BSC-40 / CV-1, Frhk-4, LLC-MK2, HeLa, A549, HT1080, HepG2, U2OS, 9L / C6, BHK-21, CHO-K1 / CHO-S, THP-1, Raji / Daudi, K562, and NIH-3T3. / 3T3-Swiss, L929, MRC-5 / WI-38, Huh-7, RK-13, PK-15, MDCK, MDBK, Mv.1.Lu / M-9, Sf9, Sf21, Sf-RVN®, HighFive™, TniPRO™, Mimic™Sf9, primary chicken embryo fibroblasts, primary duck embryo fibroblasts, quail QT-6 / QT-35 cell lines, EPC, CHSE-214, FHM, SSN-1, BME / CT, C6 / 36, AP-61, primary bovine testicular cells, primary sheep testicular cells, ST, MARC-145, IBRS-2.

[0055] On the other hand, the present invention provides the use of the above-mentioned nucleic acid molecules, nucleic acid constructs, expression cassettes, expression vectors, vector plasmids, expression vector sets, host cells, or viral particles in T-cell-specific expression or in the preparation of T-cell-specific expression products.

[0056] In specific embodiments, the use includes not expressing the substance in B cells and hepatocytes.

[0057] In specific embodiments, the product is a nucleic acid molecule, a nucleic acid construct, an expression cassette, an expression vector, a vector plasmid, an expression vector set, or a viral particle.

[0058] In a specific embodiment, the product is a drug.

[0059] In a specific embodiment, the T cells include CD3+ T cells, αβ T cells, γδ T cells, CD4+ T cells, CD8+ T cells, naive T cells, effector T cells, memory T cells, natural killer T cells, mucosa-associated invariant T cells, and regulatory CD8+ T cells.

[0060] In specific embodiments, the memory T cells include central memory T cells, effector memory T cells, tissue-resident memory T cells, and stem cell-like memory T cells.

[0061] In a specific embodiment, the CD4+ T cells include Th1 cells, Th2 cells, Th17 cells, follicular helper T cells, regulatory T cells (natural Tregs or induced / adaptive Tregs), Th9 cells, Th22 cells, and helper T cell precursors.

[0062] In a specific embodiment, the CD8+ T cells include naive CD8+ T cells, effector CD8+ T cells, memory CD8+ T cells, exhausted CD8+ T cells, terminally differentiated effector CD8+ T cells, and memory stem cell-like CD8+ T cells.

[0063] In a specific embodiment, the nucleic acid construct further includes an enhancer with a nucleotide sequence as shown in SEQ ID NO: 2, the enhancer being operatively linked to the heterologous nucleic acid sequence; in a specific embodiment, the nucleotide sequence of the heterologous nucleic acid sequence is shown in SEQ ID NO: 3.

[0064] In a specific embodiment, the virus particles or virus vector are selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0065] The above-mentioned nucleic acid molecules, nucleic acid constructs, expression cassettes, expression vectors, vector plasmids, expression vector sets, host cells, or viral particles are used in any of the following: (1) in T-cell-specific expression of heterologous polynucleotides; (2) in a system for preparing T-cell-specific expression of heterologous polynucleotides; in a specific embodiment, the system is selected from viral delivery systems; in a specific embodiment, the system is selected from viral particles.

[0066] In specific embodiments, the host cells are selected from 293, 293T, 293A, H293SF, HK-2, Vero, BSC-1, Frhk-4, LLC-MK2, HeLa, A549, HT1080, HepG2, U2OS, 9L / C6, BHK-21, CHO-K1 / CHO-S, THP-1, K562, NIH-3T3, L929, MRC-5 / WI-38, and Hu. h-7, RK-13, PK-15, MDCK, MDBK, Mv.1.Lu / M-9, Sf9, Sf21, primary chicken embryo fibroblasts, primary duck embryo fibroblasts, quail QT-6 / QT-35 cell lines, EPC, CHSE-214, FHM, SSN-1, BME / CT, C6 / 36, AP-61, primary bovine testicular cells, primary sheep testicular cells, ST, MARC-145, IBRS-2.

[0067] In a specific embodiment, the T cells are selected from CD3. + T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, including naive T cells, effector T cells, memory T cells, natural killer T cells, mucosa-associated invariant T cells, and regulatory CD8+ T cells; in a specific embodiment, the T cells are selected from peripheral blood mononuclear cells.

[0068] In a specific embodiment, the specific expression includes no expression or low expression in B cells and hepatocytes.

