Compositions, constructs and vectors for cell reprogramming

Abstract

The present disclosure relates to compositions, vectors, constructs, cells and methods for reprogramming cells into natural killer (NK) cells or progenitor cells. In particular, the present disclosure relates to combinations of transcription factors for the reprogramming of cells.

Description

[Technical Field]

[0001] The present invention relates to the generation of autologous natural killer (NK) cells and progenitor cells by direct reprogramming using a combination of transcription factors. Furthermore, the present disclosure relates to the development of methods for generating innate lymphoid progenitors and mature cells with cytotoxic potential from differentiated, multipotent, or pluripotent stem cells by introducing and expressing isolated / synthetic transcription factors. In particular, the present disclosure provides methods for obtaining autologous innate lymphoid cells, particularly NK cells, by direct cell reprogramming using a specific combination of transcription factors.

[0002] [Background technology]

[0003] Cellular reprogramming is the process of changing the transcriptional and epigenetic networks of one cell state to those of another cell type. Mouse and human somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) by the expression of defined transcription factors (TFs) (Takahashi et al., 2006). Furthermore, somatic cells can be directly reprogrammed to become alternative somatic cell types. By using TFs capable of specifying target cell identity, somatic cells have been directly converted to other mature cell fates, such as myocytes, neurons, and hepatocytes (Pereira et al., 2012). In the hematopoietic system, direct reprogramming has been used to generate hematopoietic progenitor cells (Pereira et al., 2013; Gomes et al., 2018) and dendritic cells (Rosa et al., 2018) from mouse and human fibroblasts. Direct reprogramming is emerging as a powerful approach for studying cell identity and commitment. Intrinsic lineage selection is stochastic and dependent on the activation of lineage-specifying TFs, which lock in cell fate through cross-antagonistic interactions with alternative lineage-specifying factors (Graf and Enver, 2009). Despite recent successes in lymphocyte-based cancer immunotherapy, no cases of lymphopoiesis by direct cell reprogramming have been reported.

[0004] NK cells represent a specific group of innate lymphoid cells (ILCs) that control viral infections and cancer. Although some NK cell production is detected in the thymus, the bone marrow is the primary site of NK cell precursor and mature NK cell production in adults (Vivier et al., 2011). NK cells are present in various tissues, including peripheral blood, spleen, and lymph nodes, as well as the liver, which is the active site for fetal hematopoiesis and NK cell production (Renoux et al., 2015). Several transcription factors are important for NK cell generation and function. However, many of these transcription factors are also involved in regulating other blood lineages. Expression of ETS1 is initiated in lymphoid progenitor cells and is involved in both early lymphoid commitment and terminal NK differentiation. Together with NFIL3, ETS1 regulates the NK cell transcriptional network and activates the expression of ID2 and EOMES. Both TBET and EOMES are essential for NK cell functional properties, such as IFN-gamma production. Although TBET is expressed by other ILCs, EOMES is restricted to the NK cell lineage and TBET + Ectopic expression of EOMES in cells induces NK cell-like characteristics. Despite the importance of these individual TFs in NK biology, the primary combination that directs NK identity in other cell types is unknown. Based on computational predictions, the TFs RORα, SMAD7, FOS, JUN, and NFATC2 have been suggested to play such a role (WO2019 / 177936). Overall, successful generation of NK cells by direct cell reprogramming has not been reported.

[0005] Unlike T cells, NK lymphocytes can directly recognize and eliminate target cells without the need for antigen specification. Instead, NK cell killing depends on the balance of signals from activating and inhibitory receptors on the NK cell surface (Noriko et al., 2020). Because they are inhibited by major histocompatibility complex (MHC) class I molecules, NK lymphocytes eliminate tumor cells that downregulate MHC-I on their surface without reacting to healthy tissue, supporting their clinical utility. Preclinical data have extensively demonstrated the antitumor activity of NK cells, providing evidence of their ability to eliminate MHC-I-negative cancer cells that are not targeted by T cells, increase immune cell infiltration (Bottcher et al., 2018; Barry et al., 2018), and, importantly, prevent metastasis by eliminating circulating tumor cells (Lopez-Soto et al., 2017). Furthermore, the cytokine secretion profile of NK cells differs from that of T lymphocytes, which produce proinflammatory cytokines associated with the development of cytokine release syndrome. Therefore, NK-based immunotherapy has a safer profile than adoptive T cell strategies (Lopez-Soto et al., 2017). Adoptive transfer of NK cells isolated from peripheral blood or differentiated from umbilical cord hematopoietic progenitor cells has been shown to be safe in clinical studies and has demonstrated efficacy against hematopoietic cancers (Bachanova et al., 2014; Passweg et al., 2004). However, obtaining large quantities of primary NK cells from peripheral blood or umbilical cord blood is difficult. The NK cell line NK92 has also shown minimal side effects in clinical trials, but the need for irradiation of these cells limits the persistence of NK92 cells after injection (Tam et al., 2003). In solid tumors, the immunosuppressive microenvironment inhibits NK cell infiltration and activation. However, several strategies have been developed to utilize NK cells to maximize their infiltration and antitumor activity in solid tumors. NK cell engineering using chimeric antigen receptors (CARs) has been developed to direct NK lymphocytes to tumor cells bearing specific antigens, enhancing the antitumor activity of NK cells.In preclinical studies, allogeneic iPSC-derived CAR NK cells demonstrated the ability to prevent tumor progression and promote long-term survival in an ovarian xenograft model (Ye Li et al., 2018). Collectively, these studies demonstrate that primary NK cells and iPSC-derived allogeneic NK cells are safe and cause minimal graft-versus-host disease. Nevertheless, the protocol for differentiating NK cells from iPSCs is complex and time-consuming, requiring the addition of multiple cytokine combinations and coculture with a feeder layer. These limitations pose challenges to the rapid production and delivery of autologous iPSC-NK cells. Although allogeneic NK cells have proven safe, they can be rejected by the recipient's immune system and have limited persistence (WO2019 / 177936). MHC-I gene editing has been proposed as an alternative approach. However, MHC-I-negative cells can be rejected by host NK cells, which require cell-intrinsic MHC-I to maintain their function ( Boudreau et al., 2016 ).

