Viral vector for hematopoietic stem cells and use thereof

Abstract

This application provides a viral vector for hematopoietic stem cells and its application. A first aspect of this application proposes a lentiviral vector packaging system for hematopoietic stem cells, the lentiviral vector packaging system comprising a recombinant expression vector, the recombinant expression vector comprising nucleotide sequences encoding thrombopoietin and FMS-associated receptor tyrosine kinase 3 ligand. This invention, by constructing thrombopoietin and FMS-associated receptor tyrosine kinase 3 ligand onto nucleic acid molecules, thereby enabling their expression on the surface of a lentiviral vector, shows that compared to direct addition, it can effectively activate and enhance the stemness or immune activity of various cells, including hematopoietic stem cells, at lower dosages.

Description

Technical Field

[0001] This application relates to the field of cell biology technology, and in particular to a viral vector for hematopoietic stem cells and its application. Background Technology

[0002] Gene therapy, represented by cell therapy, has shown great promise in basic research, and some strategies have been successfully translated into clinical research and application. Approved cell therapies usually involve constructing specific functional cells in vitro and then reinfusing them into the patient. The process is complex, highly individualized, and difficult to promote. In recent years, researchers have been committed to directly transducing target cells in patients. However, this places high demands on the in vivo transduction vectors for transduced cells: (1) precise targeting to reduce off-target effects and vector depletion caused by transduction of non-target cells; (2) high transduction efficiency and stimulation and activation of cells; and (3) low immunogenicity.

[0003] Hematopoietic stem cells (HSCs) are ideal target cells for cell therapy. Their genetic information is inherited through cell division and differentiation, passing on to all lineages from which they originate. Currently, for diseases such as β-thalassemia and sickle cell disease (SCD), HSC transplantation is the only proven curative option. Therefore, developing targeted in vivo transduction vectors for HSCs can effectively reduce the treatment risks and costs for these diseases, and is of great significance for accelerating the clinical application of cell therapy.

[0004] Currently proven effective eukaryotic delivery vectors can be divided into viral vectors and non-viral vectors. Non-viral vectors have simpler structures and are easy to mass-produce, but they are more difficult to target in vivo. They mainly achieve non-targeted transduction in areas such as the liver and alveoli by changing the administration method. In comparison, viral vectors, especially lentiviral vectors (LV), can achieve targeted transduction to specific cells by engineering envelope glycoproteins (such as vesicular stomatitis virus glycoprotein, VSV-G). However, they still have the problem of low transduction efficiency in cell types such as hematopoietic stem cells. In addition, conventional lentiviral vectors exhibit low transduction efficiency when applied to resting hematopoietic stem cells or lymphocytes, and increasing the viral dosage may affect the stemness or immune activity of the target cells. Therefore, there is an urgent need for a vector that can balance targeted transduction efficiency and stimulation effect. Summary of the Invention

[0005] The main objective of this invention is to propose a viral vector for hematopoietic stem cells and its application.

[0006] To achieve the above objectives, a first aspect of this application provides a lentiviral vector packaging system for hematopoietic stem cells, the lentiviral vector packaging system comprising a recombinant expression vector, the recombinant expression vector comprising nucleotide sequences encoding thrombopoietin (TPO) and FMS-like receptor tyrosine kinase 3 ligand (FLT3L), the nucleotide sequence encoding thrombopoietin being shown in SEQ ID NO. 2, and the nucleotide sequence encoding FMS-like receptor tyrosine kinase 3 ligand being shown in SEQ ID NO. 3.

[0007] In some embodiments, the recombinant expression vector further includes a nucleotide sequence encoding a stem cell factor (SCF).

[0008] In some embodiments, the nucleotide sequence encoding the stem cell factor is shown in SEQ ID NO.1.

[0009] In some embodiments, the amino acid sequence of the stem cell factor is shown in SEQ ID NO.4.

[0010] In some embodiments, the amino acid sequence of thrombopoietin is shown in SEQ ID NO.5.

[0011] In some embodiments, the amino acid sequence of the fms-associated receptor tyrosine kinase 3 ligand is shown in SEQ ID NO. 6.

[0012] In some embodiments, the recombinant expression vector further includes at least one of an enhancer, a promoter, an intron, a Kozak concordant sequence, and a polyadenylation signal.

[0013] In some embodiments, the enhancer includes a cytomegalovirus enhancer.

[0014] In some implementations, the promoter includes a cytomegalovirus promoter.

[0015] In some embodiments, the intron includes the human β-globin intron.

[0016] In some embodiments, the polyadenylation signal includes the human β-globin polyadenylation signal (β-globin poly A).