[0069] In a specific embodiment, the specific expression includes a viral transduction rate ≤ 5.9%; in a specific embodiment, the viral transduction rate is ≤ 5.4%, ≤ 2.9%, ≤ 2.5%, ≤ 2.3%, ≤ 1.9%, ≤ 1.7%, ≤ 1.3%, ≤ 1.0%, 0.9% ≤ 0.9%, ≤ 0.3%, ≤ 0.2%; in a specific embodiment, the viral MOI value is 1, 2, or 4.

[0070] The term “expression” refers to, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0071] The term "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, the coding sequence being operatively linked to a suitable control sequence capable of influencing DNA expression in a suitable host. Such control sequences may include promoters influencing transcription, optional operon sequences controlling transcription, sequences encoding suitable ribosome-binding sites on mRNA, enhancers, and sequences controlling the termination of transcription and translation. In a specific embodiment, the vector is a pLVX-IRES-Puro vector.

[0072] The term "fragment" refers to a polypeptide lacking one or more amino acids at its amino and / or carboxyl terminus, wherein the fragment possesses phytase activity, or wherein the fragment is an ALAB fragment that forms a functional regulatory subunit of the lactose synthase (LS) heterodimer. The fragment may arise naturally during polypeptide expression and / or purification, or may be the result of expression of a modified nucleotide sequence expressing the fragment, or the result of targeted removal of amino acids from the amino and / or carboxyl terminus.

[0073] The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0074] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid," "polynucleotide," and "nucleic acid molecule" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of this invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0075] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a way that does not originally exist in nature to contain segments of nucleic acid, or that is synthesized and contains one or more control sequences operatively linked to the nucleic acid sequence.

[0076] The term "operably linked" refers to a relationship (including but not limited to juxtaposition) in which specified components are positioned to allow them to function in the intended manner. For example, a regulatory sequence (such as a promoter or enhancer) is operably linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence. Specifically, it can refer to a functional link between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. It can also refer to a functional link between other regulatory elements (such as enhancers) and a target gene to regulate the transcription and / or expression of the target gene.

[0077] The term "promoter" generally refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and assists or promotes the transcription of polynucleotides. Promoters can be synthesized, modified, or derived from known or naturally occurring promoters or other promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters disclosed herein can include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences known or provided herein.

[0078] Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0079] The sequence information involved in this application is shown in Table 9.

[0080] Table 9. Related sequence information

[0081] In Example 1, the 498 bp Mus musculus CD3d promoter (nucleotides as shown in SEQ ID NO: 1), the 433 bp Homo sapiens CD3 delta enhancer sequence (nucleotides as shown in SEQ ID NO: 2), and the BCMA sequence (nucleotides as shown in SEQ ID NO: 3) were cloned into the third-generation self-inactivated lentiviral vector pLVX-IRES-Puro (TAKARACAT NO. 632186), respectively, to obtain the EF1a-BCMA vector, pHiT-BCMA vector, and pHiTE-BCMA vector. The EF1a-BCMA vector was obtained by inserting the sequence shown in SEQ ID NO: 4 between the CMV and WPRE of the pLVX-IRES-Puro vector; the pHiT-BCMA vector was obtained by inserting the sequence shown in SEQ ID NO: 5 between the CMV and WPRE of the pLVX-IRES-Puro vector; and the pHiTE-BCMA vector was obtained by inserting the sequence shown in SEQ ID NO: 6 between the CMV and WPRE of the pLVX-IRES-Puro vector. The vector was obtained using the above method and the following studies were performed. The nucleotide sequence of BCMA is shown from position 499 to 1608 of SEQ ID NO: 6.

[0082] a) Comparison of TU packaging using three vectors: EF1a-BCMA, pHiT-BCMA, and pHiTE-BCMA: Lentiviral virus was packaged using the 293T (Sigma-Aldrich VP001) cell line, with three different promoters: EF1a (corresponding to EF1a-BCMA during viral transfection), pHiTE (corresponding to pHiT-BCMA during viral transfection), and pHiT (corresponding to pHiTE-BCMA during viral transfection).

[0083] The lentiviruses tested in this study were packaged using a third-generation lentivirus and a four-plasmid system. Specific information regarding the packaging cells, envelope plasmids, packaging plasmids (including packaging plasmid-1 and packaging plasmid-2), and shuttle plasmids is shown in Table 1 below.

[0084] The nucleotide sequence of the EF1a promoter is shown in positions 1-1246 of SEQ ID NO: 4.