[0006] Therefore, new approaches are needed that allow the direct generation of NK cells that can be simultaneously genetically engineered and hold compelling value in cancer immunotherapy. Summary of the Invention

[0007] The present inventors have developed a novel method for generating NK cells that is rapid and reproducible. In particular, this method involves the generation of autologous NK cells that are highly persistent in vivo and suitable for use in immunotherapy. The present invention also relates to a combination of transcription factors shown to be enriched in NK cells that can be used to reprogram cells into NK cells.

[0008] In one aspect, the present invention provides at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of ETS1, TBET, NFIL3, EOMES, ID2, GATA3, ZFP105, IKZF3, ETS1, TOX, RUNX3, KLF12, ZEB2, IRF2, STAT5, IKZF1, ELF4, ZBTB16, GATA2, and ELF1.

[0009] In another aspect, the present invention relates to at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of ETS1, TBET, NFIL3 and EOMES.

[0010] In one aspect, the present invention relates to a construct or vector comprising at least one polynucleotide described herein.

[0011] In one aspect, the present invention provides a cell comprising at least one polynucleotide, and / or construct or vector described herein.

[0012] In one aspect, the present invention relates to a pharmaceutical composition comprising at least one polynucleotide, construct or vector, and / or cell described herein.

[0013] In one aspect, the present invention provides a method for reprogramming or inducing any cell into a natural killer cell or progenitor cell, the method comprising: a. transducing a cell with at least one polynucleotide, or construct or vector described herein; b. culturing and growing the transduced cells in cell culture medium; c. obtaining the reprogrammed cell.

[0014] In one aspect, the invention provides induced natural killer cells obtained by the methods described herein. [Brief explanation of the drawings]

