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

Abstract

The present invention provides a method for producing human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines (102) from adult CD34 + HSPC (100) capable of maturation into functional megakaryocytes (103) producing functional platelets (104), said method comprising at least the steps of: producing functional platelets (104) by transduction, in particular lentiviral (LV) transduction; an expression cassette comprising at least one gene is introduced into the CD34 + HSPC (100) to effectuate forced expression of the at least one gene wherein the transduced CD34 + HSPC (100) is configured to express at least one gene from the HOXL subclass of the ANTP homologous cassette family and / or the TALE homologous cassette family upon activator-induced expression, and constitutively expressing 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 ANTP homologous cassette family and / or the HOXL subclass of the TALE homologous cassette family, preferably wherein the at least one gene from the HOXL subclass comprises a combination of one of the HOXB8, HOXA7, HOXA10 or HOXB4 genes and at least one of the MEIS1, MEIS2, MEIS3, PBX1, PBX2, PBX3 and / or PBX4 genes, and the at least one anti-apoptosis gene from the BCL2 family comprises BCL2L1 or BCL2L2. The invention further provides a method of 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. Further, the present invention provides a pharmaceutical composition comprising functional platelets obtained by the above method and its therapeutic use.

Description

[0001] This invention belongs to the field of methods and technical areas for obtaining functional platelets, specifically functional clinical-grade platelets, from human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines.

[0002] Specifically, the present invention relates to a method for generating 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.

[0003] The present invention further relates to a method for producing functional platelets, specifically functional clinical-grade platelets, using immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines obtained by the above method.

[0004] Furthermore, the present invention relates to a functional platelet obtained by the above method.

[0005] Finally, the present invention relates to a pharmaceutical composition comprising functional platelets obtained by the above method and its therapeutic use.

[0006] Diseases such as thrombocytopenia can cause patients to have low platelet levels.

[0007] In such cases, platelet transfusion is the only treatment option to avoid life-threatening bleeding.

[0008] Platelet transfusions can also be used to treat other clinically relevant conditions, such as leukemia, bone marrow transplantation, and cancer treatment.

[0009] Platelets can be separated during voluntary blood donation.

[0010] However, their short shelf life (ranging from about 5 to 7 days) negatively impacts their availability.

[0011] This becomes even more problematic during health crises such as pandemics.

[0012] In addition, platelets from donors always contain human plasma, red blood cells, and residual white blood cells, which can lead to adverse events after transfusion unless the concentrate is treated with devices such as filters or pathogen reduction techniques.

[0013] In the body, platelets are formed and released into the bloodstream by precursor cells called megakaryocytes (MKs), which are derived from pluripotent hematopoietic stem cells and progenitor stem cells (HSPCs) residing in the bone marrow (BM) (Wang and Zheng, 2016).

[0014] Megakaryocyte formation, also known as megakaryocyte production or platelet formation, is the process by which megakaryocytes (and eventually platelets) develop from HSPCs.

[0015] Platelet differentiation from hematopoietic stem cells (HSCs) occurs through multiple cell differentiation steps coordinated by numerous transcriptional programs regulated by a network of transcription factors.

[0016] In principle, the hierarchical megakaryocyte generation model is as follows: HSCs differentiate into pluripotent progenitor cells, pluripotent progenitor cells differentiate into ordinary myeloid progenitor cells, ordinary myeloid progenitor cells differentiate into megakaryocyte-erythroid progenitor cells, and megakaryocyte-erythroid progenitor cells differentiate into megakaryocyte blast cells.

[0017] Megakaryocytes mature into megakaryocytes in the bone marrow.

[0018] Mature megakaryocytes migrate into sinusoidal vessels and form pseudopodia-like extensions (proplatelets) through vascular endothelial cells to produce platelets.

[0019] Over the years, tremendous efforts have been made to develop technologies for generating platelets in vitro from various sources in order to maintain the ongoing demand for fresh platelet concentrates, specifically: (1) HSPCs, (2) reprogrammed cells, (3) non-hematopoietic sources, and (4) artificial platelets.

[0020] Mass production of platelets from HSPC for therapeutic use is a challenge, primarily for the following reasons.

[0021] First, MK from external sources produces fewer platelets than MK from within the body.

[0022] In addition, the in vitro expansion of megakaryocytes or their progenitor cells has limited self-renewal capacity.

[0023] Ultimately, the source of megakaryocytes or their progenitor cells is limited, as they are isolated from peripheral blood (PB) or BM punctures from volunteer donors.

[0024] Specifically, even if large-scale donation of megakaryocyte progenitor cells and / or platelets is possible, proper isolation, maturation, and platelet production protocols would be too expensive and would introduce unresolved variables in platelet production and quality, potentially negatively impacting patient safety and efficacy.

[0025] Therefore, mass blood donation is not suitable for industrial use for therapeutic purposes.

[0026] New technologies for identifying functional platelets produced in vitro will meet clinical needs, help reduce the cost of safety testing, and support patient treatment.

[0027] Developing megakaryocyte cell lines as a single source of platelet production would allow for the production of an unlimited number of platelets of equivalent quality, thereby improving patient safety and transfusion efficiency.

[0028] To overcome the limited self-renewal capacity of HSPCs, scientists have developed human induced pluripotent stem cells (iPSCs). Under appropriate growth conditions (including but not limited to a mixture of growth factors), iPSCs have the potential to differentiate into megakaryocytes and produce platelet-like granules (Chen, Sugimoto and Eto, 2023).

[0029] This solution provides an inexhaustible, self-renewing source of megakaryocytes that can be genetically modified to become universal donor platelets (Figueiredo et al., 2010) and can be grown under serum-free conditions (Pick et al., 2013).

[0030] However, the clinical benefits of this technology have not yet been proven.

[0031] One limitation is that iPSCs are more prone to tumorigenesis after transplantation, and current methods for generating iPSCs cannot guarantee reproducibility and maintenance because the raw materials come from differentiated tissues (Prochazkova et al., 2015). As seen in experimental work, the maturation of iPSCs into megakaryocytes is suboptimal (Mookerjee et al., 2020). Furthermore, due to their embryonic-like biology, iPSCs may lead to the generation of primitive megakaryocytes, resulting in low levels of key receptors such as CD9 or GPIV expressed by megakaryocytes and platelets, thereby reducing their functionality (Mattia et al., 2002; Bluteau et al., 2013).

[0032] EP2708597B1 discloses a method for generating polyploid megakaryocytes. Specifically, this known method comprises the following steps: forcibly expressing an oncogene selected from the MYC gene family and multicomb genes (such as BMI1) in cells at any differentiation stage from hematopoietic progenitor cells to pre-polyploidized megakaryocytes (MKs); forcibly expressing BCL-XL in pre-polyploidized MKs; and culturing and proliferating the resulting cells to obtain polyploid megakaryocytes. A method for generating platelets from the obtained polyploidized MKs is further disclosed therein.

[0033] Other cell types have also been developed for the in vitro production of megakaryocytes and platelets.

[0034] Endothelial cells and fibroblasts can transdifferentiate into megakaryocytes under specific conditions (Ono et al., 2012; Lis et al., 2017).

[0035] However, the most promising cell type is adipose tissue-derived stromal cells, specifically because of their availability, despite the limitation that each cell produces less than one platelet (Tozawa et al., 2019).

[0036] Furthermore, although described as functional, these megakaryocytes and the resulting platelets are not derived from their natural progenitor cells, namely HSPCs. Therefore, they may lack important characteristics such as hemostatic efficacy.

[0037] To overcome the limitations of platelet progenitor cells, scientists are also attempting to develop artificial platelets using biosynthetic particle constructs that functionally mimic various hemostatic mechanisms of platelets.

[0038] Several approaches exist for developing these artificial platelets, such as protein-modified artificial platelet systems, peptide-modified artificial platelet systems, and heterologous multivalent modifications (Luc et al., 2022).

[0039] Although artificial platelets appear to be an excellent alternative solution, specifically in terms of their versatility, precise studies are still needed to assess the risk of thrombosis, scalability, potential immune response in the event of multiple infusions, and total cost.

[0040] In view of the foregoing, one object of the present invention is to provide an improved method for generating human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines, the method allowing the acquisition of high-quality functional platelets from adult CD34+ HSPCs, the functional platelets being as similar as possible to circulating natural platelets in clinically relevant quantities.

[0041] Another object of the present invention is to provide a method for producing functional platelets using human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines obtained by the above method.