[0017] In some implementations, the recombinant expression vector is a plasmid vector.

[0018] In some embodiments, the lentiviral vector packaging system further includes at least one of an envelope protein particle, a packaging plasmid, and a transfer plasmid.

[0019] In some embodiments, the envelope protein particle includes an insertion site region.

[0020] In some implementations, the insertion site region includes a target antibody coding region.

[0021] In some embodiments, the amino acid sequence of the target antibody encoded by the target antibody coding region is shown in SEQ ID NO. 11.

[0022] In some embodiments, the nucleotide sequence of the target antibody coding region is shown in SEQ ID NO.12.

[0023] In some embodiments, the insertion site region includes a rhabdoviral glycoprotein coding region.

[0024] In some embodiments, the amino acid sequence of the rhabdovirus glycoprotein encoded by the rhabdovirus glycoprotein coding region is shown in SEQ ID NO.13.

[0025] In some embodiments, the nucleotide sequence of the coding region of the rhabdovirus glycoprotein is shown in SEQ ID NO.14.

[0026] In some embodiments, the insertion site region includes the target antibody coding region and the rhabdovirus glycoprotein coding region.

[0027] In some embodiments, the target antibody coding region and the rhabdovirus glycoprotein coding region are connected by a 2A peptide linker region.

[0028] In some embodiments, the insertion site region includes a transmembrane region.

[0029] In some embodiments, the transmembrane region is a PDGFR transmembrane region (TMD).

[0030] In some embodiments, the amino acid sequence of the transmembrane region is shown in SEQ ID NO.7.

[0031] In some embodiments, the nucleotide sequence of the transmembrane region is as shown in SEQ ID NO.8.

[0032] In some embodiments, the insertion site region includes a connector region.

[0033] In some embodiments, the amino acid sequence of the adapter region is shown in SEQ ID NO.9.

[0034] In some embodiments, the nucleotide sequence of the adapter region is shown in SEQ ID NO.10.

[0035] In some embodiments, the insertion site region includes a PDGFR transmembrane region, a linker region, a single-chain antibody coding region, a 2A peptide linker region, and a rhabdovirus glycoprotein mutant coding region.

[0036] In some embodiments, the insertion site region includes, in sequence, a PDGFR transmembrane region, a linker region, a single-chain antibody coding region, a 2A peptide linker region, and a rhabdovirus glycoprotein mutant coding region.

[0037] In some embodiments, the enveloped protein particle further includes at least one of an enhancer, a promoter, an intron, a Kozak concordant sequence, and a polyadenylation signal.

[0038] In some embodiments, the enhancer includes a cytomegalovirus enhancer.

[0039] In some implementations, the promoter includes a cytomegalovirus promoter.

[0040] In some embodiments, the introns include human β-globin introns.

[0041] In some embodiments, the polyadenylation signal includes the human β-globin polyadenylation signal.

[0042] In some embodiments, the enveloped protein grains further include enhancers, promoters, introns, Kozak concordant sequences, and polyadenylation signals.

[0043] In some embodiments, the enveloped protein particle includes, in sequence, an enhancer, a promoter, an intron, a Kozak concordant sequence, an insertion site region, and a polyadenylation signal.

[0044] In some embodiments, the envelope protein particle includes, in sequence, an enhancer, a promoter, an intron, a Kozak co-occurrence sequence, a PDGFR transmembrane region, a linker region, a single-chain antibody coding region, a 2A peptide linker region, a rhabdovirus glycoprotein mutant coding region, and a polyadenylation signal.

[0045] In some embodiments, the 2A peptide linker region includes any one of P2A, T2A, E2A, F2A, etc.

[0046] In some embodiments, the nucleotide sequence of the 2A peptide linker region is shown in SEQ ID NO.16.

[0047] In some embodiments, the backbone of the envelope protein particle is pMD2.G.

[0048] In some embodiments, the packaging plasmid is the helper plasmid psPAX2.

[0049] In some embodiments, the transfer plasmid is the shuttle plasmid pCDH.

[0050] A second aspect of this application provides a recombinant packaging cell for hematopoietic stem cells, said recombinant packaging cell comprising the aforementioned lentiviral vector packaging system.

[0051] In some implementations, the recombinant packaging cells are eukaryotic cells.

[0052] In some implementations, the recombinant packaging cells are animal cells.

[0053] In some implementations, the recombinant packaging cells are mammalian cells.

[0054] In some embodiments, the recombinant packaging cells are either HEK293T cells or HEK293FT cells.

[0055] A third aspect of this application provides a lentivirus for hematopoietic stem cells, which is obtained by transfecting packaging cells with the aforementioned lentivirus vector packaging system.