[0085] Table 1

[0086] The virus packaging process is shown in Figure 1. The specific operation is as follows: Cells are resuscitated from the 293T working cell bank (WCB) according to the parameters in Table 2 to obtain resuscitated 293T cells. The 293T cells used in this application were purchased from Sigma-Aldrich VP001.

[0087] Table 2 Control Parameters for Cell Processing During the Recovery Phase

[0088] After resuscitation, following the process control parameters for the amplification stage in Table 3, the resuscitated 293T cells were transferred to 125 mL disposable sterile Erlenmeyer flasks and amplified using HiLenti 293T suspension medium (Hillgene HG-MD001-1000) (the initial concentration of 293T cells was (0.5±0.1)×10⁻⁶). 6 The cells / mL were amplified under the following conditions: 37℃, 5% CO2, 160 rpm. After 72 h of amplification, amplified 293T cells were obtained.

[0089] Table 3 Process control parameters for the amplification stage

[0090] The expanded 293T cells were divided into 2×10 6 Cells / mL were seeded in 50 ml of HiLenti 293T suspension medium. After seeding, samples were taken daily and cell density and viability were measured using a Countstar Rigel S2 analyzer. After 3 days of culture, the viable cell density reached (6.0 ± 1.0) × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability not less than 90%, to obtain 293T cells to be transfected and lentivirus packaging, the specific method is as follows: Preparation of PEI solution: Add 150ul of PEI pro (Sartorius 101000026) PEI solution to 1.1ml of HiLenti 293T suspension medium to obtain PEI solution.

[0091] Preparation of Group 1 DNA solution: According to Table 1, add EF1α-BCMA vector, pHi001, pHi002 and pHi003 to 1.18 ml of HiLenti 293T suspension medium in a molar ratio of 2:1:1:1 to make the final concentration of EF1α-BCMA vector 0.04 ug / ml, and obtain Group 1 DNA solution.

[0092] Preparation of Group 2 DNA solution: According to Table 1, EF1α-BCMA vector, pHi001, pHi002 and pHi003 were added to 1.18 ml of HiLenti 293T suspension medium in a molar ratio of 2:1:1:1 to make the final concentration of pHiT-BCMA vector 0.04 ug / ml, thus obtaining the Group 2 DNA solution.

[0093] Preparation of Group 3 DNA solution: According to Table 1, pHiTE-BCMA vector, pHi001, pHi002 and pHi003 were added to 1.18 ml of HiLenti 293T suspension medium in a molar ratio of 2:1:1:1 to make the final concentration of pHiT-BCMA vector 0.04 ug / ml, thus obtaining the Group 3 DNA solution.

[0094] In a biosafety cabinet, prepare PEI and DNA solutions (preparation method as described above). After both solutions stand at room temperature for 5 minutes, slowly add the PEI solution to the group 1 DNA solution, group 2 DNA solution, and group 3 DNA solution respectively through tubing at a mass ratio of 2:1. Keep the DNA solution agitated during the addition process. Let stand at room temperature for 15 minutes, then slowly add it to the reactor for transfection. 6-8 hours after transfection, add 20% of the packaged volume of HiLenti® transfection enhancer (Hillgene HG-MD005-125) and 2% of the packaged volume of 200 mM glutamine solution (Gibco 35050061). After 48±2 hours of transfection, collect the virus by centrifuging the solution at 4000 g for 5 min and collecting the supernatant to obtain group 1 virus, group 2 virus, and group 3 virus.

[0095] The transduction titer of group 1 virus, group 2 virus and group 3 virus was tested as follows: 1. Cell preparation: Jurkat cells (ATCC TIB-152) were cultured in a 37℃, 5% CO2 incubator for 1-3 days until the cells were in good condition and sufficient in number.

[0096] 2. Prepare the infection culture medium: 90% FBS (ATCC® 30-2020™) + 1640 (ATCC® 30-2001™) + 0.1% Polybrene (10 mg / ml).

[0097] 3. Plate preparation: Add 400 μL of Jurkat cell suspension to each well of a 24-well plate, with a cell count of 1 E+05 cells per well.

[0098] 4. Virus inoculation: Dilute the virus with infection medium, at least 3 dilutions (samples were taken at serial dilution ratios of 100, 400, and 800), add 100 μl of virus dilution to each well, and add 100 μl of infection medium to the biological control.

[0099] 5. Replenishing fluid: 20-24 hours after viral infection, add 500 μL of 1640 medium containing 10% FBS to each well.

