Virus vector for hematopoietic stem cells and application thereof

Abstract

The invention provides a virus vector for hematopoietic stem cells and application of the virus vector. The first aspect of the application provides a lentiviral vector packaging system for hematopoietic stem cells, the lentiviral vector packaging system comprises a recombinant expression vector, and the recombinant expression vector comprises nucleotide sequences encoding thrombopoietin and a fms associated receptor tyrosine kinase 3 ligand. The thrombopoietin and the fms related receptor tyrosine kinase 3 ligand are constructed on the nucleic acid molecule, so that the thrombopoietin and the fms related receptor tyrosine kinase 3 ligand are expressed on the surface of a lentiviral vector and the like; the stemness or immunocompetence of various cells including hematopoietic stem cells can be effectively activated and improved under the condition of lower dosage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell biology, and particularly relates to a viral vector for hematopoietic stem cells and application thereof. BACKGROUND

[0002] Gene therapy represented by cell therapy has shown broad prospects in basic research, and some strategies have been successfully translated into clinical research and application. The approved cell therapy is usually constructed by specific functional cells through ex vivo transduction and then reinfused into patients, which is complex and highly individualized, and is difficult to promote. In recent years, researchers have been committed to directly transducing target cells in patients, but this requires a higher requirement for the in vivo transduction vector of the transduced cells: (1) precise targeting to reduce off-target and vector depletion caused by transduction of non-target cells; (2) high transduction efficiency and stimulation and activation of cells; (3) low immunogenicity.

[0003] Hematopoietic stem cells (HSCs) are ideal target cells for cell therapy, and their genetic information is inherited to all lineages derived from them as they divide and differentiate. At present, for diseases such as β-thalassemia and sickle cell disease (SCD), HSC transplantation is the only choice that has been proven to have a cure. Therefore, developing an in vivo transduction vector with targeting for HSCs can effectively reduce the treatment risk and cost of such diseases, and has important significance for accelerating the application of cell therapy in clinical practice.

[0004] Currently, the effective eukaryotic delivery vectors can be divided into viral vectors and non-viral vectors. The non-viral vector has a relatively simple structure, and is easy to scale up, but it is difficult to achieve in vivo targeting, and is mainly achieved by changing the administration method to achieve non-targeted transduction in the liver, lung alveoli and the like. In comparison, the viral vector, especially the lentiviral vector (LV), can achieve targeted transduction of specific cells by engineering the envelope glycoprotein (such as vesicular stomatitis virus glycoprotein, VSV-G), but there is still a problem of low transduction efficiency in cell types such as hematopoietic stem cells. In addition, when the conventional lentiviral vector is applied to resting hematopoietic stem cells or lymphocytes, the problem of low transduction efficiency occurs, and increasing the amount of virus may affect the stemness or immune activity of the target cells, so there is an urgent need for a vector that can balance the targeting transduction efficiency and stimulation effect. SUMMARY

[0005] The main purpose of the embodiment of the present application is to provide a viral vector for hematopoietic stem cells and application thereof.

[0006] To achieve the above object, the first aspect of the present 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 a nucleotide sequence encoding Thrombopoietin (TPO) and Fms-like Tyrosine Kinase 3 Ligand (FLT3L), the nucleotide sequence encoding Thrombopoietin being as shown in SEQ ID NO. 2, and the nucleotide sequence encoding Fms-like Tyrosine Kinase 3 Ligand being as shown in SEQ ID NO. 3.

[0007] In some embodiments, the recombinant expression vector further comprises a nucleotide sequence encoding Stem Cell Factor (SCF).

[0008] In some embodiments, the nucleotide sequence encoding Stem Cell Factor is as shown in SEQ ID NO. 1.

[0009] In some embodiments, the amino acid sequence of Stem Cell Factor is as shown in SEQ ID NO. 4.

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

[0011] In some embodiments, the amino acid sequence of Fms-like Tyrosine Kinase 3 Ligand is as shown in SEQ ID NO. 6.

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

[0013] In some embodiments, the enhancer comprises a cytomegalovirus enhancer (CMV enhancer).

[0014] In some embodiments, the promoter comprises a cytomegalovirus promoter (CMV promoter).

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

[0016] In some embodiments, the polyadenylation signal comprises a human β-globin poly A.

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

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

[0019] In some embodiments, the envelope protein plasmid comprises an insertion site region.

