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CN-121986158-A - Method for producing immortalized megakaryocyte progenitor cells from adult hematopoietic stem cells and progenitor cells for in vitro mass production of functional platelets

CN121986158ACN 121986158 ACN121986158 ACN 121986158ACN-121986158-A

Abstract

The present invention provides a method for generating human immortalized megakaryocytes and/or human immortalized megakaryocyte progenitor cells and/or human megakaryocyte cell lines (102) from adult cd34+hspcs (100), said adult cd34+hspcs (100) being capable of maturing into functional megakaryocytes (103) producing functional platelets (104), said method comprising at least the steps of introducing an expression cassette comprising at least one gene into said cd34+hspcs (100) by transduction, in particular Lentiviral (LV), wherein the transduced cd34+hspcs (100) is configured to express at least one gene from the HOXL subclass of the ANTP homeobox family and/or TALE homeobox family upon activator-induced expression, and constitutively expressing at least one anti-apoptotic gene from the BCL2 family, wherein said transduced cd34+hspcs (100) by expression of said at least one gene from said homobox family and/or said 3, b 2 is preferably from at least one of the PBX2, 3h 2 is, b 1 and b 2 is preferably from said 3, 3h 2 is or at least one of the 3h 2 is, b 2 is preferably a combination of said PBX, 3h 2 is or 3h 2 is, b 2 is preferably selected from said at least one of the 3h 2 is. The present invention further provides a method for producing functional platelets by using the immortalized megakaryocytes and/or immortalized megakaryocyte progenitor cells and/or immortalized megakaryocyte cell lines obtained by the above method. Still further, the present invention provides a pharmaceutical composition comprising functional platelets obtained by the above method and therapeutic uses thereof.

Inventors

  • Laurent Bunier
  • Marjorie Amber
  • CELINE ROCCA
  • Panoyot Bifsha

Assignees

  • 海默斯特股份有限公司

Dates

Publication Date
20260505
Application Date
20240630
Priority Date
20230809

Claims (15)

  1. 1. A method of producing human immortalized megakaryocytes and/or human immortalized megakaryocyte progenitor cells and/or human megakaryocyte cell lines (102) from adult cd34+ hematopoietic stem cells and progenitor cells (HSPCs) (100) capable of maturing into functional megakaryocytes (103) that produce functional platelets (104), the method comprising at least: S1, forced expression of at least one gene is carried out by introducing an expression cassette comprising the at least one gene into the CD34+HSPC (100) by transduction, in particular Lentiviral (LV) transduction, Wherein the transduced cd34+ HSPCs (100) are configured to express at least one gene from the HOXL subclasses of the antennapedia homeoprotein (ANTP) homeobox family and/or the tri-amino acid loop extension (TALE) homeobox family upon activator-induced expression, and to constitutively express at least one anti-apoptotic gene from the BCL2 family; Wherein the transduced CD34+HSPC (100) is immortalized by expression of the at least one gene from the HOXL subclasses of the ANTP and/or TALE homology cassette families, Preferably wherein said at least one gene from said HOXL subclass comprises a combination of one of the HOXB8, HOXA7, HOXA10 or HOXB4 genes with at least one of the MEIS1, MEIS2, MEIS3, PBX1, PBX2, PBX3 and/or PBX4 genes, and said at least one anti-apoptotic gene from said BCL2 family comprises BCL2L1 or BCL2L2.
  2. 2. The method according to claim 1, characterized in that the immortalized megakaryocytes and/or immortalized megakaryocyte progenitor cells and/or human megakaryocyte cell lines (102) produced are capable of providing mature functional platelets (104), in particular functional clinical grade platelets (104), upon differentiation and maturation in the absence of the activator.
  3. 3. The method according to claim 1, wherein the activator is one of tetracycline or an estrogen receptor modulator and/or isopropyl β -D-1-thiogalactopyranoside (IPTG) and/or lactate and/or 4 isopropylbenzoate.
  4. 4. The method according to one of the preceding claims, characterized in that the method further comprises: s2, culturing the transduced CD34+HSPC (100), Wherein culturing the transduced cd34+ HSPCs (100) comprises amplifying the transduced cd34+ HSPCs (100) in an amplification medium composition in the presence of the activator and at least one growth factor mixture.
  5. 5. The method of claim 4, wherein the at least one growth factor mixture comprises at least one of Thrombopoietin (TPO), TPO receptor agonist, stem Cell Factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 9 (IL-9), and interleukin 11 (IL-11).
  6. 6. The method according to one of the preceding claims, characterized in that the method further comprises: s3 differentiating and maturing said transduced cd34+ HSPCs and/or said immortalized megakaryocytes and/or said immortalized megakaryocyte progenitor cells and/or said immortalized cell line (102) into mature functional megakaryocytes (103) in the absence of said activator and in the presence of said at least one growth factor mixture.
  7. 7. The method of claim 6, wherein differentiation and maturation is performed in a differentiation and maturation medium composition that is different from the amplification medium composition.
  8. 8. The method according to one of the preceding claims, characterized in that the method further comprises: s4 culturing said immortalized megakaryocyte and/or said immortalized megakaryocyte progenitor and/or said immortalized megakaryocyte cell line in the absence of said activator (102).
  9. 9. The method according to one of claims 4 to 8, wherein the step of culturing the transduced adult cd34+ HSPCs (100) and/or the immortalized megakaryocytes and/or the immortalized megakaryocyte progenitor cells and/or the immortalized megakaryocyte cell lines (102) is performed in the absence of serum and/or in the absence of feeder cells.
  10. 10. The method according to one of the preceding claims, wherein the adult cd34+ HSPCs (100) are derived from human donors, in particular adult peripheral blood or bone marrow of a single human donor.
  11. 11. A method of producing functional platelets (104), the method comprising: s11 obtaining immortalized megakaryocytes and/or immortalized megakaryocyte progenitor cells and/or immortalized megakaryocyte cell lines (102) by performing the method according to any one of claims 1 to 10; S12, culturing the obtained immortalized megakaryocyte and/or immortalized megakaryocyte progenitor cells and/or immortalized megakaryocyte cell lines (102); S13 differentiating and maturing said immortalized megakaryocyte and/or said immortalized megakaryocyte progenitor and/or said immortalized megakaryocyte cell line (102) into mature functional megakaryocytes (103), and S14 functional platelets (104) are obtained by mechanical manipulation of the mature functional megakaryocytes (103), preferably in a bioreactor or a microfluidic device.
  12. 12. A functional platelet (104) obtained by implementing the method according to claim 11.
  13. 13. A pharmaceutical composition comprising the functional platelets (104) of claim 12.
  14. 14. Use of a pharmaceutical composition according to claim 13 for the treatment of a disease, in particular for target-specific treatment of a disease, preferably wherein the disease is one of thrombocytopenia or hemorrhagic disorders.
  15. 15. Use of the pharmaceutical composition according to claim 13 for regenerative medicine.