[0042] Another object of the present invention is to provide a pharmaceutical composition comprising functional platelets obtained by the above method and its therapeutic use therein.

[0043] The above objective is achieved by providing the method as defined in claim 1.

[0044] According to the present invention, a method for generating 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 includes at least: S1: By transduction, specifically lentiviral (LV) transduction, an expression cassette containing at least one gene is introduced into the CD34+ HSPC (100) to force expression of at least one gene. The transduced CD34+ HSPCs were configured to express at least one gene from the HOXL subclass of the antennal foot homeotype protein (ANTP) homeobox family and / or the triamino acid ring extension (TALE) homeobox family upon activator-induced expression, and constitutively express at least one anti-apoptotic gene from the BCL2 family. The transduced CD34+ HSPCs are immortalized by expressing at least one gene from the HOXL subclass of the ANTP homeobox family and / or the TALE homeobox family. Preferably, the at least one gene from the 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 the at least one anti-apoptotic gene from the BCL2 family comprises BCL2L1 or BCL2L2.

[0045] This invention provides a method for producing human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines.

[0046] Specifically, according to the present invention, human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines are obtained from adult CD34+ hematopoietic stem cells and progenitor cells (HSPCs) capable of maturing into functional megakaryocytes.

[0047] Specifically, these functional megakaryocytes are able to produce functional platelets.

[0048] The method includes at least the following steps: introducing an expression cassette containing at least one gene into a CD34+ HSPC via transduction, specifically lentivirus (LV) transduction, to perform forced expression of at least one gene.

[0049] Transduction using lentiviruses allows expression cassettes to be integrated into the cell's genome, thereby avoiding loss of gene expression.

[0050] Preferably, second- or third-generation lentiviruses are used for transduction.

[0051] Specifically, the box contains a constitutive promoter (e.g., an elongation factor (EF) 1a) and / or an inductive promoter, such as a tetracycline-dependent promoter (e.g., a tetracycline response element (TRE)).

[0052] The transduced CD34+ HSPCs were configured to express at least one gene from the HOXL subclass of the antennal foot homeotype protein (ANTP) homeobox family and / or the triamino acid ring extension (TALE) homeobox family upon activator-induced expression, and constitutively express at least one anti-apoptotic gene from the BCL2 family.

[0053] The transduced CD34+ HSPC is immortalized by expressing at least one gene from the HOXL subclass of the ANTP homeobox family and / or the TALE homeobox family.

[0054] Preferably, the at least one gene from the 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.

[0055] Preferably, the at least one anti-apoptotic gene from the BCL2 family includes BCL2L1 or BCL2L2.

[0056] This invention is based on the fundamental idea of ​​immortalizing human adult CD34+ HSPCs (the natural source of megakaryocytes and platelet-derived stem cells and progenitor cells) by blocking the differentiation process, while ensuring continuous proliferation through transduction of at least one gene, the expression of which is controlled by an activator and at least one constitutively expressed gene. The transduced CD34+ HSPCs are immortalized and can differentiate into mature megakaryocytes by reducing the expression of at least one transduced gene, resulting in an immortalized megakaryocyte cell line with almost unlimited proliferative capacity while preserving the megakaryocyte differentiation potential.

[0057] Therefore, it provides a virtually unlimited source of cells for the in vitro production of clinically relevant platelets.

[0058] Specifically, this near-unlimited proliferative capacity allows for the acquisition of a potentially unlimited reserve of immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or human megakaryocyte cell lines, without the need to constantly search for new sources of megakaryocyte progenitors.

[0059] This, in particular, allows for increased uniformity between batches of platelets produced.

[0060] The method allows for the production of standardized platelets that function similarly to naturally occurring platelets circulating in the body.

[0061] Furthermore, the method allows for a reduction in total production costs.

[0062] Immortalized cell lines can be defined as populations derived from multicellular organisms that do not normally proliferate indefinitely, but are genetically modified to avoid cellular senescence and maintain division. Therefore, these cells can grow in vitro for extended periods. The genetic modifications required for immortality can occur naturally or can be intentionally introduced or induced.

[0063] CD34 is a transmembrane phosphoglycoprotein encoded by the CD34 gene in humans, mice, rats, and other species.

[0064] CD34 protein is a member of the single-pass transmembrane sialic acid mucin protein family that is shown to be expressed on early hematopoietic and vascular-associated progenitor cells.

[0065] Immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or megakaryocyte cell lines obtained by the method of the present invention can provide functional platelets, specifically functional clinical-grade platelets, upon maturation into megakaryocytes.

[0066] As previously described, an unlimited cell source for in vitro platelet production is provided, which allows for the production of enough platelets for transfusion, obtains platelets with similar function to naturally occurring platelets in vivo, achieves versatility, and reduces overall production costs.

[0067] As described below, mature megakaryocytes can be mechanically manipulated to produce platelets on demand.

[0068] The method of the present invention allows for overcoming the aforementioned disadvantages and limitations of existing technical solutions.

[0069] Immortalized megakaryocyte cell lines derived from human HSPCs, specifically adult CD34+ HSPCs, have the advantage of being able to grow for a longer period of time in basal growth medium compared to megakaryocyte progenitor cells derived from human HSPCs, specifically adult CD34+ HSPCs.

[0070] Immortalized CD34+ HSPCs and / or mature megakaryocytes derived from immortalized CD34+ HSPCs provide the possibility of freezing and thawing immortalized CD34+ HSPCs and / or mature megakaryocytes derived from immortalized CD34+ HSPCs as needed, representing an unlimited source of megakaryocytes for producing improved functional platelets at minimal cost.

[0071] Members of the B-cell lymphoma 2 (BCL2) family are well known for their role in regulating apoptosis.

[0072] Anti-apoptotic proteins from the BCL2 family, specifically MCL1, BCL2L1, and BCL2L2, are known to increase proplatelet formation in mature megakaryocytes (Kodama et al., 2012; Bhatlekar et al., 2019), and BCL2L1 expression has a beneficial effect on platelet lifespan (Mason et al., 2007; Zhang et al., 2007; Debrincat et al., 2012).

[0073] In summary, leukemia patients treated with Venetoclax (an inhibitor of the BCL2 family of anti-apoptotic members) are prone to thrombocytopenia (10.6004 / jadpro.2022.13.4.4).

[0074] Key transcription factors that coordinate hematopoiesis are members of the homeobox family.

[0075] The HOXL subclass of ANTP homeobox family genes (HOX genes) is known to be highly involved in the regulation of hematopoiesis, and when aberrantly expressed in acute myeloid leukemia, it is associated with abnormal proliferation and inhibition of differentiation.

[0076] HOXB4, HOXA9, HOXA10 and HOXB8 can immortalize hematopoietic progenitor cells in a growth-dependent manner, but when used alone, they reduce megakaryogenesis and erythropoiesis potential (Ferrell et al., 2005; Redecke et al., 2013; Stahlhut et al., 2021).

[0077] Several HOX genes, such as HOXB8, HOXA10, or HOXA7, are expressed in the CD34+ subset that allows CFU-GEMM to proliferate and in the K562 cell line with megakaryocyte differentiation potential, and are important for cell fate determination (Sauvageau et al., 1994; So et al., 2004; Ferrell et al., 2005).

[0078] As described above, in the method of the present invention, the preferred HOXL subclasses are HOXB8, HOXA7, HOXA10 or HOXB4.

[0079] The transcription factor members of the TALE family are atypical homologous domain proteins, characterized by the insertion of three residues between helix 1 and 2, and play an important role in embryonic development through a transcription factor network that interacts with the HOX gene.

[0080] Members of the myeloid troponin-binding virus integration site (MEIS) homeobox family, along with pre-B-cell leukemia transcription factor (Pbx), act as cofactors for the HOX gene to promote DNA binding and maintain hematopoietic cell proliferation, while inhibiting myeloid differentiation in the presence of growth factors (Calvo et al., 2001; Fujino et al., 2001; Wong et al., 2007; Bessa et al., 2008; Jolma et al., 2015).

[0081] Specifically, MEIS1 has been described as an important regulator of early hematopoiesis and megakaryocyte differentiation, and constant MEIS1 expression is essential for progenitor fate selection to the MK lineage (Cai et al., 2012).

[0082] Preferably, the resulting immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or human megakaryocyte cell lines are capable of differentiating and maturing to provide mature functional platelets, specifically functional clinical-grade platelets, in the absence of the activator.