[0020] In some embodiments, the insertion site region comprises a targeting antibody coding region.

[0021] In some embodiments, the targeting antibody coding region encodes a targeting antibody having an amino acid sequence as set forth in SEQ ID NO. 11.

[0022] In some embodiments, the targeting antibody coding region has a nucleotide sequence as set forth in SEQ ID NO. 12.

[0023] In some embodiments, the insertion site region comprises a rhabdovirus glycoprotein coding region.

[0024] In some embodiments, the rhabdovirus glycoprotein coding region encodes a rhabdovirus glycoprotein having an amino acid sequence as set forth in SEQ ID NO. 13.

[0025] In some embodiments, the rhabdovirus glycoprotein coding region has a nucleotide sequence as set forth in SEQ ID NO. 14.

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

[0027] In some embodiments, the targeting 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 comprises a transmembrane region.

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

[0030] In some embodiments, the transmembrane region has an amino acid sequence as set forth in SEQ ID NO. 7.

[0031] In some embodiments, the transmembrane region has a nucleotide sequence as set forth in SEQ ID NO. 8.

[0032] In some embodiments, the insertion site region comprises a linker region.

[0033] In some embodiments, the linker region has an amino acid sequence as set forth 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-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 helper plasmid psPAX2.

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

[0050] In a second aspect of the present application, there is provided a recombinant packaging cell for hematopoietic stem cells, comprising the aforementioned lentiviral vector packaging system.

[0051] In some embodiments, the recombinant packaging cell is a eukaryotic cell.

[0052] In some embodiments, the recombinant packaging cell is an animal cell.

[0053] In some embodiments, the recombinant packaging cell is a mammalian cell.

[0054] In some embodiments, the recombinant packaging cell is any one of HEK293T cell, HEK293FT cell.

[0055] In a third aspect of the present application, there is provided a lentivirus for hematopoietic stem cells, which is obtained after transfecting the aforementioned lentiviral vector packaging system into packaging cells.

[0056] In some embodiments, the transfected packaging cell is a eukaryotic cell.

[0057] In some embodiments, the transfected packaging cell is an animal cell.

[0058] In some embodiments, the transfected packaging cell is a mammalian cell.

[0059] In some embodiments, the transfected packaging cell is any one of HEK293T cell, HEK293FT cell.

[0060] The present application also relates to a method for preparing the aforementioned lentivirus, comprising the following steps: constructing the aforementioned lentiviral vector packaging system; transferring the lentiviral vector packaging system into packaging cells, and obtaining the lentivirus after culture.

[0061] In a fourth aspect of the present application, there is provided a composition for hematopoietic stem cells, comprising the aforementioned lentiviral packaging system, the aforementioned recombinant packaging cell, or the aforementioned lentivirus.

[0062] In some embodiments, the composition is a pharmaceutical composition.

[0063] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or diluent.

[0064] In a fifth aspect of the present application, a target cell is provided, wherein the target cell comprises hematopoietic stem cells, and the target cell comprises the aforementioned lentivirus.

[0065] In some embodiments, the target cell comprises CD34 + hematopoietic stem cells.

[0066] In a sixth aspect of the present application, the aforementioned lentivirus vector packaging system, the aforementioned recombinant packaging cell, the aforementioned lentivirus, the aforementioned composition, or the aforementioned target cell is used in the preparation of a cell therapy drug.

[0067] In some embodiments, the cell therapy comprises hematopoietic stem cell therapy.

[0068] The beneficial effects of the present application are: The present application constructs thrombopoietin and fms-related tyrosine kinase 3 ligand on a nucleic acid molecule, so that it is expressed on the surface of a lentivirus vector, and the results show that compared with direct addition, the stemness or immune activity of various cells including hematopoietic stem cells can be effectively activated and improved at a lower dosage. And the virus vector using the combination of thrombopoietin + Fms tyrosine kinase 3 ligand can effectively maintain the transduction positive rate during in vitro culture, has more significant long-acting, and is expected to shorten the treatment interval when applied to in vivo transduction.

[0069] In addition, by co-expressing the rhabdovirus envelope protein mutant, single-chain antibody, and stimulating factor on the surface of the lentivirus vector, the transduction efficiency and specificity of the delivery carrier tool are effectively improved, and the proportion of stimulated resting cells is also increased, which increases the safety while greatly reducing the treatment cost, has great practical value, and lays the foundation for future in vivo delivery. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 is the map of the co-stimulating factor plasmid (pCoSti) of the lentivirus vector system in Example 1 of the present application and the different compositions of the co-stimulating factor expression sequence.