[0056] In some implementations, the transfected packaging cells are eukaryotic cells.

[0057] In some implementations, the transfected packaging cells are animal cells.

[0058] In some implementations, the transfected packaging cells are mammalian cells.

[0059] In some implementations, the transfected packaging cells are either HEK293T cells or HEK293FT cells.

[0060] This application also relates to a method for preparing the aforementioned lentivirus, comprising the following steps:

[0061] Construct the aforementioned lentiviral vector packaging system;

[0062] The lentiviral vector packaging system was transferred into packaging cells, and the lentivirus was obtained after culturing.

[0063] A fourth aspect of this application provides a composition for hematopoietic stem cells, the composition comprising the aforementioned lentiviral packaging system, the aforementioned recombinant packaging cells, or the aforementioned lentivirus.

[0064] In some embodiments, the composition is a pharmaceutical composition.

[0065] In some embodiments, the composition also includes a pharmaceutically acceptable carrier or diluent.

[0066] A fifth aspect of this application provides a target cell comprising hematopoietic stem cells, the target cell containing the aforementioned lentivirus.

[0067] In some embodiments, the target cells include CD34. + Hematopoietic stem cells.

[0068] A sixth aspect of this application provides the use of the aforementioned lentiviral vector packaging system, the aforementioned recombinant packaged cells, the aforementioned lentivirus, the aforementioned composition, or the aforementioned target cells in the preparation of cell therapy drugs.

[0069] In some implementations, cell therapy includes hematopoietic stem cell therapy.

[0070] The beneficial effects of this invention are:

[0071] This invention constructs thrombopoietin and FMS-associated receptor tyrosine kinase 3 ligand onto nucleic acid molecules, enabling their expression on the surface of lentiviral vectors. Results show that, compared to direct addition, this method effectively activates and enhances the stemness or immune activity of various cell types, including hematopoietic stem cells, at lower dosages. Furthermore, the viral vector using the combination of thrombopoietin and FMS-associated receptor tyrosine kinase 3 ligand maintains a consistently high transduction positivity rate over time during in vitro culture, exhibiting significantly longer-lasting effects. Its application in in vivo transduction holds promise for shortening treatment intervals.

[0072] Furthermore, by co-expressing rhabdovirus envelope protein mutants, single-chain antibodies, and stimulating factors on the surface of lentiviral vectors, the transduction efficiency and specificity of the delivery vector tool are effectively improved, while simultaneously increasing the proportion of stimulated resting cells. This increases safety and significantly reduces treatment costs, demonstrating significant practical value and laying the foundation for future in vivo delivery. Attached Figure Description

[0073] Figure 1 This is a map of the co-stimulatory factor plasmid (pCoSti) of the lentiviral vector system in Example 1 of this application, as well as different compositions of the co-stimulatory factor expression sequence.

[0074] Figure 2 This refers to the particle size and flow cytometry results of the lentiviral vector particles detected by nanoflow cytometer in Example 1 of this application.

[0075] Figure 3 This is the result of the transduction positivity rate obtained by flow cytometry in different cells at different times in Example 3 of this application.

[0076] Figure 4In Embodiment 4 of this application, different lentiviral vectors are used to transduce CD34. + Experimental results of hematopoietic stem cells. A shows microscopic images, B and C show colony-forming unit test results, and D shows cell differentiation status.

[0077] Figure 5 These are the experimental results of transducing blood cells using different lentiviral vectors in Example 5 of this application. In the figures, A is a microscopic photograph, B shows the transduction positivity rate of different cell types, and C shows the colony-forming unit test results. Detailed Implementation

[0078] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terminology used herein is for the purpose of describing embodiments of this application only and is not intended to limit the application. Those skilled in the art will recognize that, with the evolution of technology and the emergence of new application scenarios, the technical solutions provided in the embodiments of this application are equally applicable to similar technical problems.

[0079] Example 1: Preparation and characterization of envelope protein plasmids and co-stimulatory factor plasmids

[0080] 1. Structure of enveloped protein granules and co-stimulatory factor plasmids