[0015] [Figure 1] Schematic diagram of an application of directly reprogrammed NK cells. Human somatic cells, including fibroblasts, pluripotent stem cells, hematopoietic stem cells, and other cell types isolated from patients, are reprogrammed into autologous NK cells that can be applied in personalized immunotherapy. This direct reprogramming approach allows for simultaneous gene editing with transduction of NK cell-identifying TF instructors to improve NK cell activity or targeting. Induced NK cells can be expanded in vitro and used to mount cytotoxic immune responses against cancer and viral infections. [Figure 2] Figure 1 shows the identification of 19 candidate transcription factors for programming NK cells. The heat map shows the gene expression profile of the 19 selected TFs across multiple mouse tissues and cell types (GeneAtlas MOE430). The candidate TFs are highly enriched in NK cells compared to more than 75 other tissues or cell types. [Figure 3A] We demonstrate that the combined forced expression of 19 transcription factors activates an NK-specific reporter. We demonstrate the experimental strategy for screening NK-inducible TFs. NCR1-tdTomato double transgenic mouse embryonic fibroblasts (MEFS) were cotransduced with lentiviral particles encoding candidate TFs and M2rtTA and cultured in the presence of Dox for 12 days. tdTomato expression was monitored by fluorescence microscopy. [Figure 3B] We show that the combined forced expression of 19 transcription factors activates an NK-specific reporter. We show the gene expression profile of NCR1 across several mouse tissues and cells (GeneAtlas MOE430). [Figure 3C]Forced expression of 19 transcription factors combined activates an NK-specific reporter. Fluorescence micrographs of MEFS transduced with M2rtTA alone (left) or co-transduced with M2rtTA and all 19 candidate TFs (right) 6 days after Dox addition. Entire wells of a 6-well plate were acquired on an automated Zeiss CD7. The status of tdTomato-positive colonies generated with combined expression of all TFs at day 6 is highlighted at higher magnification. Scale bar is 200 μm. [Figure 3D] This shows that the combined forced expression of 19 transcription factors activates an NK-specific reporter. The number of tdTomato cells per colony is shown from day 3 to day 12. The mean + SEM of 13 colonies is shown. [Figure 4A] Figure 1 shows the increased number of tdTomato-positive colonies using a limited pool of transcription factors. MEFs were transduced with control M2rtTA or co-transduced with pools of TFs (TF pools A to D). MEFs were transduced with control M2rtTA or co-transduced with pools of TFs (TF pools A to D). Quantification of tdTomato-positive colonies per TF combination at day 6 (left) and day 12 (right). Means + SD of two independent experiments are shown. *p<0.05. [Figure 4B] Figure 1. Fluorescence micrographs of MEFs transduced with M2rtTA (left) or cotransduced with M2rtTA and ETS1, NFIL3, ID2, TBET, and EOMES (right) at days 6 (top) and 12 (bottom) after DOX addition, showing the increased number of tdTomato-positive colonies using a limited pool of transcription factors. [Figure 5A] Fluorescence micrographs of tdTomato-positive colonies after transduction with ETS1, NFIL3, ID2, TBET, and EOMES on days 6 (top) and 12 (bottom) after DOX addition show that tdTomato-positive colonies expand in culture. [Figure 5B]tdTomato-positive colonies expand in culture. Number of tdTomato cells per colony from days 3 to 12. Mean + SEM of 10 colonies is shown. [Figure 5C] Flow cytometry plot showing expansion of tdTomato-positive colonies in culture. [Figure 5D] tdTomato-positive colonies expand in culture. Quantification of tdTomato-positive cells 12 days after DOX addition. Mean ± standard deviation of two independent experiments. [Figure 6] Figure 1 shows the improved reprogramming efficiency achieved by the combination of ETS1, NFIL3, TBET, and EOMES. MEFs were transduced with control M2rtTA, co-transduced with ETS1, NFIL3, ID2, TBET, and EOMES, or with additional combinations in which one transcription factor was individually removed from this five-TF pool. (A) Number of tdTomato-positive colonies quantified by immunofluorescence and (B) percentage of tdTomato-positive cells quantified by flow cytometry on day 12 of reprogramming (mean ± standard deviation, n = 2). [Figure 7] Figure 1 shows that ETS1, NFIL3, TBET, and EOMES are necessary and sufficient to efficiently activate the NCR1 reporter. MEFs were transduced with control M2 rtTA, cotransduced with ETS1, NFIL3, TBET, and EOMES, or with an additional combination in which one transcription factor was individually removed from the four TF pools. (A) Number of tdTomato-positive colonies quantified by immunofluorescence and (B) percentage of tdTomato-positive cells quantified by flow cytometry at day 12 of reprogramming (mean ± standard deviation, n = 2 cases). [Figure 8]Figure 1. Combined expression of ETS1, NFIL3, TBET, and EOMES is enriched in NK cells. Gene expression enrichment scores for the combination of ETS1, NFIL3, TBET, and EOMES in 96 mouse tissues and cell types. Gene expression data (GeneAtlas MOE430) were log-transformed and normalized to a range of 0 to 1 for each gene, after which the highest mean expression of ETS1+NFIL3+TBET+EOMES was determined. [Figure 9A] Figure 1 shows that the combination of RORα, SMAD7, FOS, JUN, and NFATC2 does not activate the NCR1-tdTomato reporter. The transcription factor combination of the present invention (ETS1, NFIL3, TBET, and EOMES) was compared with the combination disclosed in Vivier et al., 2011 (RORa, SMAD7, FOS, and JUN). The ability of the combination to activate the NCR1-tdTomato reporter was analyzed. The number of tdTomato-positive colonies after transduction with the combination of RORα, SMAD7, FOS, JUN, and NFATC2, or the combination of ETS1, NFIL3, TBET, and EOMES, was measured at days 6 (left) and 12 (right) of reprogramming. M2rtTA-transduced MEFs were included as a control. [Figure 9B] This shows that the combination of RORα, SMAD7, FOS, JUN, and NFATC2 does not activate the NCR1-tdTomato reporter. The transcription factor combination of the present invention (ETS1, NFIL3, TBET, and EOMES) was compared with the combination disclosed in Vivier et al., 2011 (RORα, SMAD7, FOS, and JUN). The ability of the combination to activate the NCR1-tdTomato reporter was analyzed. Gene expression enrichment scores for the combination of RORα, SMAD7, FOS, JUN, and NFATC2 in 96 mouse tissues and cell types. Gene expression data (GeneAtlas MOE430) were log-transformed and normalized to a range of 0 to 1 for each gene, and the highest mean expression of RORα, SMAD7, FOS, JUN, and NFATC2 was determined. [Figure 10]The combined expression of ETS1, NFIL3, TBET, and EOMES induces global gene expression changes. NCR1-tdT+ MEFS were FACS-sorted on days 3, 6, and 12 of reprogramming and profiled by single-cell RNA sequencing (RNA-seq) using 10x Genomics. Untransduced MEFs were included as controls. t-SNE visualization of MEFS on days 3, 6, and 12, and of reprogrammed cells (left). Expression of the mouse NK-specific marker Itga2 (right). Arrows indicate the reprogramming trajectory. [Figure 11] Fibroblast-associated genes are downregulated during NK reprogramming. t-SNE plots showing gene expression heatmaps for the fibroblast-associated genes Col1a2, Lox, and Fbnl2 in MEFS and reprogrammed cells at days 3, 6, and 12. [Figure 12] Figure 1 shows that the natural killer transcriptional network is induced by ETS1, NFIL3, TBET, and EOMES transcription factors. Violin plot quantification of the endogenous expression of 19 candidate transcription factors (TFs) (left) and the endogenous expression of Ets1, Nfil3, Tbx21 (encoding Tbet), and EOMES (right) during MEFS and the reprogramming process. [Figure 13A] Figure 1 shows that the immature NK cell program is specified by forced expression of ETS1, NFIL3, TBET, and EOMES. Figure 2 shows t-SNE plots of single-cell transcriptomes showing the immature NK gene signature (left) and mature NK gene signature (right) on NCR1-tdT+ MEFs at 3, 6, and 12 days after transduction with the four transcription factors (Bezman et al., 2012). [Figure 13B] Figure 1 shows that the immature NK cell program is specified by forced expression of ETS1, NFIL3, TBET, and EOMES. Boxplots showing the expression distribution of immature and mature NK gene signatures in single cells during NK reprogramming. [Figure 13C] Figure 1 shows that the immature NK cell program is specified by forced expression of ETS1, NFIL3, TBET, and EOMES. t-SNE gene expression heatmap of NK progenitor genes Cd38, Cd34, and Mme (encoding CD10) during reprogramming. [Figure 14] Figure 1 shows that ETS1, NFIL3, TBET and EOMES induce expression of NK chemokine and cytokine receptors. Boxplot quantification of ccl5, Ifih1 and Il15ra in MEFS at days 3, 6 and 12 of reprogramming. [Figure 15] Schematic diagram of the protocol for inducing NK-like cells from human cells. Human embryonic fibroblasts (HEF) were co-transduced with lentiviral particles encoding M2rtTA (UbC-M2rtTA) and Ets1 (tetO-Ets1), Nfil3 (tetO-Nfil3), and Tbet (tetO-Tbet) and Eomes (tetO-Eomes). Alternatively, constitutive lentiviral-driven expression using the SFFV promoter (SFFV-Ets1, SFFV-Nfil3, SFFV-Tbet, and SFFV-Eomes) was tested. After overnight incubation with lentiviral particles, the medium was replaced with fresh growth medium. For FUW-TetO-transduced cells, the growth medium was supplemented with doxycycline (Dox). A cytokine cocktail containing stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (FLT3L), interleukin-3 (IL-3), IL-7, and IL-15 was added to the cultures at the times indicated. NK reprogramming was assessed by flow cytometry 6 and 12 days after transduction. [Figure 16A]Forced expression of ETS1, NFIL3, TBET, and EOMES in human fibroblasts generates CD34+ cells. Representative flow cytometry plots of human embryonic fibroblasts (HEF) 6 and 12 days after transduction with four transcription factors individually cloned into inducible (FUW-TetO) or constitutive (SFFV) lentiviral vectors. FUW-M2rtTA and empty vector (SFFV-MCS) were used as controls. [Figure 16B] Figure 1 shows that forced expression of ETS1, NFIL3, TBET, and EOMES in human fibroblasts generates CD34+ cells. Quantification of CD34+ cells induced with four TFs in two lentiviral expression systems on days 6 and 12 (mean + standard deviation, n=2). [Figure 17A] Cytokine signaling and forced expression of transcription factors improve reprogramming efficiency. Representative flow cytometry plots of human embryonic fibroblasts (HEF) 6 and 12 days after transduction with four TFs, with or without the addition of cytokines SCF, FLT3L, IL-3, IL-7, and IL-15. SFFV-MCS was used as a control. [Figure 17B] Cytokine signaling and forced expression of transcription factors enhance reprogramming efficiency. Quantification of CD34+ cells induced with four TFs in the presence or absence of a cytokine cocktail (mean + standard deviation, n=2). [Figure 18A] Figure 1 shows that combined expression of ETS1, NFIL3, TBET, and EOMES induces human CD56-positive cells. Representative flow cytometry plots of human embryonic fibroblasts (HEF) 6 days after transduction with the four TFs. Empty vector (SFFV-MCS) was used as a control. [Figure 18B] Figure 1 shows that combined expression of ETS1, NFIL3, TBET, and EOMES induces human CD56-positive cells. Quantification of CD56+ cells induced with the four TFs (mean + standard deviation, n=2). DETAILED DESCRIPTION OF THE INVENTION