[0071] Figure 2 is the particle size of the lentivirus vector particles detected by the nano-flow instrument and the flow cytometry results in Example 1 of the present application.

[0072] Figure 3 is the transduction positive rate results obtained by flow cytometry in different cells at different times in Example 3 of the present application.

[0073] Figure 4 is the transduction of CD34 +The experimental results of hematopoietic stem cells. Among them, A is the microscopic observation photo, B and C are the results of colony forming unit test, and D is the cell differentiation.

[0074] Figure 5 The experimental results of blood cells transduced by different lentiviral vectors in Example 5 of the present application. Among them, A is the microscopic observation photo, B is the results of transduction positive rate of different types of cells, and C is the results of colony forming unit test. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application and are not intended to limit the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by those skilled in the art to which the present application belongs. The terms used herein are only for the purpose of describing the embodiments of the present application and are not intended to limit the present application. Those skilled in the art can know that as technology evolves and new application scenarios appear, the technical solutions provided by the embodiments of the present application are also applicable to similar technical problems.

[0076] Example 1: Preparation and characterization of envelope protein plasmid and costimulatory factor plasmid 1. Structure of envelope protein plasmid and costimulatory factor plasmid The recombinant lentiviral vector envelope protein of the present embodiment is a rhabdovirus envelope protein mutant fused with an antibody. An amino acid site mutation is introduced by primers, and the 331st isoleucine (I) is mutated to alanine (A) to construct a protein mutant (G Mutant) targeting the candidate key amino acid site. The constituent elements of the envelope protein plasmid of the co-stimulating recombinant lentiviral vector include: cytomegalovirus enhancer (CMV enhancer), cytomegalovirus promoter (CMV promoter), human β-globin intron (β-globin intron), Kozak, PDGFR transmembrane region (TMD), linker region (Linker), single-chain antibody (scFv) coding region sequence, 2A peptide linker region (2A), rhabdovirus glycoprotein mutant (G Mutant) coding region sequence, human β-globin polyadenylation signal (β-globin poly A). The constituent elements of the co-stimulating factor plasmid include: cytomegalovirus enhancer (CMV enhancer), cytomegalovirus promoter (CMV promoter), human β-globin intron (β-globin intron), Kozak, co-stimulating factor coding region (CoSti), human β-globin polyadenylation signal (β-globin poly A). The co-stimulating factor coding region (CoSti) includes stem cell factor (SCF), thrombopoietin (TPO), and Fms-like tyrosine kinase 3 ligand (FLT3L), and the respective gene sequences are shown in SEQ ID NO. 1-3, and the amino acid sequences are shown in SEQ ID NO. 4-6.

[0077] The nucleotide sequence of the stem cell factor (SCF) is: ATGGAAGGGATCTGCAGGAATCGTGTGACTAATAATGTAAAAGACGTCACTAAATTGGTGGCAAATCTTCCAAAAGACTACATGATAACCCTCAAATATGTCCCCGGGATGGATGTTTTGCCAAGTCATTGTTGGATAAGCGAGATGGTAGTACAATTGTCAGACAGCTTGACTGATCTTCTGGACAAGTTTTCAAATATTTCTGAAGGCTTGAGTAATTATTCCATCATAGACAAACTTGTGAATATAGTCGATGACCTTGTGGAGTGCGTCAAAGAAAACTCATCTAAGGATCTAAAAAAATCATTCAAGAGCCCAGAACCCAGGCTCTTTACTCCTGAAGAATTCTTTAGAATTTTTAATAGATCCATTGATGCCTTCAAGGACTTTGTAGTGGCATCTGAAACTAGTGATTGTGTGGTTTCTTCAACATTAAGTCCTGAGAAAGGGAAGGCCAAAAATCCCCCTGGAGACTCCAGCCTACACTGGGCAGCCATGGCATTGCCAGCATTGTTTTCTCTTATAATTGGCTTTGCTTTTGGAGCCTTATACTGGAAGAAGAGACAGCCAAGTCTTACAAGGGCAGTTGAAAATATACAAATTAATGAAGAGGATAATGAGATAAGTATGTTGCAAGAGAAAGAGAGAGAGTTTCAAGAAGTG (SEQ ID NO. 1).