[0100] 6. Harvesting: 68-72 hours after viral infection, harvest the cells and transfer them to pre-labeled 5ml flow cytometry tubes.

[0101] 7. Flow cytometry: a) Centrifuge at 400g for 3 minutes. Discard the supernatant of the culture medium in the flow cytometry tube, and place the flow cytometry tube on a vortex mixer to disperse the cell pellet.

[0102] b) Add 1 ml of PBS to each tube, centrifuge at 400 g for 3 minutes. Discard the supernatant and vortex for 5 seconds to disperse the cells.

[0103] c) Add 2 μl of “Goat-IgG-FITC” to the ISO group and 2 μl of “FITC-Labeled Human BCMA,Fc Tag (ARCO BCA-HF254)” to the experimental group. After shaking and mixing, incubate at 4°C in the dark for 30 minutes.

[0104] d) Add 1 ml of PBS to each tube and mix well. Centrifuge at 400 g for 3 minutes. Discard the supernatant and vortex for 5 seconds to disperse the cells. Repeat the washing once.

[0105] e) Discard the supernatant, add 400 μl of PBS to the flow cytometer, and vortex for 2 seconds to mix.

[0106] f) On-machine detection: Based on the biological blank control, adjust the voltage and gate to detect the "CAR" positivity rate of the sample tube.

[0107] 8. Data processing a) Sample CAR positivity rate = Sample CAR positivity rate detection value - Blank control CAR positivity rate detection value.

[0108] b) Transduction titer (TU / ml) = dilution factor * CAR positivity rate * cell count / virus sample volume (ml).

[0109] c) The CAR positivity rate is taken as the result within the linear range (5-30%); the cell count is 1E+5; the virus sample volume is 0.1ml.

[0110] 9. Result Interpretation a) When the CAR positivity rate of the biological blank control is less than 5%, the experimental results are valid.

[0111] b) Report the results of the transduction titer test as the average titer within the linear interval of the CAR positivity rate.

[0112] Table 4b) Comparison of the effects of transducer PBMC:

[0113] The virus was packaged using the EF1a-BCMA vector, pHiT-BCMA vector, and pHiTE-BCMA vector obtained in a), and transduced into human PMBC. Two donors were transduced, and the proportion of CAR-positive T cells to total T cells was measured. The procedure is shown in Figure 2. Specific operations are as follows: D0) uses fresh peripheral blood from a healthy donor. D0 uses Ficoll (GE)... 17-1440-03) Isolation of PBMCs, the specific method is as follows: (1) Centrifuge 1000×g of peripheral blood for 15min; (2) Transfer the upper plasma layer after centrifugation to a new centrifuge tube to obtain human lymphocyte separation solution, and dilute the remaining 0.9% precipitate with 10ml of sodium chloride injection (Foshan Haolang National Drug Approval Number H20034174) to obtain diluted peripheral blood; (3) Add 15ml of human lymphocyte separation solution to a 50ml centrifuge tube, and slowly add 20ml of diluted peripheral blood along the tube wall to the separation solution; (4) Centrifuge at 400×g (increase 3, decrease 0) for 30min, and use a pipette to aspirate the white membrane layer (white membrane layer is PBMC cells) to a new centrifuge tube, and add complete culture medium to (X-VIVO15 (Lonza BEBP04-054Q) + 200IU / ml) (5) Centrifuge at 400×g for 10 min, discard the supernatant, add complete culture medium to 40 ml, and mix well by pipetting; (6) Centrifuge at 400×g for 10 min, discard the supernatant, add complete culture medium (200 IU / ml IL-2 (Beijing Sihuan Biopharmaceutical Co., Ltd., National Drug Approval Number S20040018) + X-VIVO15 (Lonza BEBP04-054Q)) to resuspend the cells, and mix well by pipetting; (7) Take 2 samples for counting, and adjust the cell density of PBMC to 1-2E+06 cells / ml (±10%) according to the counting results; (8) Transfer the adjusted PBMC to a 6-well plate and place it in a carbon dioxide incubator at 37℃ and 5% CO2 for culture.The following procedures were performed: D1: After culturing for 24-36 hours, PBMC cells were transfected with complete culture medium (200 IU / ml IL-2 (Beijing Sihuan Biopharmaceutical Co., Ltd., National Drug Approval Number S20040018) + X-VIVO15 (Lonza BEBP04-054Q)) to a density of 1E5 cells / mL. 400 μL of this medium was added to each well of a 24-well plate (i.e., 1E5 cells per well). Group 1 virus, Group 2 virus, and Group 3 virus were added to a MOI of 2.0 for transfection. Each virus was repeated twice (two Donor blood samples were used for transduction testing of each virus). The MOI (Multiple of Infection) was calculated as: MOI = (Virus titer × Virus volume) / Cell number. D2: 24 hours after transfection, serum culture medium (X-VIVO15 medium with added IL-2) was added to each well. The final concentration was adjusted to 200 IU / ml; 3% volume of inactivated plasma was added to 2 ml; D3: 48 h after transfection, samples were taken for positivity rate detection. The specific operation was as follows: Cells were stained with BV786 Mouse Anti-Human CD3 (BD ​​Biosciences 563800) and PE-Labeled Human BCMA Protein, His Tag (ACRO BCA-HP2H2), respectively. Flow cytometry was used for detection. First, CD3+ cells were circled as T cells, and then BCMA+ (CAR+) CAR-positive T cells were circled among the T cells. The proportion of CAR-positive T cells to total T cells was detected, and the results are shown in Table 6. The results show that the proportion of CAR-positive T cells to total T cells was basically the same after transduction of PBMCs by the three promoter combinations.