[0081] In this embodiment, the recombinant lentiviral vector envelope protein is expressed by fusing a rhabdovirus envelope protein mutant with an antibody. An amino acid mutation is introduced via primers, with isoleucine (I) at position 331 mutated to alanine (A) to construct a protein mutant (G Mutant) targeting a candidate key amino acid site. The components of the envelope protein particle of the co-stimulated recombinant lentiviral vector include: a cytomegalovirus enhancer, a cytomegalovirus promoter, a human β-globin intron, a Kozak concordant sequence, a PDGFR transmembrane region (TMD), a linker, a single-chain antibody (scFv) coding region sequence, a 2A peptide linker (2A), a rhabdovirus glycoprotein mutant (GMutant) coding region sequence, and a human β-globin poly A signal. The components of the co-stimulatory factor plasmid include: a cytomegalovirus enhancer, a cytomegalovirus promoter, a human β-globin intron, a Kozak concordance sequence, a co-stimulatory factor coding region (CoSti), and a human β-globin poly A signal. The co-stimulatory factor coding region (CoSti) includes stem cell factor (SCF), thrombopoietin (TPO), and Fms-like tyrosine kinase 3 ligand (FLT3L), the gene sequences of which are shown in SEQ ID NO. 1-3, and the amino acid sequences in SEQ ID NO. 4-6.

[0082] The nucleotide sequence of stem cell factor (SCF) is as follows:

[0083] ATGGAAGGGATCTGCAGGAATCGTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTCGATGACCTTGTGGAGTGCGTCAAAGAAAACTCATCTAAGGATCTAAAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTTAGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGCATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGAAAGGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCATTGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTATACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATATACAAATTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAGAGAGAGAGTTTCAAGAAGTG (SEQ ID NO.1).

[0084] The nucleotide sequence of thrombopoietin (TPO) is as follows:

[0085] ATGAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGCGGGCCCCACCCACCACAGCTGTCCCCAGCAGAACCTCTCTAGTCCTCACACTGGAATTCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO.2).

[0086] The nucleotide sequence of fms-related receptor tyrosine kinase 3 ligand (FLT3-L) is as follows:

[0087] ATGACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGCCACCCCCATGGAGTCCCCGGCCCCTGGAGGCCACAGCCCCGACAGCCCCGCAGCCCCCTCTGCTCCTCCTACTGCTGCTGCCCGTGGGCCTCCTGCTGCTGGCCGCTGCCTGGTGCCTGCACTGGCAGAGGACGCGGCGGAGGACACCCCGCCCTGGGGAGCAGGTGCCCCCCGTCCCCAGTCCCCAGGACCTGCTGCTTGTGGAGCAC (SEQ ID NO.3).

[0088] The amino acid sequence of stem cell factor is:

[0089] MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (SEQ ID NO.4).

[0090] The amino acid sequence of thrombopoietin is:

[0091] MSPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHK DPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPSRTSLVLTLEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO.5).

[0092] The amino acid sequence of the FMS-related receptor tyrosine kinase 3 ligand is:

[0093] MTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQ ETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH (SEQ IDNO.6).

[0094] The amino acid sequence of the transmembrane region of PDGFR is as follows:

[0095] RPKKQWLMILIILSIITLVVLALIASIVVVTG (SEQ ID NO. 7).

[0096] The nucleotide sequence of the transmembrane region of PDGFR is as follows:

[0097] AGACCAAAGAAGCAGTGGTTGATGATCCTCATCATACTTTCTATCATCACACTGGTGGTGCTGGCCCTGATCGCATCTATCGTGGTCGTAACTGGG (SEQ ID NO. 8).

[0098] The amino acid sequence of the linker region is GGGGSGGGGSGGGGS (SEQ ID NO.9).

[0099] The nucleotide sequence of the linker region is as follows:

[0100] GGTGGAGGTGGCTCTGGTGGAGGAGGCTCTGGAGGTGGTGGATCA (SEQ ID NO.10)

[0101] The amino acid sequence of the single-chain antibody is as follows:

[0102] AAQPAQIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKAEDTAVYFCARGYGNYARGAWLAYWGQGTLVTVSSGGGGSG GGGSGGGGSDVLLTQSPLSLPVTLGQPASISCRSSQTIVHSNGNTYLEWFQQRPGQSPRLLIYQVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKRTVALERGS (SEQ ID NO.11).

[0103] The nucleotide sequence of the single-chain antibody is as follows:

[0104] GCGGCCCAGCCGGCCCAGATCCAACTCGTCCAGTCCGGCTCAGAGCTAAAGAAACCCGGAGCCAGTGTAAAGGTGAGCTGCAAAGCATCTGGGTACACCTTTACAAACTATGGTATGAATTGGGTTCGCCAAGCGCCAGGCCAGGGACTGAAGTGGATGGGGTGGATAAACACGAATACTGGTGAACCGAAATACGCTGAGGAGTTCAAGGGCCGATTCGCCTTATCCTTGGACACCTCAGTCTCGACAGCATATCTTCAAATTAACAGTCTCAAAGCGGAGGATACGGCTGTATACTTTTGTGCCCGGGGATATGGGAATTACGCAAGAGGTGCGTGGCTCGCTTATTGGGGCCAGGGAACTCTGGTGACCGTTTCTTCTGGAGGCGGAGGATCTGGCGGAGGGGGATCCGGGGGAGGCGGATCTGACGTCTTGCTTACCCAGTCCCCCCTCTCACTACCAGTAACACTGGGCCAACCGGCCTCGATCAGTTGCCGCAGCTCTCAGACGATAGTGCACTCCAACGGAAATACTTACTTAGAGTGGTTCCAACAGCGACCTGGGCAATCACCGCGGTTGCTTATTTATCAGGTTTCGAACAGATTTAGTGGTGTCCCTGATAGGTTCAGCGGCTCTGGCTCCGGGACCGACTTTACACTCAAGATCTCACGTGTAGAAGCAGAGGATGTGGGTGTTTACTATTGTTTCCAAGGCTCGCATGTCCCCCGCACGTTTGGAGGGGGTACTAAAGTAGAAATAAAGCGAACTGTGGCTCTCGAGAGAGGTTCT (SEQ ID NO.12).

[0105] The amino acid sequence of the rhabdovirus glycoprotein mutant is as follows:

[0106] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAAIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDGELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPVFTIINGTLKYFETRYARVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSLGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIEFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO.13).

[0107] The nucleotide sequence of the rhabdovirus glycoprotein mutant is as follows: <000,0253>

[0109] The amino acid sequence of the T2A peptide linker region is as follows:

[0110] EGRGSLLTCGDVEENPGP (SEQ ID NO. 15).

[0111] The nucleotide sequence of the T2A peptide linker region is as follows:

[0112] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO. 16).

[0113] 2. Preparation process of co-stimulatory factor plasmids

[0114] This co-stimulatory factor plasmid is based on a third-generation lentiviral vector and performs pseudotype substitution on the viral envelope protein. The lentiviral envelope protein backbone vector is pMD2.G (Addgene, 12259), which is linearized using restriction endonucleases PmlI (New England Biolabs, R0532L) and PstI (New England Biolabs, R3140M). Gibson assembly primers are then designed based on the terminal sequence of the linearized vector to amplify the coding region sequence of the co-stimulatory factor. Then, 0.2 pmol of the amplified product was mixed with 0.05 pmol of the linearized vector, and the total volume was brought up to 10 μL with deionized water. 10 μL of 2×Gibson Assembly Master Mix (New England Biolabs, E2611L) was added and mixed thoroughly. The mixture was incubated at 50 °C for 1 hour, immediately cooled on ice, and then heat-shocked transformed using *E. coli* Stbl3 competent cells. The cells were cultured overnight at 37 °C on solid LB agar plates containing ampicillin. Single colonies were selected for colony PCR identification, and positive colonies were verified by first-generation sequencing. Plasmids with correct sequencing were uniformly amplified and extracted. The synthesis and sequencing of all viral protein coding regions and corresponding primer sequences were completed by Beijing BGI Genomics Co., Ltd.

[0115] For wild-type rhabdovirus glycoproteins, broad-spectrum receptor binding was eliminated by targeting a key amino acid site (I331A); simultaneously, a targeting protein containing an anti-CD34 single-chain antibody was expressed on the viral envelope surface, achieving targeting against CD34. + Targeted gene delivery to hematopoietic stem cells.

[0116] To further enhance the transduction efficiency of lentiviral vectors targeting resting hematopoietic stem cells (HSCs), which constitute the majority of the bone marrow microenvironment, it was hypothesized that adding HSC-stimulating factors to the viral envelope could improve the transduction efficiency of the vector for resting HSCs. Therefore, a series of cytokines commonly used in HSC in vitro culture, such as stem cell factor (SCF), thrombopoietin (TPO), and fms-associated receptor tyrosine kinase 3 ligand (FLT3-L), were investigated and their envelope surface modified to be displayed on the viral envelope surface after packaging. The co-stimulating factor plasmid map is shown below. Figure 1 As shown, the costimulatory factor coding region (CoSti) of the costimulatory factor plasmid is designed and synthesized with various factor combinations during construction, such as... Figure 1 As shown.

[0117] 3. Characterization of co-stimulatory factor plasmids

[0118] Viral vector characterization was performed using a Beckmem CytoFLEX nanometer. First, buffer and antibody preparation were performed. 1×PBS buffer was filtered through a 20 nm filter using a syringe. 50 μL of the antibody stock solution was centrifuged at 16000 g for 30 min. An appropriate amount of supernatant was taken according to the sample quantity and mixed with the filtered buffer to prepare the staining working solution. The viral vector was diluted with buffer to a concentration of 10% before staining. 11 Particles / mL, then incubate in staining working solution in the dark for 60 min, then dilute to 10. 8 Particles / mL sample loading and detection.