[0078] The thrombopoietin (TPO) nucleotide sequence is: ATGAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCGTGACTCCCATGTCCTTCACAGCAGACTGAGCCAGTGCCCAGAGGTTCACCCTTTGCCTACACCTGTCCTGCTGCCTGCTGTGGACTTTAGCTTGGGAGAATGGAAAACCCAGATGGAGGAGACCAAGGCACAGGACATTCTGGGAGCAGTGACCCTTCTGCTGGAGGGAGTGATGGCAGCACGGGGACAACTGGGACCCACTTGCCTCTCATCCCTCCTGGGGCAGCTTTCTGGACAGGTCCGTCTCCTCCTTGGGGCCCTGCAGAGCCTCCTTGGAACCCAGCTTCCTCCACAGGGCAGGACCACAGCTCACAAGGATCCCAATGCCATCTTCCTGAGCTTCCAACACCTGCTCCGAGGAAAGGTGCGTTTCCTGATGCTTGTAGGAGGGTCCACCCTCTGCGTCAGGCGGGCCCCACCCACCACAGCTGTCCCCAGCAGAACCTCTCTAGTCCTCACACTGGAATTCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO. 2).

[0079] The nucleotide sequence of the fms-related receptor tyrosine kinase 3 ligand (FLT3-L) is: ATGACCCAGGACTGCTCCTTCCAACACAGCCCCATCTCCTCCGACTTCGCTGTCAAAATCCGTGAGCTGTCTGACTACCTGCTTCAAGATTACCCAGTCACCGTGGCCTCCAACCTGCAGGACGAGGAGCTCTGCGGGGGCCTCTGGCGGCTGGTCCTGGCACAGCGCTGGATGGAGCGGCTCAAGACTGTCGCTGGGTCCAAGATGCAAGGCTTGCTGGAGCGCGTGAACACGGAGATACACTTTGTCACCAAATGTGCCTTTCAGCCCCCCCCCAGCTGTCTTCGCTTCGTCCAGACCAACATCTCCCGCCTCCTGCAGGAGACCTCCGAGCAGCTGGTGGCGCTGAAGCCCTGGATCACTCGCCAGAACTTCTCCCGGTGCCTGGAGCTGCAGTGTCAGCCCGACTCCTCAACCCTGCCACCCCCATGGAGTCCCCGGCCCCTGGAGGCCACAGCCCCGACAGCCCCGCAGCCCCCTCTGCTCCTCCTACTGCTGCTGCCCGTGGGCCTCCTGCTGCTGGCCGCTGCCTGGTGCCTGCACTGGCAGAGGACGCGGCGGAGGACACCCCGCCCTGGGGAGCAGGTGCCCCCCGTCCCCAGTCCCCAGGACCTGCTGCTTGTGGAGCAC (SEQ ID NO. 3).

[0080] The amino acid sequence of the stem cell factor is: MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV (SEQ ID NO. 4).

[0081] The amino acid sequence of the thrombopoietin is: MSPAPPACDLRVLSKLLRDSHVLHSRLSQCPEVHPLPTPVLLPAVDFSLGEWKTQMEETKAQDILGAVTLLLEGVMAARGQLGPTCLSSLLGQLSGQVRLLLGALQSLLGTQLPPQGRTTAHKDPNAIFLSFQHLLRGKVRFLMLVGGSTLCVRRAPPTTAVPSRTSLVLTLEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 5).

[0082] The amino acid sequence of the fms-related receptor tyrosine kinase 3 ligand is: MTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH (SEQ ID NO. 6).

[0083] The amino acid sequence of the PDGFR transmembrane region is: RPKKQWLMILIILSIITLVVLALIASIVVVTG (SEQ ID NO. 7).

[0084] The nucleotide sequence of the PDGFR transmembrane region is: AGACCAAAGAAGCAGTGGTTGATGATCCTCATCATACTTTCTATCATCACACTGGTGGTGCTGGCCCTGATCGCATCTATCGTGGTCGTAACTGGG (SEQ ID NO. 8).

[0085] The amino acid sequence of the linker region is GGGGSGGGGSGGGGS (SEQ ID NO. 9).