[0114] CAR-positive T cells as a percentage of total T cells = number of CAR-positive T cells / total number of T cells.

[0115] Table 5

[0116] Table 6

[0117] Transduction efficiency of different promoter combinations on B cells and hepatocytes: C) Nalm6 cell line (ATCC CRL-3273 human B lymphocytic leukemia cells) was selected as the test B cells and HepG2 cell line (ATCC HB-8065) ​​was selected as the test hepatocytes. Different MOIs and viruses were used to transduce the two cell lines respectively.

[0118] The experimental procedure is shown in Figure 3. The specific operations are as follows: D1: According to Table 7, adjust the density of the two cell lines (Nalm6 and HepG2) to 1E5 cells / mL using culture medium (RPMI-1640 Medium (ATCC® 30-2001™) + 10% Fetal Bovine Serum (ATCC® 30-2020™). In a 24-well plate, add 400 μL of medium per well (i.e., 1E5 cells per well). Based on the MOI in Table 7, add three viruses (group 1 virus, group 2 virus, and group 3 virus) for transfection. D2: 24 hours after transfection, add 500 μL of culture medium (RPMI-1640 Medium (ATCC® 30-2001™) + 10% Fetal Bovine Serum (ATCC® 30-2020™)) to each well. 30-2020™); D3: No additional treatment was performed; cells were cultured in a CO2 incubator at 37°C with 5% CO2. D4: 72 hours after transfection, samples were taken and stained with E-Labeled Human BCMA Protein, His Tag (ACRO BCA-HP2H2) by flow cytometry to detect the BCMA positivity rate (transduction rate %). The results are shown in Table 8. The transduction positivity rate of Nalm6 and HepG2 cells using the HiPT and HipTE promoters was less than 10%, while the transduction efficiency of the EF1a promoter for HepG2 cells was greater than 50%. The HiPT and HipTE promoters did not transduce B cells or hepatocytes. In summary, the virus packaged using the HiPT and HipTE promoters, while ensuring the same viral titer as the EF1a promoter, has a stable transduction efficiency for T cells and does not transduce B cells or hepatocytes.

[0119] BCMA positivity rate (transduction rate %) = BCMA+(CAR+) cells / total cells.

[0120] Table 7

[0121] Table 8

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A biomaterial, characterized in that, The biological material is any one of the following: (1) a nucleic acid molecule, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) a nucleic acid construct, the construct comprising a promoter and a heteropolynucleotide sequence as shown in SEQ ID NO: 1, the promoter and the heteropolynucleotide sequence being operatively linked; (3) an expression cassette, the expression cassette comprising the nucleic acid molecule of (1) or the nucleic acid construct of (2); (4) an expression vector, the expression vector comprising the nucleic acid molecule of (1), the nucleic acid construct of (2) or the expression cassette of (3); (5) a vector plasmid, the expression vector comprising the nucleic acid molecule of (1), the nucleic acid construct of (2), the expression cassette of (3) or the expression vector of (4); (6) an expression vector set, the expression vector... The present invention includes (5) the vector plasmid, envelope plasmid and / or packaging plasmid; (7) the host cell, wherein the host cell comprises the nucleic acid molecule of (1), the nucleic acid construct of (2), the expression cassette of (3), the expression vector of (4), the vector plasmid of (5) or the expression vector group of (6); (8) the virus particle, wherein the virus particle comprises the nucleic acid molecule of (1), the nucleic acid construct of (2), the expression cassette of (3), or the virus particle is prepared from the expression vector of (4), the vector plasmid of (5) or the expression vector group of (6).

