CN-121472333-B - Virus vector for hematopoietic stem cells and application thereof

Abstract

The application provides a viral vector for hematopoietic stem cells and application thereof. In a first aspect the application provides a lentiviral vector packaging system for a hematopoietic stem cell comprising a recombinant expression vector comprising a nucleotide sequence encoding thrombopoietin and fms-related receptor tyrosine kinase 3 ligand. The application constructs thrombopoietin and fms related receptor tyrosine kinase 3 ligand on nucleic acid molecules, so that the ligand is expressed on the surface of a slow virus vector, and the result shows that compared with a direct adding mode, the application can effectively activate and promote the stem property or the immune activity of various cells including hematopoietic stem cells at a lower dosage.

Inventors

• XIONG YECHENG
• YIN ZEHUA
• ZHANG TINGTING
• HU DINGFANG
• OUYANG WENJIE
• LIU CHAO
• GU YING

Assignees

• 深圳华大生命科学研究院
• 深圳市禾沐基因生物技术有限责任公司

Dates

Publication Date

20260505

Application Date

20260108

Claims (6)

1. 1. A lentiviral vector packaging system for hematopoietic stem cells, the lentiviral vector packaging system comprising: the recombinant expression vector comprises a costimulatory factor coding region sequence, wherein the costimulatory factor coding region sequence comprises a nucleotide sequence for coding thrombopoietin and fms related receptor tyrosine kinase 3 ligand, the nucleotide sequence for coding thrombopoietin is shown as SEQ ID NO.2, and the nucleotide sequence for coding fms related receptor tyrosine kinase 3 ligand is shown as SEQ ID NO. 3; the envelope protein plasmid comprises an insertion site region, the insertion site region comprises a targeting antibody coding region and a rhabdovirus glycoprotein coding region, the targeting antibody coding region and the rhabdovirus glycoprotein coding region are connected through a 2A peptide connecting region, the rhabdovirus glycoprotein coded by the rhabdovirus glycoprotein coding region has an amino acid sequence shown as SEQ ID NO.13, and the targeting antibody coded by the targeting antibody coding region has an amino acid sequence shown as SEQ ID NO. 11.
2. 2. The lentiviral vector packaging system of claim 1, further comprising at least one of a packaging plasmid and a transfer plasmid.
3. 3. A recombinant packaging cell for hematopoietic stem cells, characterized in that it comprises a lentiviral vector packaging system according to any one of claims 1 to 2.
4. 4. Lentivirus for hematopoietic stem cells, characterized in that it is obtained after transfection of packaging cells with a lentivirus vector packaging system according to any one of claims 1 to 2.
5. 5. A composition for use in hematopoietic stem cells, comprising 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. 6. Use of the lentiviral vector packaging system of any one of claims 1 to 2, the recombinant packaging cell of claim 3, the lentivirus of claim 4, or the composition of claim 5 in the preparation of a cell therapy medicament.

Description

Virus vector for hematopoietic stem cells and application thereof Technical Field The application relates to the technical field of cell biology, in particular to a viral vector for hematopoietic stem cells and application thereof. Background Gene therapy represented by cell therapy has shown broad prospects in basic research, and part of strategies have been successfully transformed into clinical research and application. The obtained cell therapy is usually carried out by constructing specific functional cells through in vitro transduction and then reinjecting the cells into a patient, and has the advantages of complex flow, high individuation degree and difficult popularization. In recent years, researchers have focused on direct transduction of cells of interest in patients, but this places high demands on in vivo transduction vectors for transduced cells (1) accurate targeting to reduce off-target and carrier depletion resulting from transduction of non-target cells, (2) high transduction efficiency and stimulation and activation of cells, and (3) low immunogenicity. Hematopoietic STEM CELLS (HSC) is an ideal target cell for cell therapy, whose genetic information is inherited to all lineages derived from them as cells divide and differentiate, and HSC transplantation is currently the only choice demonstrated to have curative effects for diseases such as thalassemia (β -thalassemia) and sickle cell anemia (SICKLE CELL DISEASE, SCD). Therefore, the development of targeted in vivo transduction vectors for HSC can effectively reduce the treatment risk and cost of the diseases, and has important significance for accelerating the clinical application of cell therapy. The eukaryote delivery vectors which are proved to be effective at present can be divided into viral vectors and non-viral vectors, the non-viral vectors have simpler structure, and although the scale production is easy, the in-vivo targeting is difficult to achieve, and the non-targeting transduction at the liver, alveoli and the like is realized mainly by changing the administration mode. In contrast, viral vectors, particularly lentiviral vectors (LENTIVIRAL VECTOR, LV), are capable of achieving targeted transduction of specific cells through engineering of envelope glycoproteins (e.g., vesicular stomatitis virus glycoprotein, VSV-G), but still suffer from poor 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 cause the stem property or the immunocompetence of the target cells to be affected to some extent, so that a vector capable of achieving both targeted transduction efficiency and stimulation effect is needed. Disclosure of Invention The embodiment of the invention mainly aims at providing a virus vector for hematopoietic stem cells and application thereof. To achieve the above object, a first aspect of the present application provides a lentiviral vector packaging system for hematopoietic stem cells, comprising a recombinant expression vector comprising nucleotide sequences encoding thrombopoietin (Thrombopoietin, TPO) and Fms-related receptor tyrosine kinase 3 ligand (Fms-like Tyrosine Kinase 3 Ligand, FLT 3L), the nucleotide sequence encoding thrombopoietin being shown as SEQ ID NO.2, and the nucleotide sequence encoding Fms-related receptor tyrosine kinase 3 ligand being shown as SEQ ID NO. 3. In some embodiments, the recombinant expression vector further comprises a nucleotide sequence encoding a Stem Cell Factor (SCF). In some embodiments, the nucleotide sequence encoding a stem cell factor is set forth in SEQ ID NO. 1. In some embodiments, the stem cell factor has an amino acid sequence as set forth in SEQ ID NO. 4. In some embodiments, the amino acid sequence of thrombopoietin is shown in SEQ ID NO. 5. In some embodiments, the amino acid sequence of fms-related receptor tyrosine kinase 3 ligand is set forth in SEQ ID No. 6. 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. In some embodiments, the enhancer comprises the cytomegalovirus enhancer (CMV ENHANCER). In some embodiments, the promoter comprises a cytomegalovirus promoter (CMV promoter). In some embodiments, the intron comprises a human β -globin intron (β -globin intron). In some embodiments, the polyadenylation signal comprises a human β -globin polyadenylation signal (β -globin poly a). In some embodiments, the recombinant expression vector is a plasmid vector. 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. In some embodiments, the envelope plasmid comprises an insertion site region. In some emb