[0086] The nucleotide sequence of the linker region is: GGTGGAGGTGGCTCTGGTGGAGGAGGCTCTGGAGGTGGTGGATCA (SEQ ID NO. 10) The amino acid sequence of the single chain antibody is: AAQPAQIQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTNTGEPKYAEEFKGRFALSLDTSVSTAYLQINSLKAEDTAVYFCARGYGNYARGAWLAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVLLTQSPLSLPVTLGQPASISCRSSQTIVHSNGNTYLEWFQQRPGQSPRLLIYQVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPRTFGGGTKVEIKRTVALERGS (SEQ ID NO. 11).

[0087] The nucleotide sequence of the single chain antibody is: GCGGCCCAGCCGGCCCAGATCCAACTCGTCCAGTCCGGCTCAGAGCTAAAGAAACCCGGAGCCAGTGTAAAGGTGAGCTGCAAAGCATCTGGGTACACCTTTACAAACTATGGTATGAATTGGGTTCGCCAAGCGCCAGGCCAGGGACTGAAGTGGATGGGGTGGATAAACACGAATACTGGTGAACCGAAATACGCTGAGGAGTTCAAGGGCCGATTCGCCTTATCCTTGGACACCTCAGTCTCGACAGCATATCTTCAAATTAACAGTCTCAAAGCGGAGGATACGGCTGTATACTTTTGTGCCCGGGGATATGGGAATTACGCAAGAGGTGCGTGGCTCGCTTATTGGGGCCAGGGAACTCTGGTGACCGTTTCTTCTGGAGGCGGAGGATCTGGCGGAGGGGGATCCGGGGGAGGCGGATCTGACGTCTTGCTTACCCAGTCCCCCCTCTCACTACCAGTAACACTGGGCCAACCGGCCTCGATCAGTTGCCGCAGCTCTCAGACGATAGTGCACTCCAACGGAAATACTTACTTAGAGTGGTTCCAACAGCGACCTGGGCAATCACCGCGGTTGCTTATTTATCAGGTTTCGAACAGATTTAGTGGTGTCCCTGATAGGTTCAGCGGCTCTGGCTCCGGGACCGACTTTACACTCAAGATCTCACGTGTAGAAGCAGAGGATGTGGGTGTTTACTATTGTTTCCAAGGCTCGCATGTCCCCCGCACGTTTGGAGGGGGTACTAAAGTAGAAATAAAGCGAACTGTGGCTCTCGAGAGAGGTTCT (SEQ ID NO. 12).

[0088] The amino acid sequence of the rhabdovirus glycoprotein mutant is: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAAIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISTDITFFSEDGELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPVFTIINGTLKYFETRYARVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSLGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIEFVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK (SEQ ID NO. 13).

[0089] The nucleotide sequence of the rhabdovirus glycoprotein mutant is:

[0090] The amino acid sequence of the T2A peptide linker region is: EGRGSLLTCGDVEENPGP (SEQ ID NO. 15).

[0091] The nucleotide sequence of the T2A peptide linker region is: GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT (SEQ ID NO. 16).

[0092] 2. Preparation process of co-stimulatory factor plasmid The co-stimulatory factor plasmid is based on the third generation of lentiviral vectors, and the viral envelope protein is replaced by pseudotyping. The lentiviral envelope protein backbone vector is pMD2.G (Addgene, 12259), which is linearized by selecting restriction endonucleases PmlI (New England Biolabs, R0532L) and PstI (New England Biolabs, R3140M), and then the Gibson assembly primer is designed according to the sequence of the linearized vector end to amplify the sequence of the co-stimulatory factor coding region. Then 0.2 pmol of the amplification product is mixed with 0.05 pmol of the linearized vector, and the total volume is supplemented to 10 μL with deionized water, and then 10 μL of 2×Gibson Assembly Master Mix (New England Biolabs, E2611L) is added and mixed, and then incubated at 50 ℃ for 1 hour, immediately cooled on ice, and then transformed with E. coli Stbl3 competent cells by heat shock, and then cultured on solid LB plates containing ampicillin at 37 ℃ overnight, and then single colonies are selected for colony PCR identification, and then positive colonies are subjected to first-generation sequencing verification, and then the correct plasmid is uniformly expanded and extracted. All viral protein coding regions and corresponding primer sequences are synthesized and sequencing work is completed by Beijing Lihe Huada Gene Technology Co., Ltd.

[0093] For wild-type rhabdovirus glycoprotein, the key amino acid site (I331A) is targeted to eliminate its broad-spectrum receptor binding; at the same time, the targeting protein of anti-CD34 single-chain antibody is expressed on the surface of the viral envelope, realizing the targeting of CD34 + Targeted gene delivery of hematopoietic stem cells.