2. The biomaterial as described in claim 1, characterized in that, (2) The nucleic acid construct further comprises an enhancer with a nucleotide sequence as shown in SEQ ID NO: 2, the enhancer being operatively linked to the heterologous polynucleotide sequence; preferably, the nucleotide sequence of the heterologous nucleic acid sequence is shown in SEQ ID NO:

3.

3. The biomaterial as described in claim 2, characterized in that, The nucleic acid construct comprises, in sequence, a promoter, a heteropolynucleotide, and an enhancer; preferably, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO:

6.

4. The biomaterial as described in claim 1, characterized in that, (8) The virus particles are selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus or baculovirus.

5. A method for preparing virus particles, characterized in that, The method includes the step of preparing the virus particles from the biomaterial of claim 1.

6. Use of the biomaterial according to any one of claims 1-4 in any of the following: (1) use of the biomaterial in T-cell-specific expression of heteropolynucleotides; (2) use of the biomaterial in the preparation of a T-cell-specific expression system for heteropolynucleotides; preferably, the system is selected from a viral delivery system; more preferably, the system is selected from a viral vector; even more preferably, the viral vector is selected from adeno-associated virus, lentivirus, adenovirus, retrovirus, herpes simplex virus, vaccinia virus or baculovirus.

7. The biomaterial as described in any one of claims 1-4 or the use as described in claim 6, characterized in that, The host cells were selected from 293, 293T, 293A, H293SF, HK-2, Vero, BSC-1, Frhk-4, LLC-MK2, HeLa, A549, HT1080, HepG2, U2OS, 9L / C6, BHK-21, CHO-K1 / CHO-S, THP-1, K562, NIH-3T3, L929, MRC-5 / WI-38, and Huh-7. RK-13, PK-15, MDCK, MDBK, Mv.1.Lu / M-9, Sf9, Sf21, primary chicken embryo fibroblasts, primary duck embryo fibroblasts, quail QT-6 / QT-35 cell lines, EPC, CHSE-214, FHM, SSN-1, BME / CT, C6 / 36, AP-61, primary bovine testicular cells, primary sheep testicular cells, ST, MARC-145, IBRS-2.

8. The use as described in claim 6, characterized in that, The T cells are selected from CD3. + T cells, αβ T cells, γδ T cells, CD4 + T cells, CD8 + T cells, naive T cells, effector T cells, memory T cells, natural killer T cells, mucosa-associated invariant T cells, regulatory CD8+ + T cells; preferably, the T cells are selected from peripheral blood mononuclear cells; preferably, the memory T cells are selected from central memory T cells, effector memory T cells, tissue-resident memory T cells, and stem cell-like memory T cells; preferably, the CD4+ T cells are selected from Th1 cells, Th2 cells, Th17 cells, follicular helper T cells, regulatory T cells, Th9 cells, Th22 cells, and helper T cell precursors; preferably, the CD8+ T cells are selected from Th1 cells, Th2 cells, Th17 cells, follicular helper T cells, regulatory T cells, Th9 cells, Th22 cells, and helper T cell precursors; preferably, the CD8+ T cells are selected from Th1 cells, Th2 cells, Th17 cells, follicular helper T cells, regulatory T cells, Th9 cells, Th22 cells, and helper T cell precursors; preferably, the CD8+ T cells are selected from Th1 cells, Th2 cells, Th17 cells, Th17 cells, Th18 ... + T cells are selected from naïve CD8 cells. + T cells, effector CD8 + T cells, memory CD8 + T cells, depleted CD8 + T cells, terminal differentiation effector CD8 + T cells, memory stem cell-like CD8 + T cells; and / or, the T cells are derived from mononuclear cells; preferably, the mononuclear cells are derived from human peripheral blood.

9. The use as described in claim 6, characterized in that, The specific expression includes no expression or low expression in B cells and hepatocytes.

10. The use as described in claim 6, characterized in that, The specific expression includes a viral transduction rate of ≤5.9%; preferably, the viral transduction rate is ≤5.4%, ≤2.9%, ≤2.5%, ≤2.3%, ≤1.9%, ≤1.7%, ≤1.3%, ≤1.0%, 0.9% ≤0.9%, ≤0.3%, ≤0.2%; more preferably, the viral MOI value is 1, 2, or 4.

Images