[0083] Preferably, the activator is tetracycline or an estrogen receptor modulator.

[0084] As a non-limiting example, the activator may be tetracycline or a derivative thereof.

[0085] For example, the activator may be one of the following: chlortetracycline, oxytetracycline, demethylcycline, lysine, methacycline, minocycline, hydropyrtetracycline, doxycycline, tigecycline, eracycline, salazine, or omacycline.

[0086] Estrogen receptor modulators can be selective estrogen receptor modulators.

[0087] Specifically, estrogen receptor modulators can be tamoxifen, toremifene, raloxifene, opemifene, diacetylcholine, bardoxifene, bromoestradiol, clomiphene, cyclofennig, lasoxifene, or omexifen.

[0088] Alternatively or alternatively, the activator may be isopropyl β-D-1-thiogalactopyranoside (IPTG) and / or lactose (e.g., via a Lac operon-based system).

[0089] Alternatively or alternatively, the activator may be cumate (e.g., an operating system controlled by cumate).

[0090] Gene expression can be activated (in other words, induced) by adding an activator (e.g., transcriptional activation controlled by tetracycline).

[0091] This has the advantage that the gene is expressed only during the amplification phase and reduced during the maturation phase.

[0092] Advantageously, the method further includes the step of culturing transduced adult CD34+ HSPCs.

[0093] Specifically, the step of culturing transduced adult CD34+ HSPCs includes amplifying transduced adult CD34+ HSPCs in the presence of a mixture of activator and at least one growth factor.

[0094] Specifically, the step of culturing transduced CD34+ HSPCs is carried out in an amplification medium composition.

[0095] The presence of activators allows and / or facilitates the expansion of transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or human megakaryocyte cell lines by implementing forced gene expression regulated by inducible promoters.

[0096] For example, the inducible promoter may include a tetracycline response element (TRE) or a cumate operon (CuO).

[0097] Specific effects can be determined by selecting a combination of individual growth factors and adjusting their concentrations in the mixture.

[0098] Preferably, the at least one growth factor mixture comprises at least one of the following: 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).

[0099] Specifically, these growth factors, whether alone or in combination, promote transduced CD34+ HSPC amplification.

[0100] Advantageously, the method may further include the step of differentiating and maturing transduced CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized cell lines into mature functional megakaryocytes.

[0101] Specifically, this step is performed in the absence of an activator but in the presence of at least one mixture of growth factors.

[0102] Therefore, gene expression regulated by inducible promoters is reduced.

[0103] In addition, growth factors allow and / or enable and / or induce differentiation into mature megakaryocytes.

[0104] Preferably, this step is performed in a differentiation and maturation culture medium composition.

[0105] Differentiation and maturation culture medium compositions differ from amplification culture medium compositions.

[0106] Advantageously, the method may further include the step of culturing the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell lines.

[0107] Specifically, this step is performed in the absence of the activator.

[0108] In principle, cell lines are allowed to be kept in culture for a certain period of time.

[0109] Preferably, the step of culturing the transduced CD34+ HSPC and / or the transduced adult CD34+ HSPC and / or the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell lines is performed in the absence of serum and / or in the absence of feeder cells.

[0110] Serum is quite expensive, and prices vary from batch to batch.

[0111] Therefore, culturing under serum-free conditions allows for cost savings in production and reduces unwanted variability.

[0112] Feeder cells support the growth of cells in culture by providing an uncertain and complex mixture of extracellular matrix (ECM) components and growth factors.

[0113] Using feeder cells in cell culture is generally expensive and complex.

[0114] Therefore, by carrying out culture without using feeder cells, production costs can be saved and unwanted contamination of platelets by non-hematopoietic cells can be reduced.

[0115] Preferably, adult CD34+ HSPCs are derived from healthy adult donors, specifically peripheral blood (PB) or bone marrow (BM) from a single human donor.

[0116] A single donor is advantageous because production time and costs can be minimized, since a donor bank is not required (e.g., only one health test, only one blood collection, etc.).

[0117] The procedure for differentiating HSPCs into megakaryocytes is now well known, and several sources of CD34+ HSPCs (e.g., cord blood (CB), bone marrow (BM), and mobilized or unmobilized peripheral blood (PB)) are available.

[0118] In principle, the CD34+ HSPCs used to generate megakaryocytes according to the present invention can be derived from adult BM and / or PB.

[0119] CD34+ HSPCs from CB, BM, or PB may not be equivalent in availability and may not produce the same quality as mature megakaryocytes and / or platelets derived from megakaryocytes.

[0120] CB represents an abundant source of immature HSPCs and is widely used to generate megakaryocytes due to the high proliferation rate of CB CD34+ HSPCs.

[0121] Experimental evidence shows that megakaryocytes derived from CD34+ HSPCs isolated from CB are different from those derived from CD34+ HSPCs isolated from BM or PB because the size of megakaryocytes decreases upon maturation (Ignatz et al., 2007), which in turn leads to a decrease in platelet production, which may have a negative impact on the function of the resulting platelets (Miyazaki et al., 2000; Gaur et al., 2006).

[0122] Adult BM can be an interesting source of HSPC because CD34+ HSPC is more common in BM than in PB.

[0123] However, the invasive procedures required to obtain BM limit its accessibility.

[0124] Finally, PB might be a suitable solution.

[0125] Blood donation is an easily accessible method, and although the number of adult CD34+ HSPCs in PB is low, they can be mobilized in blood donors using mobilizing agents such as G-CSF and / or plexafor.

[0126] CD34+ HSPCs derived from PB allow for the acquisition of functionally mature megakaryocytes during in vitro differentiation, and subsequently functional platelets.

[0127] In all cases, regardless of the source, CD34+ HSPCs (i.e., non-immortalized HSPCs) have limited proliferation and self-renewal properties and are therefore not suitable for long-term on-demand platelet delivery.

[0128] The cell culture conditions serve two main purposes: the expansion / survival of CD34+ HSPCs and / or transduced CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte lines, as well as their differentiation and maturation into mature megakaryocytes.

[0129] Culture conditions with defined chemical compositions can prevent damage to the final product and reduce variability.

[0130] The steps of culturing transduced CD34+ HSPCs, immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines are preferably performed for a duration of about one day to at least three months.

[0131] Furthermore, the steps of culturing transduced CD34+ HSPCs, immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines are preferably performed in a humid incubator at 37°C and 5% CO2.

[0132] Another important aspect of platelet production is the versatility of the product.

[0133] In fact, allogeneic platelet ineffectiveness is often caused by incompatibility between human leukocyte antigen (HLA) and antibodies that bind to donor epitopes in the receptor.

[0134] Universal platelets are mainly produced through gene modification technology targeting β-2-microglobulin (B2M), which encodes the β2m-light chain component of HLAI class antigens.

[0135] B2M knockdown via RNA interference reduces HLA class I expression, while B2M knockout via endonuclease (e.g., CRISPR / Cas9) produces HLA class I-deficient cells.

[0136] Experimental evidence shows that cultured HLA-deleted or ablated megakaryocytes and platelets retain function while evading immune detection (Suzuki et al., 2020). In the suggested experimental procedures, when transducing adult CD34+ HSPCs to induce expression of the following genes, the addition of a lentiviral vector encoding miRNA B2M (SEQ ID1) may be considered.

[0137] SEQ ID1: 5'-GAATCTTTGGAGTACGCTGGGATGTTTTGGCCACTGACTGACATCCAGCGCTCCAAAGATT-3' This allows for the production of universally compatible human platelet sources that can escape destruction by natural killer cells, thus avoiding potentially harmful immune responses in patients requiring platelet transfusions.

[0138] Another important aspect is the ability to cryogenically transduced CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte cell lines and / or mature megakaryocytes derived from immortalized cells.

[0139] Specifically, the selection of the aforementioned genome and promoter can increase megakaryocyte survival after freezing and thawing, allowing the generation of transduced CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte cell lines and / or mature megakaryocyte banks.

[0140] Specifically, transduced gene members of the BCL2 family with anti-apoptotic function (such as BCL2L1) are expressed from constitutive cellular expression into the process described above.

[0141] The present invention also provides a method for producing functional platelets.

[0142] Here, functional platelets are generated by using immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines obtained by the aforementioned method.

[0143] Specifically, the method includes the step of obtaining immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines by performing the above-described method.