[0119] Characterization results as follows Figure 2 As shown in the figure, the vector particle size is distributed in the range of 100~200nm, and the proportion of particles with internal nucleic acids and displaying co-stimulatory proteins is about 26%.

[0120] Example 2: Preparation and evaluation of recombinant lentiviral particles

[0121] 1. Preparation of recombinant lentiviral particles

[0122] HEK293T cells were co-transfected with the envelope protein particles prepared in Example 1 and the third-generation lentiviral packaging plasmid system. The specific procedures are as follows:

[0123] First, the HEK293T cell density was adjusted to 4 × 10⁶ cells using DMEM high-glucose medium (containing 10 v / v% fetal bovine serum, 1 v / v% penicillin and antibiotics, 1 v / v% L-glutamine solution, and 1 v / v% non-essential amino acid solution). 5Cells were cultured at a concentration of 10 cells / mL. 2 mL of cell suspension was evenly seeded into a six-well plate and incubated at 37 °C with 5% CO2 for 24 h until confluence reached approximately 80%. The total plasmid system of the recombinant lentiviral vector was 4 μg, including helper plasmid psPAX2, shuttle plasmid pCDH, targeting envelope protein plasmid pENV, and co-stimulatory envelope protein pCoSti in a ratio of 2:4:1:1. HEK293T cells were co-transfected using DNA TransfectionReagent (POLYPLUS, CPT117). After 48 h, the cell culture supernatant was collected and filtered through a 0.45 μm filter to remove cell pellet. The supernatant was mixed with the virus concentrator Lenti-X Concentrator (TAKARA, 631232), centrifuged at 15000 g for 1 h at 4 °C, and the supernatant was removed. The pellet was resuspended in 100 μL of culture medium to prepare the virus storage solution, aliquoted, and stored at -80 °C for long-term storage.

[0124] 2. Virus titer assessment

[0125] RNA was extracted from 10 μL of viral storage medium using a viral RNA extraction kit (TIANGEN, DP315). Quantification was performed using a Lenti-X qRT-PCR Titration Kit (CLONTECH, 631235). A standard curve was constructed using serially diluted viral standards to perform absolute quantification of the lentiviral vector copy number (VCN) in the sample. The viral titer was calculated by multiplying the vector copy number by the dilution ratio of the storage medium. The viral titer calculation formula is as follows:

[0126]

[0127] The total viral particle titer was determined by quantifying p24 protein in 100 μL of viral dilution using the GenScript Lentivirus Tier p24 ELISA kit (see the instructions for GenScript Lentivirus Tier p24 ELISA kit L00938). First, the sample to be titered and the accompanying standards were diluted with cell culture medium. 10 μL of lysis buffer and 100 μL of viral dilution or standards were added to each well of the ELISA plate, and the plate was vortexed for 30–60 s and then incubated for 1 h. An appropriate amount of washing buffer was prepared, and 260 μL was added to each well. The plate was vortexed for 30–60 s and washed three times. Then, 100 μL of biotinylated anti-p24 antibody was added to each well, and the plate was incubated at 25 °C for 15 min, followed by four washes. Finally, 100 μL of streptavidin-HRP was added to each well, and the plate was incubated at 25 °C for 15 min, followed by four washes. Then, add 100 μL of substrate reaction solution to each well and incubate at 25 °C in the dark for 15 min. Finally, add 50 μL of stop solution to each well and immediately read the absorbance of each well at OD450 nm using a microplate reader. Calculate the p24 protein concentration according to the standard curve and convert it to the total viral particle titer using the formula: 1 ng p24 = 1.25 × 10⁻⁶. 7 Particles.

[0128] Example 3: Lentiviral transduction experiment

[0129] Human CD34 was cultured using serum-free lymphocyte culture medium (Bio-engine, HIPP-T009). + The hematopoietic stem cell density was adjusted to 5×10 4 Cells / mL were evenly seeded into 1 mL of cell suspension and incubated in a 37 ℃, 5% CO2 incubator for 24 h. The original culture medium was removed, and the cells were washed twice with DPBS buffer to completely remove the original culture medium. The virus stock solution was diluted with the culture medium at an MOI of 10⁻²⁰, mixed well, and human CD34 was added. + Hematopoietic stem cells were cultured in an incubator at 37 ℃ and 5% CO2 for 24 h. After removing the supernatant, the cells were washed twice with DPBS buffer before being used for subsequent testing or culture.