[0016] The inventors of the present disclosure have identified a number of transcription factors that can induce natural killer (NK) cell fate. The inventors have also developed a new method for reprogramming cells into NK cells using at least three transcription factors. NK cells represent a specific group of innate lymphoid cells (ILCs) that control viral infections and cancer.

[0017] In one aspect, the present invention provides at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of ETS1, TBET, NFIL3, EOMES, ID2, GATA3, ZFP105, IKZF3, ETS1, TOX, RUNX3, KLF12, ZEB2, IRF2, STAT5, IKZF1, ELF4, ZBTB16, GATA2, and ELF1.

[0018] As used herein, "polynucleotide" refers to a nucleic acid molecule, including genomic DNA, cDNA, RNA, mRNA, mixed polymers, recombinant nucleic acids, and fragments and variants thereof.

[0019] As used herein, the term "transcription factor" refers to a DNA-binding protein that regulates transcription from DNA to RNA, such as by activating or repressing transcription. Some transcription factors affect transcription regulation alone, while others act in concert with other proteins. Some transcription factors can activate or repress transcription under certain conditions. Generally, transcription factors bind specific target sequences or sequences that are highly similar to specific consensus sequences in the regulatory regions of target genes. Transcription factors can regulate the transcription of target genes alone or in complexes with other molecules.

[0020] In another aspect, the present invention relates to at least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of ETS1, TBET, NFIL3 and EOMES.

[0021] In one embodiment of the present invention, the combination of at least three transcription factors is a combination of four transcription factors ETS1, TBET, NFIL3 and EOMES.

[0022] In one embodiment of the invention, the combination further comprises one or more selected from the group consisting of ID2, GATA3, ZFP105, IKZF3, TOX, RUNX3, KLF12, ZEB2, IRF2, STAT5, IKZF1, ELF4, ZBTB16, GATA2 and ELF1.

[0023] In one embodiment, the combination of at least three transcription factors of at least one polynucleotide according to any one of the preceding claims comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6. In another embodiment, the combination of at least three transcription factors of at least one polynucleotide according to any one of the preceding claims is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 27, or by a sequence having at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity to said polynucleotide sequences of SEQ ID NO: 22 to SEQ ID NO: 27.

[0024] In one embodiment, the transcription factor ETS1 of at least one polynucleotide of any one of the preceding paragraphs comprises the amino acid sequence of SEQ ID NO: 1 or a biologically active variant of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the transcription factor TBET comprises the amino acid sequence of SEQ ID NO: 2 or a biologically active variant of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3. In one embodiment, the transcription factor EOMES comprises the amino acid sequence of SEQ ID NO: 4 or a biologically active variant of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the biologically active variant of EOMES comprises a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 6.

[0025] As used herein, a "biologically active variant" includes any derivative or variant of a molecule that has substantially the same functional and / or biological properties, such as binding characteristics, and / or the same structural basis, such as a peptide backbone or basic polymer unit, as the molecule. It also refers to a molecule that exhibits functional characteristics as a transcription factor as disclosed herein in a test, such as the test disclosed herein, performed by a person skilled in the art. This test may identify a transcription factor that can activate the NCR1-tdTomato reporter.

[0026] In one embodiment, the polynucleotide encoding SEQ ID NO:1 is SEQ ID NO:22, or a sequence having at least 80%, such as at least 85%, for example at least 90%, for example at least 95% sequence identity to the sequence of SEQ ID NO:22.

[0027] In one embodiment, the polynucleotide encoding SEQ ID NO:2 is SEQ ID NO:23, or a sequence having at least 80%, such as at least 85%, for example at least 90%, for example at least 95% sequence identity to the sequence of SEQ ID NO:23.

[0028] In one embodiment, the polynucleotide encoding SEQ ID NO:3 is SEQ ID NO:24, or a sequence having at least 80%, such as at least 85%, for example at least 90%, for example at least 95% sequence identity to the sequence of SEQ ID NO:24.

[0029] In one embodiment, the polynucleotide encoding SEQ ID NO:4 to SEQ ID NO:6 is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or a sequence having at least 80%, such as at least 85%, for example at least 90%, for example at least 95% sequence identity to the sequence of the SEQ ID NO:

[0030] In another embodiment, the combination of at least three transcription factors of at least one polynucleotide described herein is ETS1, TBET, NFIL3, and EOMES.

[0031] In another embodiment, the combination of at least three transcription factors of at least one polynucleotide described herein is ETS1, TBET, and NFIL3.