[0144] The method further includes the step of culturing the obtained immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines.

[0145] Furthermore, it includes the step of differentiating immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte cell lines into mature functional megakaryocytes.

[0146] Finally, the method includes the step of obtaining functional platelets by mechanically manipulating mature functional megakaryocytes.

[0147] Preferably, the latter step is carried out in a bioreactor.

[0148] Alternatively, the latter step can be performed in a microfluidic device.

[0149] The functional platelets obtained in this way have similar functions to the naturally occurring platelets circulating in the body.

[0150] Specifically, the method allows for the acquisition of clinically relevant quantities of high-quality functional platelets.

[0151] Furthermore, the method allows for the production of high-quality functional platelets at reduced production costs.

[0152] The present invention further provides a functional platelet, specifically a functional platelet produced by implementing the above method.

[0153] In addition, the present invention provides a pharmaceutical composition comprising functional platelets obtained by the aforementioned method.

[0154] The pharmaceutical composition can be used to treat diseases.

[0155] Specifically, the pharmaceutical composition can be used for targeted and specific treatment of the disease.

[0156] Specifically, the disease could be thrombocytopenia.

[0157] Alternatively, the disease may be a hemorrhagic condition.

[0158] In addition, the pharmaceutical composition can be used in regenerative medicine.

[0159] As can be seen, an advantage of this invention is that adult CD34+ HSPCs are a primary (natural) source of megakaryocytes and platelets. Compared to other techniques using different cell sources, the megakaryocytes and / or platelets produced by the method according to this invention are physiologically closer to those found in vivo. During the immortalization process, adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or mature megakaryocytes can be modified to produce universal platelets. In summary, the method of this invention can practically and economically provide a solution to support clinical needs in transfusion units, therapeutic and / or preventative applications, and / or regenerative medicine.

[0160] Further details and advantages of the invention will now be disclosed in conjunction with the accompanying drawings, which are specifically illustrated in the drawings: Figure 1 This is a block diagram illustrating a method for generating human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines from CD34+ HSPCs capable of maturing into functional megakaryocytes, according to embodiments of the present invention. Figure 2 This is a block diagram illustrating a method for generating functional platelets according to an embodiment of the present invention; Figure 3 It is a display Figure 1 (See the area enclosed by the dashed line) and Figure 2 (Complete diagram) of the method; Figure 4 It is a diagram showing the plasmid maps of TRE-HOXB8, TRE-MEIS1, EF1a-rtTA and EF1a-BCL2L1 used in the described experiment; Figure 5 This is a list of cloning primers suitable for the described experiments. Specifically, the accompanying figure provides a list of primers and restriction endonucleases suitable for the cloning constructs; Figure 6 AI is a graph showing the experimental results of the described experiment, specifically demonstrating the advantages of transducing adult CD34+ HSPCs with HOXB8, MEIS1, BCL2L1 and rtTA to obtain long-term proliferating cells that can maintain the potential to differentiate into mature megakaryocytes. Figure 7 It is a summary of Figure 6 A table showing the different transduction conditions tested in the HI experiment; Figure 8 AH is a figure illustrating experimental evidence demonstrating the clinical quality of platelets produced using mature megakaryocytes derived from adult CD34+ HSPCs transduced with HOXB8-MEIS1-BCL2L1-rtTA. Figure 9 The figure shows the reduction of B2M in transduced adult CD34+ HSPCs and its effect on HLA-I surface expression.

[0161] Figure 1 A block diagram illustrating steps S1 to S4 of a method for generating human immortalized megakaryocytes and / or human immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines from adult CD34+ hematopoietic stem cells and progenitor cells (HSPCs) 100 according to an embodiment of the present invention is provided.

[0162] Specifically, CD34+ HSPCs can mature into functional megakaryocytes 103 that produce functional platelets 104.

[0163] The method includes step S1, which involves introducing an expression cassette containing at least one gene into a CD34+ HSPC 100 via transduction, specifically lentivirus (LV) transduction, to enforce the expression of at least one gene.

[0164] The transduced CD34+ HSPC 100 was configured to express at least one gene from the HOXL subclass of the antennal foot homeotype protein (ANTP) homeobox family and / or the triamino acid ring extension (TALE) homeobox family upon activator-induced expression.

[0165] In embodiments of the present invention, the activator may be a manipulation system controlled by tetracycline or estrogen receptor modulator and / or isopropyl β-D-1-thiogalactopyranoside (IPTG) and / or 4-isopropylbenzoate (cumate).

[0166] Different types of activators can also be used, as long as they are suitable for the stated purpose.

[0167] Transduced CD34+ HSPC 100 constitutively express at least one anti-apoptotic gene from the BCL2 family.

[0168] The transduced CD34+ HSPC 100 is immortalized by expressing at least one gene from the HOXL subclass of the ANTP homeobox family and / or the TALE homeobox family.

[0169] In embodiments of the present invention, the at least one gene from the HOXL subclass includes 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.

[0170] Furthermore, in the embodiments of the invention, the at least one anti-apoptotic gene from the BCL2 family includes BCL2L1 or BCL2L2.

[0171] The resulting immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or human megakaryocyte cell lines 102 are able to provide functional platelets 104 during differentiation and maturation in the absence of the activator, specifically functional clinical-grade platelets 104.

[0172] In an embodiment of the present invention, the method further includes step S2 of culturing transduced CD34+ HSPC 100.

[0173] Specifically, the transduced CD34+ HSPC 100 is cultured by amplifying transduced adult CD34+ HSPC 100 in the presence of a mixture of activator and at least one growth factor.

[0174] Here, step S2 is performed in the amplification culture medium composition.

[0175] In embodiments of the present invention, the at least one growth factor mixture comprises at least one of the following: 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).

[0176] In an embodiment of the present invention, the method further includes step S3 of differentiating and maturing transduced and / or amplified CD34+ HSPC100 and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells 102 and / or immortalized cell lines into mature functional megakaryocytes 103.

[0177] Here, step S3 is performed in the absence of an activator but in the presence of at least one mixture of growth factors.

[0178] In this embodiment of the invention, differentiation and maturation are carried out in a differentiation and maturation culture medium composition.

[0179] Specifically, the differentiation and maturation culture medium composition differs from the amplification culture medium composition.

[0180] In an embodiment of the present invention, the method further includes step S4 of culturing the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell lines.

[0181] Here, step S4 is performed in the absence of the activator.

[0182] In this embodiment of the invention, the steps of culturing the transduced CD34+ HSPC 100 (step S2), the immortalized megakaryocytes 102 and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell line 102 (step S4) are performed in the absence of serum and / or in the absence of feeder cells.

[0183] Not explicitly shown, in this embodiment, CD34+ HSPC 100 is derived from adult peripheral blood or adult bone marrow from a single adult donor.

[0184] Also not explicitly shown, in embodiments of the invention, a single adult donor is a single human adult donor.

[0185] Figure 2 A block diagram illustrating steps S11 to S14 of a method for generating functional platelets 104 according to an embodiment of the present invention is provided.

[0186] Specifically, the method includes implementing the aforementioned method ( Figure 1 Step S11: Obtaining immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines 102.

[0187] The method further includes step S12, which involves culturing the obtained immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell line 102.

[0188] Furthermore, the method includes step S13 of differentiating immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines 102 into mature functional megakaryocytes 103.

[0189] Finally, the method includes step S14 of obtaining functional platelets 104 by mechanically manipulating mature functional megakaryocytes 103.

[0190] The method allows for the acquisition of high-quality functional platelets 104 in clinically relevant quantities, similar to those of naturally occurring platelets circulating in the body.

[0191] Figure 3 Provided Figure 1 The method (the area enclosed by the dashed line) and Figure 2 An exemplary illustration of the method (complete diagram).

[0192] In embodiments of the present invention, such as Figure 3 As shown, step S14 can be conveniently performed in a bioreactor (or microfluidic device).

[0193] The starting material is adult CD34+ HPSC 100 from peripheral blood or bone marrow of a single donor.

[0194] All blood cell lineages arise from the functional maturation of a rare population of multipotent HPSCs, which can proliferate through self-renewal and differentiation.

[0195] HSPCs can be found in several organs, such as peripheral blood, bone marrow, and umbilical cord blood.

[0196] The first lentivirus LV is used to transduce cells using the first expression cassette.

[0197] The second lentivirus LV is used to transduce cells using a second expression cassette.