Description

Method for producing immortalized megakaryocyte progenitor cells from adult hematopoietic stem cells and progenitor cells for in vitro mass production of functional platelets The present invention belongs to the field of functional platelet obtaining method and technology, and is especially functional clinical grade platelet obtained from human immortalized megakaryocyte and/or human immortalized megakaryocyte progenitor cell and/or human megakaryocyte cell line. In particular, the present invention relates to a method of producing human immortalized megakaryocytes and/or human immortalized megakaryocyte progenitor cells and/or human megakaryocyte cell lines from adult cd34+ hematopoietic stem cells and progenitor cells (HSPCs) capable of maturing into functional megakaryocytes that produce functional platelets. The invention further relates to a method for producing functional platelets, in particular functional clinical grade platelets, using the immortalized megakaryocytes and/or immortalized megakaryocyte progenitor cells and/or immortalized megakaryocyte cell lines obtained by the above method. Still further, the present invention relates to a functional platelet obtained by the above method. Finally, the present invention relates to a pharmaceutical composition comprising functional platelets obtained by the above method and to the therapeutic use thereof. Diseases such as thrombocytopenia result in lower platelet levels in the affected patient. In such cases, platelet infusion is the only treatment option to avoid life threatening bleeding. Platelet infusions to patients can also be used to treat other clinically relevant conditions, such as leukemia, bone marrow transplantation, anti-cancer therapies, and the like. Platelets can be isolated during the gratuitous donation. However, their short shelf life (in the range of about 5 to 7 days) has a negative impact on their usability. This is even more problematic in case of health crisis like epidemic. In addition, platelets from donors always contain human plasma, red blood cells and residual white blood cells, which can lead to post-transfusion adverse events unless the concentrate is treated with devices such as filters or pathogen reduction techniques. In vivo, platelets are formed from precursor cells called Megakaryocytes (MK) derived from pluripotent hematopoietic stem cells and progenitor stem cells (HSPCs) residing in the Bone Marrow (BM) (Wang and Zheng, 2016) and released into the blood. Megakaryocytogenesis, also known as megakaryocytogenesis or thrombopoiesis, is the process by which megakaryocytes (and ultimately platelets) develop from HSPCs. Differentiation of platelets from Hematopoietic Stem Cells (HSCs) proceeds through multiple cell differentiation steps, coordinated by a number of transcriptional programs regulated by a network of transcription factors. In principle, the hierarchical megakaryopoiesis model is the differentiation of HSCs into pluripotent progenitors, the differentiation of pluripotent progenitors into common myeloid progenitors, the differentiation of common myeloid progenitors into megakaryocyte-erythroid progenitors, and the differentiation of megakaryocyte-erythroid progenitors into megakaryoblasts. Megakaryocyte cells mature into megakaryocytes in the bone marrow. Mature megakaryocytes migrate to the sinus vessels and form pseudopodic extensions (pre-platelets) through vascular endothelial cells to produce platelets. For many years, great efforts have been put into developing techniques for producing platelets in vitro from different sources to maintain a continuous need for fresh platelet concentrates, in particular, (1) HSPCs, (2) reprogrammed cells, (3) non-hematopoietic sources, and (4) artificial platelets. Mass production of platelets from HSPCs for therapeutic use is a challenge, mainly for the following reasons. First, MK derived in vitro produces less platelets than in vivo. In addition, in vitro expansion of megakaryocytes or their progenitors has limited ability to self-renew. Finally, the sources of megakaryocytes or their progenitors are limited because they are isolated from Peripheral Blood (PB) or BM punctures from volunteer donors. In particular, even though large-scale donation of megakaryocyte progenitor cells and/or platelets is possible, the correct isolation, maturation, and platelet generation protocols would be prohibitively expensive and would result in unresolved variables in platelet generation and quality with potential negative impact on patient safety and effectiveness. Therefore, large-scale blood donation is not suitable for industrialization for therapeutic use. New techniques for identifying functional platelets produced ex vivo (in vitro) would meet clinical needs, help reduce the cost of safety testing, and support patient treatment. Development of megakaryocyte cell lines as a single source of platelet production would allow for the production of unlimited numbers of platelets