[0032] In another embodiment, the combination of at least three transcription factors of at least one polynucleotide described herein is ETS1, TBET, and EOMES.

[0033] In another embodiment, the combination of at least three transcription factors of at least one polynucleotide described herein is ETS1, NFIL3, and EOMES.

[0034] In another embodiment, the combination of at least three transcription factors of at least one polynucleotide described herein is TBET, NFIL3, and EOMES.

[0035] In one aspect, the present invention relates to a construct or vector comprising at least one polynucleotide described herein.

[0036] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, examples include: a reference to a "vector" includes two or more vectors as well as a single vector; a reference to a "polynucleotide" includes two or more polynucleotides as well as one polynucleotide.

[0037] As used herein, "vector" refers to a nucleic acid sequence that includes a replication origin.Vector can be vector, bacteriophage, bacteria, artificial chromosome or yeast artificial chromosome.Vector can be DNA vector or RNA vector.Vector can be self-replicating extrachromosomal vector or DNA plasmid.

[0038] In one embodiment, the expression vector is a viral vector, hi one embodiment, the viral vector is selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, a herpesvirus vector, a poxvirus vector, a flavivirus vector, a rhabdovirus vector, a paramyxovirus vector, an adeno-associated virus vector, a reovirus vector, a papillomavirus vector, a picornavirus vector, a calicivirus vector, a hepadnavirus vector, a togavirus vector, a coronavirus vector, a hepatitis virus vector, an orthomyxovirus vector, a bunyavirus vector, and a filovirus vector.

[0039] As described herein, a "lentiviral vector" or a "member of the lentivirus family" is a viral particle that contains a viral genome derived from a lentivirus, lacks the ability to self-renew, and has the ability to introduce nucleic acid molecules into a host. Specifically, these vectors have a lentiviral backbone. The phrase "having a lentiviral backbone" means that the nucleic acid molecules contained in the viral particles that constitute the vector are based on the lentiviral genome.

[0040] In one embodiment, the vector is an oncolytic vector. An oncolytic virus is generally defined herein to refer to a virus that kills tumor or cancer cells more frequently than it kills normal cells.

[0041] In one embodiment, the construct or vector is a synthetic mRNA, a naked alphavirus RNA replicon, or a naked flavivirus RNA replicon.

[0042] In one embodiment, the construct or vector is a plasmid. The term "plasmid" is used interchangeably herein with the term "plasmid DNA" and encompasses various forms of plasmids, i.e., nicked plasmid DNA and open circular (oc) plasmid DNA, also known as supercoiled (ccc) plasmid DNA.

[0043] In one embodiment, the vector further comprises a post-translational regulatory element (PRE) sequence. In one embodiment, the PRE sequence is selected from the group consisting of a woodchuck PRE (WPRE) or a hepatitis B virus (HBV) PRE (HPRE).

[0044] In one embodiment, the vector may further comprise other nucleic acid elements for regulation, expression, or stabilization of the construct or other vector genetic elements, such as promoters, enhancers, TATA boxes, ribosome binding sites, IRES, as known to those skilled in the art.

[0045] In one embodiment, the vector may further comprise an internal ribosome entry site (IRES). The IRES may function as the only ribosome binding site or as one of multiple ribosome binding sites of an mRNA. An mRNA containing two or more functional ribosome binding sites may encode several peptides or polypeptides, such as the NK inducers described herein, that are independently translated by ribosomes ("multicistronic mRNA"). When an IRES is provided in the nucleic acid, a second translatable region may also optionally be provided. Examples of IRES sequences that may be used in accordance with the present disclosure include, but are not limited to, sequences derived from picornaviruses (e.g., FMDV), vermin viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C virus (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SW), or cricket paralysis viruses (CrPV).

[0046] In one embodiment, the vector further comprises a promoter sequence controlling transcription of at least one polynucleotide described herein. In one embodiment, the promoter sequence is selected from the group consisting of a spleen focus-forming virus (SFFV) promoter, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing the myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1α promoter, or a murine stem cell virus (MSCV) promoter.

[0047] In one embodiment, the viral vector contains a tetracycline response element (TRE) and a minimal CMV promoter controlling expression of the TF coding region (pFUW-TetO). This vector may be used in combination with a vector containing the reverse tetracycline transactivator (rtTA) under the control of the constitutively active human ubiquitin C promoter (FUW-M2rtTA).

[0048] In one embodiment, the vector contains a tetracycline response element (TRE) that controls expression of the TF coding region in the same construct as the rtTA transactivator under the control of a constitutively active PGK promoter.

[0049] In one embodiment, the construct or vector is a CRISPR / CAS activation system.Those skilled in the art will understand that this system can activate endogenous transcription factors, allowing the use of plasmids that do not contain transcription factors.

[0050] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, TBET, NFIL3, EOMES.

[0051] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, ETS1, NFIL3, EOMES.

[0052] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, ETS1, TBET, EOMES.

[0053] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, NFIL3, TBET, EOMES.

[0054] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, NFIL3, ETS1, EOMES.

[0055] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, TBET, ETS1, EOMES.

[0056] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, TBET, EOMES, ETS1.

[0057] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, NFIL3, EOMES, ETS1.

[0058] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, NFIL3, TBET, ETS1.

[0059] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, EOMES, TBET, ETS1.

[0060] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, EOMES, NFIL3, ETS1.

[0061] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, TBET, NFIL3, ETS1.

[0062] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, ETS1, NFIL3, TBET.

[0063] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, EOMES, NFIL3, TBET.

[0064] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, EOMES, ETS1, TBET.

[0065] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, NFIL3, ETS1, TBET.

[0066] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, NFIL3, EOMES, TBET.

[0067] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, ETS1, EOMES, TBET.

[0068] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, ETS1, EOMES, NFIL3.

[0069] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, TBET, EOMES, NFIL3.

[0070] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, TBET, ETS1, NFIL3.

[0071] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, EOMES, ETS1, NFIL3.

[0072] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, EOMES, TBET, NFIL3.

[0073] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, ETS1, TBET, NFIL3.

[0074] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, TBET, and NFIL3.