[0198] The first expression box contains a constitutive promoter (EF1a).

[0199] The second expression cassette contains a tetracycline-dependent promoter (TRE).

[0200] In principle, lentivirus LVs can contain more than one expression cassette or an expression cassette with constitutive and inducible promoters.

[0201] Transduction using lentivirus LV allows expression cassettes to be integrated into the genome of transduced cells, thereby avoiding loss of gene expression.

[0202] The cell culture conditions serve two purposes: to expand / enable transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or megakaryocyte cell line 102, and to differentiate them into mature megakaryocytes 103.

[0203] This needs to be carried out under chemically defined conditions to improve process standardization and prevent damage to the final product.

[0204] Not explicitly shown, in this embodiment, a specific gene combination for immortalizing CD34+ HSPC 100 was used, namely HOXB8 along with MEIS1, BCL2L1 and rtTA.

[0205] HOXB8 protein, along with MEIS1 protein, are sequence-specific transcription factors that have the ability to block bone marrow differentiation and allow progenitor cells to divide indefinitely, while BCL2L1 protein is a key anti-apoptotic protein and improves platelet quality.

[0206] In this experimental setup, rtTA and BCL2L1 were constitutively expressed under the control of the EF1a promoter, while HOXB8 and MEIS1 were expressed under the control of the TER promoter, which depended on the cotransduction of the inverse tetracycline-controlled trans-activator (rtTA) (LV-EF1a-rtTA) and the presence of the activator to be expressed, doxycycline.

[0207] The present invention further provides functional platelets 104, specifically functional platelets 104 produced by implementing the above method. Figure 3 ).

[0208] The present invention further provides a pharmaceutical composition, specifically a pharmaceutical composition comprising functional platelet 104 obtained by the above method. Figure 3 ).

[0209] The pharmaceutical composition is suitable for the treatment of diseases, preferably for targeted and specific treatment of diseases.

[0210] As a non-limiting example, the disease could be thrombocytopenia.

[0211] As another example, the disease could be a hemorrhagic condition.

[0212] Alternatively, the pharmaceutical composition can be used in regenerative medicine.

[0213] Experimental data The inventors have conducted laboratory activities, including implementing the above-described method of the present invention. Figures 1 to 3 ).

[0214] Now refer to Figures 4 to 9 The experimental evidence obtained through discussion.

[0215] Figure 4 The plasmid maps used in the described experiment are provided.

[0216] Here, the MEIS1 and HOXB8 coding sequences are placed next to the tetracycline response element (TRE) promoter. The rtTA and BCL2L1 coding sequences are placed next to the EF1A promoter.

[0217] In the presence of an activator, the TRE promoter is activated by rtTA.

[0218] In the experiment, doxycycline was used as an activator.

[0219] Upon activation of the TRE promoter, the expression of HOXB8 and MEIS was increased in transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or megakaryocyte cell lines.

[0220] Without an activator, rtTA is inactive as a TRE promoter.

[0221] After the TRE promoter was inactivated, the expression of HOXB8 and MEIS1 decreased.

[0222] The EF1a promoter allows constitutive expression of genes.

[0223] BCL2L1 and rtTA are persistently expressed in transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or megakaryocyte cell lines and / or mature megakaryocytes.

[0224] Figure 5 A list of cloning primers suitable for experimental contexts is provided (see also...) Figure 6 H).

[0225] Specifically, Figure 5 A list of primers and restriction enzymes that can be used to clone constructs is provided.

[0226] Figure 6 A shows experimental evidence regarding the transduction of adult CD34+ HSPCs using lentiviral (LV) vectors encoding proteins HOXB8, rtTA, and BCL2L1 or HOXB8, MEIS1, rtTA, and BCL2L1 (see also...). Figure 3 The part defined by the dashed line and Figure 4 (Plasmid map).

[0227] After transducing CD34+ HSPCs and amplifying them in the presence of an activator (doxycycline) for several weeks (more than 100 days in this case), the surface expression of CD41 and CD42b was measured by flow cytometry on different days during the culture period.

[0228] During culture, the corresponding untransduced cells (control cells, cultured under the same conditions) did not survive (data not shown), indicating that only the cells transduced with the gene were immortalized. Furthermore, the vector containing rtTA also contains a puromycin resistance cassette, allowing selection of rtTA-expressing cells by treatment with puromycin (0.25 µg / mL, for three days).

[0229] Figure 6 Evidence shown in B demonstrates that, in the presence of the activator, increased MEIS1 expression in transduced adult CD34+ HSPCs resulted in a greater number of megakaryocyte progenitor cells (CD45RA-CD9+CD49f+). The transduced adult CD34+ HSPCs remained amplified for over a month in the presence of the activator. The expression of surface markers CD45RA, CD9, and CD49f was measured by flow cytometry.

[0230] Figure 6 C demonstrates CD34+ HSPCs transduced with HOXB8, rtTA, and BCL2L1 or HOXB8, MEIS1, rtTA, and BCL2L1, which maintained expansion for over a month in the presence of doxycycline (DOX on) and then differentiated into megakaryocytes for one week in the absence of doxycycline (DOX off). CD41 and CD42b surface expression were measured by flow cytometry. It should be noted that cells forcibly expressed MEIS1 during the expansion phase (DOX on) were more CD41+CD42b+ double-positive during the differentiation phase (DOX off) compared to cells not transduced with an LV vector expressing MEIS1.

[0231] Figure 6 DG demonstrates adult CD34+ HSPCs amplified in the presence of doxycycline (DOX on) or cultured for one week in the absence of doxycycline (DOX off), transduced with a lentiviral vector encoding the proteins BCL2L1, HOXB8, MEIS1, and rtTA.

[0232] Figure 6 D shows evidence confirming reduced expression of doxycycline-induced genes MEIS1 and HOXB8 one week after doxycycline removal (DOX off) compared to transduced CD34+ HSPCs maintained at amplification with doxycycline (DOX on). MEIS1 and HOXB8 are also expressed in non-transduced cells, so complete suppression is not expected.

[0233] Figure 6Evidence presented in E demonstrates that, compared to transduced adult CD34+ HSPCs that maintained expansion in the presence of doxycycline (DOX on), the CD41+CD42b+ population increased one week after doxycycline removal (DOX off). Cells expanded for more than a month in the presence of doxycycline and were then induced to differentiate by doxycycline removal.

[0234] Figure 6 F shows evidence confirming surface expression of GPVI in transduced adult CD34+ HSPCs one week after doxycycline removal (DOX off) and in transduced adult CD34+ HSPCs that maintain amplification in the presence of doxycycline (DOX on).

[0235] Figure 6 G showed evidence that the amplified transduced adult CD34+ HSPC remained small and mononuclear in the presence of doxycycline (DOX on), while one week after the removal of doxycycline (DOX off), the transduced adult CD34+ HSPC could be polyploid and larger than when DOX was on.

[0236] In short, Figure 6 AG demonstrated that co-expression of HOXB8, MEIS1, BCL2L1, and rtTA in transduced adult CD34+ HSPCs in the presence of doxycycline allowed for long-term culture. Figure 6 AB), while maintaining the differentiation potential of megakaryocytes through cell proliferation ( Figure 6 CG).

[0237] Figure 6 HI provides evidence that the combination of HOXB8, MEIS1, BCL2L1, and rtTA in transduced adult CD34+ HSPCs provides the optimal combination for generating immortalized megakaryocytes and / or immortalized megakaryocyte progenitors.

[0238] Figure 6 H shows the specific use of doxycycline (DOX-on) in the presence of doxycycline. Figure 7The combination shown in the paper represents the amplification of transduced adult CD34+ HSPCs. Following transduction, CD34+ HSPCs were amplified over several weeks (over 100 days in this case) in the presence of the activator doxycycline. CD41 and CD42b surface expression was measured by flow cytometry at different time points during culture. During culture, the corresponding untransduced cells (control cells, cultured under the same conditions) did not survive (data not shown), indicating that only the cells transduced with the gene were immortalized. Furthermore, the vector containing rtTA also contains a puromycin resistance cassette, allowing selection of rtTA-expressing cells by treatment with puromycin (0.25 µg / mL, three days).

[0239] Figure 6 I illustrates exemplary conditions of expansion maintained for up to 100 days in the presence of doxycycline, followed by culture in the absence of doxycycline to allow cells to mature for 7 days. As observed in flow cytometry analysis, the different conditions showed only relatively low amounts of CD41+CD42b+ cells after doxycycline removal (DOX off) compared to the expansion phase with doxycycline present (DOX on).