[0094] To further enhance the targeting lentiviral vector for the majority of bone marrow microenvironment in the resting state HSC, it is assumed that the addition of HSC stimulating factors on the virus envelope can improve the transduction efficiency of the vector for resting HSC. For this purpose, a series of cytokines commonly used in the in vitro culture of HSCs, such as stem cell factor (SCF), thrombopoietin (TPO) and fms-related receptor tyrosine kinase 3 ligand (FLT3-L), were investigated, and they were surface-modified to be displayed on the surface of the virus envelope after packaging. The co-stimulatory factor plasmid map is shown in Figure 1 The co-stimulatory factor coding region (CoSti) of the co-stimulatory factor plasmid was designed and synthesized in various combinations when constructing, and the combination types are shown in Figure 1 .

[0095] 3. Characterization of co-stimulatory factor plasmid Virus vector characterization was analyzed by a nano-flow cytometer (Beckmem CytoFLEX nano). First, buffer and antibody preparation was performed, 1x PBS buffer was filtered through a 20 nm filter with a syringe for standby; 50 μL of antibody stock solution was centrifuged at 16000 g for 30 min, and according to the sample quantity, the appropriate amount of supernatant was mixed with the filtered buffer to prepare the staining working solution. The virus vector was diluted to 10 11 particles / mL before staining, then incubated in the staining working solution for 60 min in the dark, and then diluted to 10 8 particles / mL for detection.

[0096] The characterization results are shown in Figure 2 From the results in the figure, it can be seen that the average size of the vector particles is distributed in the range of 100~200 nm, and the proportion of particles with internal nucleic acids and displaying co-stimulatory proteins is about 26%.

[0097] Example 2: Preparation and evaluation of recombinant lentiviral particles 1. Preparation of recombinant lentiviral particles The envelope protein plasmid prepared in Example 1 and the third-generation lentiviral packaging plasmid system were co-transfected into HEK293T cells. The specific operation is as follows: First, the DMEM high glucose medium (containing 10 v / v% fetal bovine serum, 1 v / v% double antibody, 1 v / v% L-glutamine solution and 1 v / v% non-essential amino acid solution) was used to adjust the density of HEK293T cells to 4x10 5The total system of plasmids of the recombinant lentivirus vector was 4 μg, including the helper plasmid psPAX2, the shuttle plasmid pCDH, the targeting envelope protein plasmid pENV and the co-stimulation envelope protein pCoSti, the ratio was 2:4:1:1, and the HEK293T was co-transfected using the transfection reagent DNA Transfection Reagent (POLYPLUS, CPT117). After 48 h, the cell culture supernatant was collected and filtered through a 0.45 μm filter membrane to remove cell precipitates, mixed with the virus concentrator Lenti-X Concentrator (TAKARA, 631232), centrifuged at 4 ℃ and 15000 g for 1 h, the supernatant was removed, and the precipitate was resuspended into virus storage solution using 100 μL of culture medium, and then appropriately aliquoted and stored at -80 ℃ for long-term preservation.

[0098] 2. Virus titer evaluation The RNA in 10 μL of the virus storage solution was extracted by using the virus RNA extraction kit (TIANGEN, DP315), and quantified using the Lenti-X qRT-PCR Titration Kit (CLONTECH, 631235). The absolute quantification of the lentivirus vector copy number (VCN) in the sample was performed by using the gradient-diluted virus standard as the standard curve, and the virus titer was calculated by multiplying the vector copy number by the dilution ratio of the storage solution. The virus titer calculation formula is as follows:

[0099] The p24 protein in 100 μL virus dilution was quantified by lentivirus titer p24 ELISA kit (see GenScript Lentivirus Titer p24 ELISA Kit L00938 Instructions for details) to determine the total virus particle titer. First, dilute the sample to be determined with the titer and the matching standard with cell culture medium, add 10 μL lysate and 100 μL virus dilution or standard to the ELISA well plate, rotate and shake for 30-60 s, and then incubate for 1 h. Prepare an appropriate amount of washing solution, add 260 μL washing solution to each well, rotate and shake for 30-60 s, and continue to repeat the washing for 3 times, add 100 μL biotin anti-p24 antibody to each well, incubate at 25 ℃ for 15 min, and then wash 4 times. Next, add 100 μL streptavidin-HRP to each well, incubate at 25 ℃ for 15 min, and then wash 4 times. Then, add 100 μL substrate reaction solution to each well, and incubate at 25 ℃ in the dark for 15 min. Finally, add 50 μL stop solution to each well, and immediately use the microplate reader to read the absorbance of each well at OD450 nm. According to the standard curve, the p24 protein concentration is calculated, and the total virus particle titer is converted, the conversion formula is 1 ng p24 = 1.25 × 10 7 particles.