[0075] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, ETS1, NFIL3.

[0076] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, ETS1, TBET.

[0077] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, NFIL3, TBET.

[0078] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, NFIL3, ETS1.

[0079] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, TBET, ETS1.

[0080] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, TBET, EOMES.

[0081] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, ETS1, EOMES.

[0082] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, ETS1, TBET.

[0083] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, EOMES, TBET.

[0084] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, EOMES, ETS1.

[0085] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, TBET, ETS1.

[0086] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, NFIL3, EOMES.

[0087] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, ETS1, EOMES.

[0088] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, ETS1, NFIL3.

[0089] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': ETS1, EOMES, NFIL3.

[0090] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, EOMES, ETS1.

[0091] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, NFIL3, ETS1.

[0092] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, NFIL3, EOMES.

[0093] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, TBET, EOMES.

[0094] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, TBET, NFIL3.

[0095] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': TBET, EOMES, NFIL3.

[0096] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': NFIL3, EOMES, TBET.

[0097] In one embodiment, the at least three transcription factors of the at least one polynucleotide are in the following order from 5' to 3': EOMES, NFIL3, TBET.

[0098] In one aspect, the present invention provides a cell comprising at least one polynucleotide and / or construct or vector described herein.

[0099] In one aspect, the present invention relates to a pharmaceutical composition comprising at least one polynucleotide, construct or vector, and / or cell described herein. In one embodiment, the pharmaceutical composition further comprises a suitable carrier. These compositions may be prepared using pharmaceutically acceptable carriers composed of materials deemed safe and effective, and can be administered to an individual without causing unwanted biological side effects or undesirable interactions. A carrier is any component present in a pharmaceutical formulation other than the active ingredient(s). As generally used herein, "carrier" includes, but is not limited to, plasticizers, crystallization inhibitors, wetting agents, bulk fillers, solubilizers, bioavailability enhancers, solvents, pH adjusters, and combinations thereof.

[0100] In one aspect, the present invention provides a method for reprogramming or inducing any cell into a natural killer cell or progenitor cell, the method comprising: a. transducing a cell with at least one polynucleotide, or construct or vector described herein; b. Cultivating and growing the transduced cells in cell culture medium; and c. obtaining a reprogrammed cell.

[0101] In one embodiment, the induced progenitor cells or reprogrammed progenitor cells are CD34 positive, CD38 positive, and / or CD10 positive. Those skilled in the art will understand that these are immature cell markers. The reprogrammed cells of the present invention may transition from progenitor cells to mature cells in culture. Those skilled in the art will understand that this facilitates expansion in culture. The mature cell state may be characterized by the lack of expression of CD34 and CD10, and the expression of CD8, CD38, and DX5 (encoded by the ITGA2 gene).

[0102] As used herein, "+", + " and "positive" are used interchangeably.

[0103] In one embodiment, the method further comprises culturing the transduced cells in medium comprising one or more cytokines, hi another embodiment, the one or more cytokines are selected from the group consisting of SCF, FLT3L, IL-3, IL-7, and IL-15.

[0104] In one embodiment of the invention, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is selected from the group consisting of a stem cell, a differentiated cell, a cancer cell, or a tumor cell.

[0105] In one embodiment, the stem cells are selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, and hematopoietic stem cells.

[0106] In some embodiments of the invention, the somatic cells are fibroblasts.

[0107] Essentially any primary somatic cell type can be used to produce induced NK cells or reprogram somatic cells into induced NK cells in accordance with the compositions, constructs, vectors, cells, and methods described herein. Some non-limiting examples of primary somatic cells useful in various aspects and embodiments of the methods described herein include, but are not limited to, fibroblasts, epithelial cells, endothelial cells, neural cells, adipocytes, cardiac cells, skeletal muscle cells, hematopoietic or immune cells, liver cells, spleen cells, lung cells, circulating blood cells, gastrointestinal cells, kidney cells, bone marrow cells, pancreatic cells, and the stem cells from which they are derived. Cells can be primary cells isolated from any somatic tissue, including, but not limited to, spleen, bone marrow, blood, brain, liver, lung, intestine, stomach, small intestine, fat, muscle, uterus, skin, spleen, endocrine organs, and bone. The term "somatic cell" further encompasses, in some embodiments, primary cells grown in culture, provided that the somatic cells are not immortalized. When cells are maintained under in vitro conditions, conventional tissue culture conditions and methods can be used and are known to those skilled in the art. Methods for isolating and culturing various primary somatic cells are well within the capabilities of those skilled in the art.

[0108] In some embodiments, the somatic cell can be a hematopoietic lineage cell.

[0109] In some embodiments of the invention, the somatic cells to be reprogrammed or made into induced NK cells are cells of hematopoietic origin. As used herein, the terms "hematopoietic-derived cells," "hematopoietic-derived differentiated cells," "hematopoietic lineage cells," and "cells of hematopoietic origin" refer to cells derived from or differentiated from multipotent hematopoietic stem cells (HSCs). Thus, hematopoietic lineage cells for use with the compositions, constructs, vectors, cells, and methods described herein include multipotent hematopoietic progenitor cells, oligopotent hematopoietic progenitor cells, lineage-restricted hematopoietic progenitor cells, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, and lymphocytes (e.g., T lymphocytes bearing a T cell receptor (TCR), B lymphocytes or B cells that express immunoglobulins and produce antibodies, NK cells, NKT cells, and innate lymphocytes). As used herein, the term "hematopoietic progenitor cells" refers to multipotent hematopoietic cells, oligopotent hematopoietic cells, and lineage-restricted hematopoietic cells that can differentiate into two or more cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, B cells, and T cells. Hematopoietic progenitor cells include multipotent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), common lymphoid progenitor cells (CLPs), granulocyte-monocyte progenitors (GMPs), and promegakaryocyte-erythroid progenitors. Lineage-restricted hematopoietic progenitor cells include megakaryocyte-erythroid progenitors (MEPs), pro-B cells, pre-B cells, pre-Pro-B cells, pro-T cells, double-negative T cells, pro-NK cells, pre-granulocyte / macrophage cells, granulocyte / macrophage progenitor (GMP) cells, and pro-mast cells (ProMCs).