[0240] Figure 7 The different gene combinations tested were summarized, such as Figure 5 As demonstrated in H, the combination HOXB8-MEIS1-BCL2L1-rtTA is optimally suited for generating mature megakaryocytes from transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte cell lines.

[0241] Figure 8 This study demonstrates the generation of functional platelets using transduced adult CD34+ HSPCs and / or immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or immortalized megakaryocyte cell lines that differentiated into mature megakaryocytes one week after doxycycline removal.

[0242] Figure 8 A shows the platelet (CD42b+calcein AM+) yield in a modified bioreactor at different time points (up to 120 minutes). The platelet yield per megakaryocyte (cell CD42b+calcein AM+) was calculated as follows: (number of platelets at T(t) - number of platelets at T(0)) / megakaryocyte T(0).

[0243] Figure 8 B shows the yield at each time point.

[0244] Figure 8 The evidence provided in the CD confirms this. Figure 8The data presented in AB specifically use CD41 and CD42b as platelet surface markers.

[0245] Figure 8 EH respectively show the use of convulxin ( Figure 8 F), thrombin ( Figure 8 G) or a mixture of thrombin and convulxin ( Figure 8 H), activation of platelets produced by adult CD34+ HSPCs transduced with HOXB8-MEIS1-BCL2L1-rtTA.

[0246] Figure 9 A demonstrates how universal platelets can be generated by reducing B2M expression in adult CD34+ HSPCs transduced with HOXB8-MEIS1-BCL2L1-rtTA using lentiviral (LV) particles. Figure 9 Evidence for A).

[0247] Figure 9 The evidence presented in B demonstrates that, compared to transduced adult CD34+ HSPCs with HOXB8-MEIS1-BCL2L1-rtTA that do not express siRNA, siRNA use reduced B2M in 50.28% of transduced adult CD34+ HSPCs with HOXB8-MEIS1-BCL2L1-rtTA.

[0248] Experimental setup Materials and methods Human embryonic kidney (HEK) 293T cell culture HEK 293T cells were cultured at 37°C in a humidified incubator containing 5% CO2 and maintained in Duchenne Modified Eagle's Medium (DMEM) (Gibco, 41966-029) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. For lentivirus production, cells were cultured at 2 * 10⁻⁶ cells / year. 6 Cells were plated at a density of 10 cm in pedigree dishes pretreated with 0.002% poly-L-lysine solution (Sigma-Aldrich, P407) at room temperature for 30 minutes.

[0249] Lentiviral vector cloning Lentiviral clones expressing rtTA, HOXB8, and E6 / E7 were purchased from VectorBuilder (pLV[Exp]-EF1A>rtTA(ns):T2A:Puro (VB220725-1177cms), pLV[Exp]TRE>HPV16E6(ns):T2A:HPV16E7(ns):P2A:EGFP (VB2207271102pym), pLV[Exp]TRE>hHOXB8[NM_024016.4](ns):T2A:EGFP (VB2207271200nbb).

[0250] For subsequent construction, pLV[Exp]-EF1A>rtTA(ns):T2A:Puro and pLV[Exp]TRE>hHOXB8[NM_024016.4](ns):T2A:EGFP were modified to obtain pLV[Exp]-EF1A>MCS (multiple cloning site) and pLV[Exp]-TRE>MCS, respectively. Using data from... Figure 5 Primers amplified BCL2L1, ERG, FLI1, MEIS1, and CDK4. R24C -CCND1, hTERT, MLL-AF9, JAK2 V617F YAP S127A The open reading frame. The PCR product was purified by gel electrophoresis before digestion with the specified restriction enzyme. Figure 5 The digested fragments are then cloned into a lentiviral backbone containing either extension factor-1a (EF1a) or a tetracycline response element (TRE). Figure 5 Ligation is performed using T3 DNA ligase (NEB). Vectors containing rtTA contain puromycin and antibiotic resistance genes to facilitate selection of transduced mammalian cells.

[0251] Lentiviral generation and concentration Third-generation self-inactivating glycoprotein-pseudovirus particles of vesicular stomatitis virus (VSV-G) were generated via transient co-transfection with packaging plasmids pMD.G (VSV-G), pMDLg / p.RRE (gag and pol), and pRSV-Rev (Rev gene) and a lentiviral transfer vector. Viral supernatant was collected 48 hours later. Viral particles were concentrated using either PEG8000 or a sucrose gradient. Viral titers were determined by infecting HEK293T cells, and Psi copy number integration was quantified by quantitative polymerase chain reaction (PCR).

[0252] Gene integration quantitative PCR Genomic DNA (gDNA) was isolated using QuickExtract (Biosearchtechnologies) as recommended by the manufacturer. Fluorescence detection was performed using a Mic real-time PCR system with a total reaction solution of 10.5 µL containing PrimeTime gene expression master mixture, primers and probes for Psi (236481330, IDT), primers and probes for albumin (236892403, IDT), and 10 ng gDNA.

[0253] The carrier copy number (VCN) is calculated as follows: VCN (per cell) = (nb molecules Psi / average number of albumin genes) × 2.

[0254] Then extract the lentivirus titer as follows: Titer (IP / mL) = (nb of cells on the day of transduction × VCN) / viral volume in mL.

[0255] CD34 + Artificial blood cell isolation and transduction CD34 + Human hematopoietic stem cells and progenitor cells (HSPCs) were isolated from granulocyte colony-stimulating factor (GCSF)-mobilized leukocytes (Jackson) via immunomagnetic sorting (Miltenyi Biotec) according to the manufacturer's protocol. Briefly, the cells were diluted 1:1 in D-PBS. 30 mL of the diluted cells were carefully added to 10 mL of Ficoll and centrifuged at 300 g (accelerator 4, interrupt 0) for 30 minutes at room temperature. The leukocytes were then collected and washed twice in D-PBS. The cells were then sorted at 30 x 10⁻⁶ cm⁻¹. 8 Cells were resuspended in buffer (PBS 0.5% BSA 2 mM EDTA) at a concentration of / mL. Cells were then incubated with Fc Block and CD34 beads at 4°C for 30 min and purified using an LS column (Miltenyi). Purification was confirmed by flow cytometry using CD34 PerCPCy5.5 (Biolegend). The purified CD34 was then... + Cells were frozen in a freezing solution (40% IMDM, 50% FBS, 10% DMSO) and stored in liquid nitrogen.

[0256] Thaw cells 24 hours before transduction and use 0.3 * 10⁻⁶ cells per cell line. 6The solutions were resuspended at a concentration of 1 / mL in 4 Cell® Nutri-TGMP medium (Sartorius), 10 nM thrombopoietin receptor agonist peptide (TPOR-AP, Sigma-Aldrich, IEGPTLRQWLAARA-βAla)2-K-NH2), 5.37 U / mL SCF (Miltenyi Biotec), 4 U / mL IL-3 (Miltenyi Biotec), and 1% penicillin-streptomycin (Sigma-Aldrich), and cultured in 24-well plates (Sarstedt) in a humidified incubator at 37°C with 5% CO2.

[0257] For transduction, cells were centrifuged at 200 g for 5 minutes and placed in complete medium supplemented with lentiviral particles containing 100 µg / mL poloxamer P470, 4 µg / mL protamine sulfate, 10 µM prostaglandin E2, 8 µM cyclosporine H, and MOI 10 (see [link to original text]). Figure 7 ) with 1 * 10 6 Cells were resuspended at a concentration of 1 cell / mL in 50 µL of a 96-well U-shaped plate or 500 µL of a 24-well plate for 24 hours. The medium was changed, and after 48 hours, doxycycline (DOX) at a concentration of 1 µg / mL was added to activate the expression of genes regulated by the TRE promoter. Untransduced cells were then killed by 0.25 ng / mL puromycin for 72 hours after 3 weeks of culture in a humidified incubator at 37°C with 5% CO2.

[0258] Before differentiation, cells were cultured in DOX-supplemented amplification medium for at least 60 days.