[0130] Assessment of target gene expression and transduction positivity rate

[0131] The size and number of different types of CFU clones in transduced cells were observed and photographed using an inverted microscope (OLYMPUS, IX73), and the cell transduction positivity rate was assessed by flow cytometry (Beckmem CytoFLEX). The original culture medium was first aspirated, and the cells were washed twice with DPBS buffer to completely remove the original culture medium. The cells were then pipetted into a cell suspension, and the cell density was adjusted to 1×10⁻⁶. 5After the cells were collected at a density of 1 / mL, 1 mL was transferred to a 1.5 mL centrifuge tube, centrifuged at 500 g for 5 min, and the supernatant was discarded. The cells were then washed once with 0.5 mL of DPBS buffer. After centrifugation at 500 g for 5 min again, the cells were stained with flow cytometry antibodies such as CD34-PE (BIOLEGEND, 343606), CD69-APC (BIOLEGEND, 310909), CD14 (BIOLEGEND, 982510), CD15 (BIOLEGEND, 301908), CD45 (BIOLEGEND, 304023), and CD235a (BIOLEGEND, 306603) for half an hour. After centrifugation at 500 g for 5 min, the supernatant was discarded, and the cells were washed once with 0.2 mL of DPBS buffer. After centrifugation at 500 g for 5 min again, the cells were resuspended and filtered through a cell filter to the bottom of the flow cytometer tube. The cell suspension was mixed by pipetting and then the flow cytometer tube was placed in the sample loading slot of the flow cytometer for sample loading. Adjust the position, size, and shape of the gates in the FSC / SSC plot to enclose the target cell population. Adjust the voltage of the fluorescence channels to distinguish individual channel signals. The transduction positivity rate is the percentage of GFP-positive cells in the total cell count.

[0132] The results are as follows Figure 3 As shown, the NC group was a blank control group without lentiviral transfection. The WT group used a packaging system for lentivirus transfected without the co-stimulatory envelope protein pCoSti, the target envelope protein pENV lacking the coding region of the single-chain antibody (scFv), and the rhabdovirus glycoprotein coding region using a non-mutant. All three groups were supplemented with SCF, TPO, and FLT3L (each cytokine's final concentration was 100 ng / mL) in the culture medium. The TarV group used a packaging system for lentivirus transfected with only the co-stimulatory envelope protein pCoSti omitted. The TarV-aCD34(CoSti) group (i.e., Tarv(X)) referenced... Figure 1 According to the abbreviation X in parentheses, different cytokines are inserted into the co-stimulatory envelope protein plasmid pCoSti in the packaging system used by the transfected lentiviruses. The co-stimulatory vector TarV-aCD34(CoSti) inserts different cytokines into CD34. + The transduction efficiency of TarV-aCD34 (CoSti) in hematopoietic stem cells is generally higher than that of conventional TarV and WT vectors, and the efficiency continues to increase with the extension of transduction time. In activated CD69-positive cells, the efficiency of TarV-aCD34 (CoSti) is not significantly different from that of conventional TarV-aCD34 after 1 day of transduction, but it still maintains the same transduction efficiency as CD69-positive cells after 7 days of transduction. This indicates that the stimulating factors on the vector surface have a positive effect on transduced CD69-aCD34. +Hematopoietic stem cell transplantation has a certain maintenance effect. Among them, the TarV(TPO+FLT3L) group (marked with a dashed box) showed better overall performance 1-7 days post-transplantation compared to other groups. (Regarding CD34...) + In cells, the positivity rate 7 days after TarV(T+F) transduction was 8 times that of the TarV group and 1.5 times that of the TarV(S+T+F) group; in CD69 + In cells, the positive rate 7 days after transduction was 3 times that of the TarV group and 1.2 times that of the TarV(S+T+F) group. Therefore, the TarV(T+F) group was selected as TarV-aCD34(CoSti) for subsequent testing.

[0133] Example 4: CD34 + Hematopoietic stem cell stem cell stemness and differentiation capacity test

[0134] To ensure CD34 after transduction of the co-stimulatory targeted lentiviral vector. + The stemness and differentiation capacity of hematopoietic stem cells are not affected, utilizing CD34. + Hematopoietic stem cells underwent colony-forming unit (CFU) testing after transduction to quantify the proliferation and differentiation capacity of the transduced cells. The specific procedure is as follows:

[0135] CD34 of human transduced lentivirus + Hematopoietic stem cells were centrifuged at 500 g for 5 min to remove the original culture medium. The cells were washed twice with DPBS buffer to completely remove the original culture medium. The cells were then pipetted into a cell suspension and the cell density was adjusted to 1×10⁻⁶. 6 After the cell count was reduced to 1 / mL, 10 μL was mixed with 3 mL of methylcellulose medium (STEMCELL, H4434) and then plated into 35 mm culture dishes. The dishes were incubated at 37 ℃ in a 5% CO2 incubator for 7–14 days. After incubation, different cell types were counted and identified. A blank control (Ctrl) without viral transfection and a TarV-aCD34 group containing TPO+FLT3L (each cytokine concentration 100 ng / mL) added to the medium but without the CoSti co-stimulatory factor plasmid were included.