[0100] Example 3: Lentivirus transduction experiment The human CD34 + hematopoietic stem cells were adjusted to a density of 5 × 10 4 Take 1 mL of cell suspension and evenly inoculate it in a twelve-hole culture plate, and place it in a 37 ℃, 5% CO2 incubator for 24 h. Remove the original culture medium, wash twice with DPBS buffer to completely remove the original culture medium, dilute the virus stock with culture medium at a MOI of 10-20, mix well, add human CD34 + hematopoietic stem cells, and place it in a 37 ℃, 5% CO2 incubator for 24 h. Remove the supernatant, wash twice with DPBS buffer, and use for subsequent testing or culture.

[0101] Evaluation of target gene expression and transduction positive rate Observe and take pictures of different types of CFU clone size and number in the cells after transduction by inverted microscope (OLYMPUS, IX73), and then evaluate the cell transduction positive rate by flow cytometry (Beckmem CytoFLEX). First, remove the original culture medium, wash twice with DPBS buffer to completely remove the original culture medium, blow the cells into a cell suspension, and adjust the cell density to 1 × 10 5After 1 mL / mL was taken to 1.5 mL centrifuge tube, 500 g, 5 min centrifugation, the supernatant was discarded and the cells were washed once with 0.5 mL DPBS buffer; again 500 g, 5 min centrifugation, the cells were stained with CD34-PE (BIOLEGEND, 343606), CD69-APC (BIOLEGEND, 310909), CD14 (BIOLEGEND, 982510), CD15 (BIOLEGEND, 301908), CD45 (BIOLEGEND, 304023), CD235a (BIOLEGEND, 306603) and other flow cytometry antibodies for half an hour, 500 g, 5 min centrifugation, the supernatant was discarded and the cells were washed once with 0.2 mL DPBS buffer, again 500 g, 5 min centrifugation, the cells were resuspended, filtered through a cell strainer to the bottom of the flow tube, and the flow tube was placed in the sample slot of the flow cytometer after the cell suspension was mixed evenly by blowing. Wait for loading. Adjust the position, size and shape of the gate in the FSC / SSC graph, enclose the target cell population, adjust the voltage of the fluorescence channel to distinguish the channel signals, and the transduction positive rate is the percentage of GFP positive cells in the total cells.

[0102] The results are shown in Table 1. Figure 3 The results are shown in Table 1. Figure 1 The results are shown in Table 1. + The transduction efficiency of hematopoietic stem cells in the hematopoietic stem cells was generally higher than that of the conventional TarV and WT vectors, and the efficiency was continuously improved with the extension of the transduction time. In CD69 positive cells in the activated state, TarV-aCD34(CoSti) had little difference in transduction efficiency compared with conventional TarV-aCD34 after 1 day of transduction, but maintained the transduction efficiency in CD69 positive cells after 7 days of transduction, which indicated that the stimulating factors on the surface of the vector could maintain the transduction efficiency of CD34 +Hematopoietic stem cells have certain maintenance effect. Among them, TarV(TPO+FLT3L) group (dashed box) is better than other groups in 1-7 days after transformation. In CD34 + cells, the positive rate of TarV(T+F) 7 days after transformation is 8 times that of TarV group and 1.5 times that of TarV(S+T+F) group; in CD69 + cells, the positive rate 7 days after transformation is 3 times that of TarV group and 1.2 times that of TarV(S+T+F) group, so TarV(T+F) group is selected as TarV-aCD34(CoSti) for subsequent testing.