[0259] Cell characterization Flow cytometry The following antibodies were used for immunophenotyping: anti-human CD41 conjugated with phycocyanin (APC) (1 / 400, catalog number: 303710, Biolegend), anti-human CD42b conjugated with phycoerythrin (PE) (1 / 100, catalog number: 303906, Biolegend), anti-human CD45RA conjugated with PE (1:200, catalog number: 304108, Biolegend), anti-human CD49f conjugated with fluorescein isothiocyanate (FITC) (1:200, catalog number: 313605, Biolegend), anti-human CD9 conjugated with PE-Cyanine 7 (1:200, catalog number: 312115, Biolegend), and anti-human GPVI conjugated with APC.

[0260] In short, cells were collected and 20 µL of cell suspension was diluted in PBS with 0.1% BSA. Then, the cells were incubated with 100 µL of antibody mixture diluted in PBS with 0.1% BSA for 20 minutes at room temperature in the dark. Cells were collected using a BD fluorescence Accuri C6 PLUS flow cytometer and analyzed using FCSalyzer software.

[0261] May Grünwald Giemsa staining Centrifuge at 500 rpm for 5 minutes and coat 12-well slides with 50 µL PBS 5% BSA. Collect 1000 to 50,000 cells, centrifuge at 200 g for 5 minutes, and resuspend in 50 µL PBS 5% BSA. Then place the cell suspension in 12-well slides and centrifuge at 500 rpm for 5 minutes. Allow the slides to air dry. Perform McGraw-Grunds staining for Giemsa staining as directed by the manufacturer. Briefly, stain the cells with McGraw-Grunds stain for 5 minutes and wash for 1.5 minutes. Then stain the cells with a 1:20 diluted Giemsa stain for 15–20 minutes. Finally, wash the slides with deionized water before microscopic evaluation.

[0262] Quantitative real-time reverse transcriptase PCR Total RNA was extracted from stem cell precipitates and converted to cDNA using the Cell-to-Ct kit (Thermofisher A35374) as recommended by the manufacturer. Fluorescence detection was performed using a Mic real-time PCR system with 10 µL of total reaction solution containing primers and probes of sensiFAST noROX mixture, MEIS1, or HOXB8. Albumin primers and probes and 2 µL of cDNA template were used as gene references.

[0263] Platelet production Small-scale platelet production system As described in WO 2022167676A1, a device for preparing a small-scale platelet production facility.

[0264] Two hundred thousand cells, in 5 mL of complete culture medium supplemented with 0.02 U / mL adenosine triphosphate diphosphatase and 25 mM HEPES, were placed in a 50 mL tube fixed to an orbital mixer (IKA MS3 basic model) located in a sealed chamber containing 5% CO2. The tube was connected to the inlet and outlet of the microfluidic chip using flexible tubing (Tygon ST R-3607, Idex Health and Science, Germany) with an inner diameter of 0.51 mm. To ensure cell circulation, a peristaltic pump (IPC8, ISMATEC, Germany) was used to flow the cell suspension through the platelet production apparatus at a rate of 1 mL / min for 90 minutes. The entire setup for small-scale platelet production was sealed within the chamber, with the temperature regulated to 37°C via an air controller (The Box, Life Imaging Services, Switzerland). Finally, cell and platelet suspension samples were collected for platelet and MK counting and characterization.

[0265] Large-scale platelet production settings Before platelet production, 50 mg of cellulose microbeads were incubated overnight at 4°C with 40 µg / mL of von Willebrand factor (VWF).

[0266] It will contain 20 * 10 6 A 40 mL cell suspension of transduced cells was diluted to a final volume of 250 mL (HEPES 25 mM and 0.02 U / mL adenosine triphosphate bisphosphatase). The cells were placed in a jacketed 500 mL glass baffled container (V2 container, SpinChem, Sweden), which in turn contained a 28 mL bed reactor (RBR S2, SpinChem, Sweden) filled with VWF-coated cellulose microbeads, and connected to a rotating shaft (rotator IKA RW 20 digital). The rotation speed was set to 900 rpm for 120 minutes. A gas mixer unit (Biobrick CO2 bio-brick, Life Imaging Services, Switzerland) supplied air containing 5% CO2 to the chamber at a rate of 22 L / h. The jacket surrounding the container was connected to a temperature control unit (Colora) set at 37°C, which circulated temperature-controlled water through the jacket.

[0267] Platelet characterization The following dyes and antibodies were used for the stated purpose: Calcein AM (final concentration: 625 nM), APC-conjugated anti-human CD41 (1 / 400, catalog number: 303710, Baijin Biotechnology Co., Ltd.), PE-conjugated anti-human CD42b (1 / 100, catalog number: 303906, Baijin Biotechnology Co., Ltd.), and APC-conjugated anti-human CD42b (1 / 200, catalog number: 303912, Baijin Biotechnology Co., Ltd.).

[0268] At the beginning and end of platelet production, culture media containing cells and platelets were sampled. 20 µL of cell suspension was diluted in PBS containing 0.1% BSA. Then, the cells were incubated for 20 minutes at room temperature in the dark with 100 µL of an antibody mixture diluted in PBS with 0.1% BSA. Two mixtures were used: calcein AM and APC-CD42b or APC-CD41 and PE-CD42b. Data were collected using a BD fluorescence Accuri C6 PLUS flow cytometer and analyzed using FCSalyzer software.

[0269] Platelet activation Platelets were activated in a self-made Tyrode buffer (137 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO3, 0.43 mM NaH2PO4, 1 mM MgCl2, 5.55 mM glucose, 5 mM HEPES, 0.35% albumin and 2 mM CaCl2, pH 7.35) and incubated with 5 ng / mL to 5000 ng / mL Convulxin (CVX), 2 U / mL thrombin (FIIa) or a combination of 10 ng / mL CVX and 0.5 U / mL FIIa.

[0270] Platelets were stained with APC-conjugated anti-human CD41 or APC-conjugated anti-human CD42b, and activation was assessed by fluorescent fibrinogen binding (AF488-conjugated fibrinogen). Data were collected using a BD fluorescence Accuri C6 PLUS flow cytometer and analyzed using FCSalyzer software.

[0271] result Transgenic combinations allow for long-term culture and megakaryocyte differentiation. With an MOI of 10, adult CD34 was transduced using different gene combinations (including DOX-inducible genes (MEIS1 and / or HOXB8) and constitutively expressed genes (BCL2L1 and rtTA)). + HSPC. After gene induction with doxycycline (DOX), the cells were compared in long-term culture. Figure 6A) and their direction to CD41 + CD42b + Cellular maturation potential ( Figure 6 C). Both cell lines (HOXB8-BCL2L1 or HOXB8-MEIS1-BCL2L1) were able to maintain their cell count for more than 3 months in serum-free, feeder-free cell culture. Figure 6 A). Next, we characterize the cell phenotype. Megakaryotic progenitor cells are CD45RA. - CD41 + CD49f + CD9 + The expression of surface markers CD45RA, CD49f, and CD9 was compared by flow cytometry, and it was found that their expression was higher in HOXB8-MEIS1-BCL2L1-expressing cells than in HOXB8-BCL2L1-expressing cells. Figure 6 B). Next, the potential for megakaryocyte maturation with two different combinations was tested. At an MOI of 10, adult CD34 cells were transduced with different gene combinations (including DOX-inducible genes (MEIS1 and / or HOXB8) and constitutively expressed genes (BCL2L1 and rtTA)). + HSPCs were amplified and maintained in the presence of doxycycline for at least one month. Doxycycline was then removed for one week, and CD41 and CD42b surface expression was assessed by flow cytometry. Compared to adult CD34+ HSPCs transduced with HOXB8-BCL2L1-rtTA, adult CD34+ HSPCs transduced with HOXB8-MEIS1-BCL2L1-rtTA showed significantly higher expression levels. + CD41 in HSPC + CD42b + The proportion of cell populations is higher ( Figure 6 C). Next, it was confirmed that DOX removal induced a reduction in genes ( Figure 6 D). Transduced adult CD34 with HOXB8-MEIS1-BCL2L1-rtTA maturation potential + HSPCs were amplified with doxycycline for more than a month and then identified as megakaryocytes one week later in the absence of doxycycline. Figure 6 EG). The cells were polyploid, as confirmed by McGraw-Gymsa staining 7 days after DOX removal. Figure 6 G), and flow cytometry confirmed that the cells had increased expression of CD41 and CD42b surface markers (G). Figure 6 E). Furthermore, 5% of the cells expressed GPVI (E) on their surface. Figure 6 F).

[0272] In summary, the gene combination of BCL2L1, HOXB8, and MEIS1 has been confirmed to allow for long-term cell proliferation and has the potential to differentiate into polyploid megakaryocytes.