[0110] Cells of hematopoietic origin for use in the compositions, constructs, vectors, cells, and methods described herein may be obtained from any source known to contain these cells, such as fetal tissue, umbilical cord blood, bone marrow, peripheral blood, mobilized peripheral blood, spleen, liver, thymus, lymph, etc. Cells obtained from these sources may be expanded ex vivo using any method acceptable to one of skill in the art prior to use with the compositions, constructs, vectors, cells, and methods for generating induced NKs. For example, cells may be sorted, fractionated, treated to remove specific cell types, or otherwise manipulated using any procedure acceptable to one of skill in the art to obtain populations of cells for use in the methods described herein.

[0111] In one embodiment of the invention, the transduced cells are cultured for at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

[0112] In one embodiment of the present invention, the reprogrammed cells are NCR1 positive.

[0113] In one embodiment of the present invention, the reprogrammed cells are CD34 positive.

[0114] In one embodiment of the present invention, the reprogrammed cells are CD56 positive.

[0115] In one aspect, the invention provides induced natural killer cells or progenitor cells obtained by the methods described herein.

[0116] In one embodiment of the invention, the cells, reprogrammed cells and / or derived cells are NCR1 positive. In another embodiment of the invention, the cells, reprogrammed cells and / or derived cells are CD34 positive. In another embodiment of the invention, the cells, reprogrammed cells and / or derived cells are CD56 positive.

[0117] In one embodiment, the present invention relates to at least one polynucleotide, construct or vector, cell, and / or induced natural killer cell as described herein for use in medicine.

[0118] In one embodiment, the present invention relates to at least one polynucleotide, construct or vector, cell, and / or induced natural killer cell as described herein for use in cancer treatment.

[0119] In one embodiment, the present invention relates to at least one polynucleotide, construct or vector, cell, and / or induced natural killer cell as described herein for use in immunotherapy.

[0120] [Example]

[0121] Example 1. Identification of candidate transcription factors for NK cell reprogramming method To identify candidate transcription factors (TFs) that guide NK cell identity and function as cytotoxic innate lymphoid cells, we used three complementary approaches: (i) the developed predictive computational tool GPSMatch, (ii) literature mining, and (iii) analysis of available gene expression datasets (GeneAtlas MOE430).

[0122] result We identified 19 NK-induced candidate TFs that are enriched in NK cells compared with other tissues and cell types, such as T and B lymphocytes ( Fig. 2 ).

[0123] conclusion Nineteen candidate TFs were identified that direct NK cell identity among other cell types.

[0124] Example 2. Screening for NK-induced transcription factors method To screen for NK-inducing factors, we expressed 19 candidate TFs in primary cultures of mouse embryonic fibroblasts (MEFs) harboring an NK-specific reporter system (Ncr1-CreX R26-stop-tdT mouse, hereafter referred to as NCR1-tdTomato) (Figure 3A). NCR1 is specifically expressed in the NK cell lineage (Figure 3B).

[0125] Isolation of MEFs Ncr1Cre / Cre ( Eckelhart et al., 2011 ) animals were crossed with Rosa26-stopflox-tdTomato reporter mice to generate double homozygous Ncr1Cre / CreRosatdTomato / tdTomato (NCR1-tdTomato) mice. All animals were housed under controlled temperature (23 ± 2°C) with a fixed 12-h light / dark cycle and food and water available ad libitum.

[0126] Primary cultures of MEFs were isolated from E13.5 embryos of Ncr1-cre mice. The head, fetal liver, and all internal organs were removed, and the remaining tissues were mechanically dissociated. The dissected tissues were enzymatically digested using a solution of 0.12% trypsin / 0.1 mM ethylenediaminetetraacetic acid (EDTA) (3 mL per embryo) and incubated at 37°C for 15 minutes. An additional 3 mL of the same solution was added per embryo, followed by an additional 15 minutes of incubation. Single-cell suspensions were obtained and seeded onto 10 cm tissue culture dishes coated with 0.1% gelatin in growth medium. Cells were grown for 2 to 3 days until confluent, dissociated with Tryple Express, and frozen in fetal bovine serum (FBS), 10% dimethyl sulfoxide (DMSO). Prior to plating for lentiviral transduction, MEFs were sorted for residual CD45 expression, which may represent cells with hematopoietic potential. + and tdTomato + The MEFs used for screening and subsequent experiments were >99% pure tdTomato - CD45 - and expanded for up to four passages.

[0127] Transduction Prior to plating for lentiviral transduction, MEFs were sorted to identify residual CD45 -expressing cells that may represent cells with hematopoietic potential. + NCR1-tdTomato MEFs were maintained in growth medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) FBS, 2 mM L-glutamine, and antibiotics (10 μg / ml penicillin and streptomycin)]. All cells were maintained at 37°C and 5% (v / v) CO2. NCR1-tdTomato MEFs were seeded at a density of 40,000 cells per well on 6-well plates coated with 0.1% gelatin. Cells were incubated overnight with FUW-TetO-TF and FUW-M2rtTA lentiviral particles at a 1:1 ratio in growth medium supplemented with 8 μg / ml polybrene. Cells were transduced twice on consecutive days. After overnight incubation, the medium was replaced with fresh growth medium. After the second transduction, the growth medium was supplemented with doxycycline (1 μg / mL), which was designated day 0. The medium was changed every 2 to 3 days during the culture period. Appearing tdTomato-positive cells were analyzed microscopically between days 3 and 12 after transduction. Whole wells of 6-well plates were acquired using an automated Zeiss CD7 microscope. For flow cytometry analysis, transduced NCR1-tdTomato MEFs were dissociated with TrypLE Express, resuspended in PBS 5% FBS, and maintained at 4°C before analysis using a BD LSRFortessa (BD Biosciences).