[0103] Example 4: CD34 + Hematopoietic stem cell stemness and differentiation ability test To ensure that the CD34 + hematopoietic stem cells after transduction of co-stimulatory type target lentivirus vector are not affected, CD34 + Hematopoietic stem cells were tested for colony forming units (CFU) after transduction to quantify the proliferation and differentiation ability of cells after transduction. The specific process is as follows: Take human CD34 + hematopoietic stem cells after transduction of lentivirus, 500 g, 5 min centrifugal suction of original culture medium, DPBS buffer solution is washed twice to remove the original culture medium completely, the cells are blown into cell suspension, and the cell density is adjusted to 1×10 6 After 10 μL is mixed with 3 mL methyl cellulose medium (STEMCELL, H4434), it is placed in a 35 mm culture dish, and cultured in a 37 ℃, 5% CO2 incubator for 7-14 days. After the end, different types of cell counting / identification are performed. Set up a blank control (Ctrl) without transduction of virus and a TarV-aCD34 group without CoSti co-stimulatory factor plasmid but with separate addition of TPO+FLT3L (the final concentration of each cytokine is 100 ng / mL) in the culture medium.

[0104] The results are shown in Figure 4 After transduction, CD34 + hematopoietic stem cells can grow into explosive erythroid colony forming units (BFU-E), granulocyte colony forming unit-monocyte colony forming unit (CFU-GM=L), and granulocyte colony forming unit-erythroid colony forming unit (CFU-MIX) after 14 days of culture in the medium, and the differentiated cells such as erythrocytes (Ery), monocytes (Mono), neutrophils (Neu) and clonogenic ability have no significant difference with the control group CD34 + hematopoietic stem cells.

[0105] Example 5: Co-stimulatory targeted lentiviral transduction of blood cells To compare whether there is a difference in the maintenance of stemness of hematopoietic stem cells by lentiviruses, referring to Example 3, blood cells were transduced by co-stimulatory targeted lentivirus and traditional wild-type lentivirus, and then CD34 + hematopoietic stem cells were sorted out, and then subjected to the colony-forming unit (CFU) test in Example 4 to quantify the stemness of the stem cells after transduction. A blank control (Ctrl) without transduction of virus and a WT (CoSti) group in which the CoSti co-stimulatory factor plasmid was omitted but TPO+FLT3L (100 ng / mL each) were added separately in the culture medium were set.

[0106] The results are shown in Figure 5 From the figure, it can be seen that the co-stimulatory targeted lentivirus transduces CD34 + hematopoietic stem cells, while the traditional wild-type lentivirus transduces both CD34 + hematopoietic stem cells and CD34 - cells; after sorting, the CD34 + hematopoietic stem cells were cultured in the culture medium for 14 days, and the burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte-monocyte (CFU-GM), and colony-forming unit-mix (CFU-MIX) produced by the hematopoietic stem cells in the co-stimulatory targeted lentivirus vector group were all higher than those in the traditional wild-type lentivirus group, which indicates that at the same level, the co-stimulatory targeted lentivirus vector preferentially transduces CD34 + hematopoietic stem cells and maintains the stemness thereof unaffected.

[0107] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A lentiviral vector packaging system for hematopoietic stem cells, characterized in that, The lentiviral vector packaging system includes a recombinant expression vector, which includes nucleotide sequences encoding thrombopoietin and an FMS-associated receptor tyrosine kinase 3 ligand. The nucleotide sequence encoding thrombopoietin is shown in SEQ ID NO.2, and the nucleotide sequence encoding the FMS-associated receptor tyrosine kinase 3 ligand is shown in SEQ ID NO.

3.

2. The lentiviral vector packaging system according to claim 1, characterized in that, The recombinant expression vector also includes a nucleotide sequence encoding a stem cell factor.

3. The lentiviral vector packaging system according to claim 2, characterized in that, The nucleotide sequence encoding the stem cell factor is shown in SEQ ID NO.

1.

4. The lentiviral vector packaging system according to any one of claims 1 to 3, characterized in that, The lentiviral vector packaging system further includes at least one of an envelope protein particle, a packaging plasmid, and a transfer plasmid.

5. 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 4.

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

7. 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 4, the recombinant packaging cell of claim 5, or the lentivirus of claim 6.

8. The target cell, characterized in that, The target cells include hematopoietic stem cells, and the target cells contain the lentivirus as described in claim 6.

9. The target cell according to claim 8, characterized in that, The target cells include CD34. + Hematopoietic stem cells.

10. The use of the lentiviral vector packaging system according to any one of claims 1 to 4, the recombinant packaged cells according to claim 5, the lentivirus according to claim 6, the composition according to claim 7, or the target cells according to any one of claims 8 to 9 in the preparation of cell therapy drugs.

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