[0273] To confirm that this method is best suited for generating clinically relevant in vitro platelets, different methods for cell immortalization were tested using genes known for immortalizing adult hematopoietic progenitor cells or other cell types. First, genes (ERG, FLI1, JAK2) were selected based on their expression in megakaryocyte lineages. V617F Genes (MLL-AF9, E6 / E7) were selected based on their ability to immortalize other hematopoietic lineages, including GATA1s. hTERT was also selected to stabilize genetically immortalized cells, and BCL2L1 was selected as an anti-apoptotic factor. YAP was added. S127A and CDK4 R24C -CCND1 was used as the standard immortalization agent. Several CD34 reactions were performed sequentially. + Transduction, including Figure 6 Combinations confirmed in AH were used to compare different gene combinations, including DOX-inducible genes (CDK4). R24C -CCND1, hTERT, MLL-AF9, JAK2 V617F YAP S127A Genes expressed constitutively (and E6 / E7) or constitutively expressed (ERG, FLI1, GATA1s) Figure 7 ).like Figure 6 As shown in HI, these methods are effective in long-term proliferation (CDK4). R24C -CCND1 and YAP S127A ) or unsuccessful in any one or both of the following aspects of the potential to mature into a mature megakaryocyte (MLL-AF9 and E6 / E7 and hTERT). Figure 6 HI).

[0274] Megakaryocytes derived from HOXB8-MEIS1-BCL2L1 can produce functional platelets. Next, the goal was to generate functional platelets from megakaryocytes derived from HOXB8-MEIS1-BCL2L1-expressing cells. HOXB8-MEIS1-BCL2L1 cells were seeded in xenogeneic-free medium containing thrombopoietin to differentiate into MK cells and transferred to a specially modified rotating bed reactor in a 200 mL bioreactor, cultured at 900 RPM for 2 hours. The reactor consisted of vesicles containing cellulose microbeads. Over time, platelet-like particle phylogeny (PMP-P) was detected by flow cytometry. Figure 8 A) Inner Calcein AM + CD42b + The event increased. After 120 minutes, each calcein AM was found to be... + CD42b+ Cellular calcium chlorophyll AM + CD42b + MK produces 20 calcein AMs + CD42b + Platelet-like granules (PLP) Figure 8 B). Using CD41 + CD42b + Staining to confirm these data ( Figure 8 CD).

[0275] An evaluation was conducted to ensure that CD41 + CD42b + PLP refers to functional platelets that bind to fibrinogen via flow cytometry in the presence of PLT agonists such as Convulxin (CVX), thrombin, or a combination of both. Figure 8 E). In the absence of agonists, 5% to 10% of in vitro generated platelets bind to fibrinogen ( Figure 8 FH). CVX titration achieved CD41. + The dose response to fibrinogen binding in PLP is increased ( Figure 8 F). Additionally, in the presence of 2 U / mL thrombin, up to 37.8% of CD41... + PLP binds to fibrinogen ( Figure 8 G). The combination of 10 ng / mL CVX and 0.5 U / mL thrombin produced 56.2% of CD42b. + PLP fibrinogen + ( Figure 8 H). In summary, in vitro platelets produced by megakaryocytes derived from HOXB8-MEIS1-BCL2L1 were able to bind to fibrinogen upon activation, without prior activation.

[0276] Inhibition of β-2-microglobulin in immortalized megakaryocyte cell lines In order to generate universal platelets in vitro ( Figure 9 A) After amplification for more than 20 days in the presence of doxycycline, β-2-microglobulin knockdown was tested in adult CD34+ HSPCs transduced with HOXB8-MEIS1-BCL2L1-rtTA. Transduction was performed using lentiviral particles based on MOI 10 transduced with HOXB8-MEIS1-BCL2L1-rtTA. β-2-microglobulin surface expression was assessed by flow cytometry two weeks later. Reduced B2M expression was potentially confirmed in 50.28% of the population. Figure 9B). This result confirms the possibility of reducing B2M in adult CD34+ HSPC transduced with HOXB8-MEIS1-BCL2L1-rtTA.

[0277] Figure Labels 100 (CD34+) hematopoietic stem cells and progenitor cells, HSPC 102 Immortalized Megakaryocytes / Immortalized Megakaryocyte Progenitor Cells / Human Megakaryocyte Cell Line 103 mature functional megakaryocytes, MK 104 platelets LV Lentivir S1 Method Steps S2 Method Steps S3 Method Steps S4 Method Steps S11 Method Steps S12 Method Steps S13 Method Steps S14 Method Steps References Bessa, J. et al. (2008) 'meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye', Development ( Development ) (Cambridge, UK) Cambridge England )), 135(5), pp. 799-803. Available at https: / / doi.org / 10.1242 / dev.011932.

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Claims

1. A method for generating 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), wherein the adult CD34+ hematopoietic stem cells and progenitor cells are capable of maturing into functional megakaryocytes (103) that produce functional platelets (104), the method comprising at least: S1: By transduction, specifically lentiviral (LV) transduction, an expression cassette containing at least one gene is introduced into the CD34+ HSPC (100) to force expression of at least one gene. The transduced CD34+ HSPC(100) was configured to express at least one gene from the HOXL subclass of the antennal foot homeotype protein (ANTP) homeobox family and / or the triamino acid ring extension (TALE) homeobox family upon activator-induced expression, and constitutively express at least one anti-apoptotic gene from the BCL2 family. The transduced CD34+ HSPC(100) is immortalized by expressing at least one gene from the HOXL subclass of the ANTP homeobox family and / or the TALE homeobox family. Preferably, the at least one gene from the 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 the at least one anti-apoptotic gene from the BCL2 family comprises BCL2L1 or BCL2L2.

2. The method according to claim 1, characterized in that, The resulting immortalized megakaryocytes and / or immortalized megakaryocyte progenitors and / or human megakaryocyte cell lines (102) are able to differentiate and mature in the absence of the activator to provide mature functional platelets (104), specifically functional clinical-grade platelets (104).

3. The method according to claim 1, characterized in that, 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. The method according to any one of the preceding claims, characterized in that, The method further includes: S2: Culturing the transduced CD34+ HSPCs (100). The transduced CD34+ HSPC (100) is cultured by amplifying the transduced CD34+ HSPC (100) in an amplification medium composition in the presence of the activator and at least one growth factor mixture.

5. The method according to claim 4, characterized in that, The at least one growth factor mixture comprises at least one of the following: 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. The method according to any one of the preceding claims, characterized in that, The method further includes: S3: In the absence of the activator and in the presence of the at least one growth factor mixture, the transduced CD34+ HSPC and / or the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitors and / or the immortalized cell line (102) are differentiated and matured into mature functional megakaryocytes (103).

7. The method according to claim 6, characterized in that, Differentiation and maturation are carried out in a differentiation and maturation culture medium composition that is different from the amplification culture medium composition described above.

8. The method according to any one of the preceding claims, characterized in that, The method further includes: S4: Culture the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell lines in the absence of the activator (102).

9. The method according to any one of claims 4 to 8, characterized in that, The steps of culturing the transduced adult CD34+HSPC (100) and / or the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell line (102) are performed in the absence of serum and / or feeder cells.

10. The method according to any one of the preceding claims, characterized in that, The adult CD34+ HSPC (100) is derived from human donors, specifically from the peripheral blood or bone marrow of a single human donor.

11. A method for producing functional platelets (104), the method comprising: S11: Obtain immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines by performing the method according to any one of claims 1 to 10 (102). S12: Culture the obtained immortalized megakaryocytes and / or immortalized megakaryocyte progenitor cells and / or immortalized megakaryocyte cell lines (102). S13: Differentiate and mature the immortalized megakaryocytes and / or the immortalized megakaryocyte progenitor cells and / or the immortalized megakaryocyte cell line (102) into mature functional megakaryocytes (103), and S14: Functional platelets (104) are obtained by mechanically manipulating the mature functional megakaryocytes (103), preferably in a bioreactor or microfluidic device.

12. A functional platelet (104) obtained by performing the method according to claim 11.

13. A pharmaceutical composition comprising the functional platelet (104) according to claim 12.

14. Use of the pharmaceutical composition according to claim 13 for treating a disease, specifically for target-specific treatment of the disease, preferably wherein the disease is one of thrombocytopenia or a hemorrhagic condition.

15. Use of the pharmaceutical composition according to claim 13 for regenerative medicine.

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