[0136] The results are as follows Figure 4 As shown, the transduced CD34 + After 14 days of culture, hematopoietic stem cells can grow into burst erythroid colony-forming units (BFU-E), granulocyte colony-forming units-monocyte colony-forming units (CFU-GM=L), and granulocyte colony-forming units-erythroid colony-forming units (CFU-MIX). Furthermore, the differentiated cells, such as erythrocytes (Ery), monocytes (Mono), and neutrophils (Neu), and their clonogenic ability are comparable to the control group CD34. +There was no significant difference in hematopoietic stem cells.

[0137] Example 5: Co-stimulatory targeted lentivirus transduction of blood cells

[0138] To compare whether there is a difference in the maintenance of hematopoietic stem cell stemness by lentiviruses, referring to Example 3, blood cells were transduced using co-stimulatory targeted lentiviruses and conventional wild-type lentiviruses, and then CD34 cells were sorted out. + Hematopoietic stem cells were then subjected to the colony-forming unit (CFU) test as described in Example 4 to quantify the stemness of transduced stem cells. A blank control (Ctrl) without viral transfection and a WT(CoSti) group were included, with the CoSti co-stimulatory factor plasmid omitted but TPO+FLT3L (each cytokine final concentration 100 ng / mL) added separately to the culture medium.

[0139] The results are as follows Figure 5 As shown in the figure, co-stimulatory lentiviruses target and transduce CD34 in blood cells. + Hematopoietic stem cells, while traditional wild-type lentiviruses simultaneously transduce CD34. + hematopoietic stem cells and CD34 - Cells; after sorting, CD34 + After 14 days of culture, hematopoietic stem cells from the co-stimulated targeted lentiviral vector group produced significantly higher numbers of burst erythroid colony-forming units (BFU-E), granulocyte colony-forming unit-monocyte colony-forming units (CFU-GM), and granulocyte colony-forming unit-erythroid colony-forming units (CFU-MIX) compared to those from the conventional wild-type lentiviral group. This indicates that, at the same level, the co-stimulated targeted lentiviral vector preferentially transduces CD34. + Hematopoietic stem cells and maintain their stemness without being affected.

[0140] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A lentiviral vector packaging system for hematopoietic stem cells, characterized in that, The lentiviral vector packaging system includes: A recombinant expression vector comprising a costimulatory factor coding region sequence, wherein the costimulatory factor coding region sequence is composed of nucleotide sequences encoding thrombopoietin and FMS-associated receptor tyrosine kinase 3 ligand, wherein the nucleotide sequence encoding thrombopoietin is shown in SEQ ID NO.2, and the nucleotide sequence encoding FMS-associated receptor tyrosine kinase 3 ligand is shown in SEQ ID NO.3; An enveloped protein particle, the enveloped protein particle including an insertion site region, the insertion site region including a target antibody coding region and a rhabdovirus glycoprotein coding region, the target antibody coding region and the rhabdovirus glycoprotein coding region being connected by a 2A peptide linker region, the amino acid sequence of the rhabdovirus glycoprotein encoded by the rhabdovirus glycoprotein coding region being shown in SEQ ID NO.13, and the amino acid sequence of the target antibody encoded by the target antibody coding region being shown in SEQ ID NO.

11.

2. The lentiviral vector packaging system according to claim 1, characterized in that, The lentiviral vector packaging system further includes at least one of a packaging plasmid and a transfer plasmid.

3. Recombinant packaging cells for hematopoietic stem cells, characterized in that, The recombinant packaging cells comprise the lentiviral vector packaging system according to any one of claims 1 to 2.

4. A lentivirus for use in hematopoietic stem cells, characterized in that, The lentiviral vector packaging system according to any one of claims 1 to 2 is obtained by transfecting packaging cells.

5. A composition for use with hematopoietic stem cells, characterized in that, The composition comprises the lentiviral vector packaging system of any one of claims 1 to 2, the recombinant packaging cell of claim 3, or the lentivirus of claim 4.

6. The use of the lentiviral vector packaging system of any one of claims 1 to 2, the recombinant packaged cells of claim 3, the lentivirus of claim 4, or the composition of claim 5 in the preparation of cell therapy drugs.

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