[0128] result Expression of all 19 candidate TFs induces tdTomato-positive (tdT+) colonies that asynchronously emerge and grow in culture between days 3 and 6 (Figure 3C and Figure 3D). The number of tdT+ colonies increased after overexpression of a smaller pool of transcription factors, particularly with a combination of five candidate TFs consisting of ETS1, NFIL3, ID2, EOMES, and TBET (Figure 4). tdT+ colonies induced by this pool of five TFs continue to grow in culture between days 3 and 12 of reprogramming (Figure 5). Furthermore, this TF pool induces 6% of tdT+ cells, suggesting that the minimal combination required to induce NCR1-tdT activation is contained within this pool (Figure 5C and Figure 5D). To define the minimal combination, we individually removed one TF from this pool of five TFs, demonstrating that ID2 is dispensable for the reprogramming process. The discovered combination of four TFs increased the number of tdT+ colonies (Figure 6A) and induced approximately 20% tdT+ cells on day 12 of reprogramming (Figure 6B). Another round of TF elimination showed that the combination of three TFs was sufficient to increase the number of tdT+ colonies and the percentage of tdT+ cells compared to the control.

[0129] conclusion Screening using the NCR1-tdTomato reporter allows the identification of NK-TF instructors. These results showed that a combination of three TFs selected from ETS1, NFIL3, EOMES, and TBET was necessary and sufficient to activate the NCR1 reporter and induce proliferative NCR1-positive colonies. The combination of four TFs (ETS1, NFIL3, TBET, and EOMES) resulted in the greatest increase in NCR1 reporter activity. NK reprogramming is fast (starting on day 3) and efficient (approximately 20% by day 12).

[0130] Example 3. ETS1, NFIL3, TBET, and EOMES are enriched in NK cells method Gene expression data (GeneAtlas MOE430) were log-transformed and normalized to a range of 0 to 1 for each gene, after which the highest mean expression of ETS1+NFIL3+TBET+EOMES was examined.

[0131] result The combined expression of ETS1, NFIL3, TBET, and EOMES was highly associated with NK cells (Fig. 8).

[0132] conclusion These results validated the identification of ETS1, NFIL3, TBET, and EOMES as a combination that induces NK cell identity.

[0133] Example 4. The combination of RORα, SMAD7, FOS, JUN and NFATC2 does not activate the NCR1-tdTomato reporter. method Screening using the NCR1-tdTomato reporter was performed as described in Example 2, and gene expression data were analyzed as described in Example 3.

[0134] result Expression of RORα, SMAD7, FOS, JUN, and NFATC2 did not activate the NCR1 reporter at days 6 or 12 of reprogramming (Figure 9A). Furthermore, gene expression analysis showed that combined expression of RORα+SMAD7+FOS+JUN+NFATC2 was not enriched in NK cells, among other tissues and cell types (Figure 9B).

[0135] conclusion These results indicate that RORα, SMAD7, FOS, JUN, and NFATC2 are not enriched in NK cells and do not activate the NCR1 reporter.

[0136] Example 5. ETS1, NFIL3, TBET, and EOMES induce global transcriptional changes to the NK cell lineage method Untransduced MEFs and MEFs transduced with ETS1, NFIL3, TBET, and EOMES were dissociated using TrypLE Express, resuspended in 5% PBS, and sorted by FACS on days 3 (tdT+), 6 (tdT+), and 12 (tdT+). Purified MEFs (5,000 cells), tdT+d3 (5,000 cells), tdT+d6 (5,000 cells), and tdT+d12 (5,000 cells) were loaded onto 10x Chromium (10x Genomics) according to the manufacturer's protocol. Single-cell RNA-Seq libraries were prepared using the Chromium Single Cell 3' v3.1 Reagent Kit (10x Genomics) according to the manufacturer's protocol. Single cells were isolated into droplets with gel beads coated with a unique primer containing a 10x unique molecular identifier (UMI) and a poly(dT) sequence. Reverse transcription reactions were performed to generate barcoded full-length cDNA. Indexed sequencing libraries were constructed using reagents from the Chromium Single Cell 3′ v3.1 Reagent Kit (10x Genomics). Library quantification and quality assessment were determined using Qubit and Agilant TapeStation. Indexed libraries were sequenced on an Illumina NovaSeq 6000 S2 100 FlowCell v1. Approximately 100,000 reads per single cell were obtained for tdT+ day 3, day 6, and day 12 samples, while 25,000 reads per single cell were obtained for MEFS samples.

[0137] result To determine whether the four TFs induce NK cell identity in MEFS, we profiled tdT+ cells at days 3, 6, and 12 of reprogramming by single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform. TdT+ cells exhibited global transcriptional changes beginning as early as day 3, gradually increasing at days 6 and 12, when fibroblast-specific genes were silenced (Figures 10A and 11). At day 12, mature NK genes such as Itga2 were also detected (Figure 10B). The combined expression of the four TFs imposed an NK-like transcriptional network, inducing the expression of 19 candidate NK-specific TFs at day 3, which gradually increased until day 12 of reprogramming (Figure 12). Endogenous expression of Ets1, Nfil3, Tbx21 (encoding Tbet), and EOMES was also activated during reprogramming. Furthermore, scRNA-seq analysis revealed that the four TFs induced global transcriptome reprogramming toward NK cell progenitor identity, inducing upregulation of NK progenitor genes, such as CD38, CD34, and MME (encoding CD10) (Figure 13C). Induced NK cells generated with the four TFs were highly enriched in immature NK cell characteristics from the early stages of reprogramming (Bezman et al., 2012). Accordingly, the mature NK gene expression signature gradually increased during later reprogramming stages (Figures 13A and 13B). Importantly, we detected increased expression of genes related to NK function, such as ccl5, Ifih1, and Il15ra (Figure 14).

[0138] conclusion These data demonstrated that ETS1, NFIL3, TBET, and EOMES direct the reprogramming of fibroblasts toward the NK cell lineage and impose progenitor NK identity.

[0139] Example 6. Forced expression of ETS1, NFIL3, TBET, and EOMES generates human CD34 and CD56 positive cells method Human embryonic fibroblasts (HEF) were seeded at a density of 30,000 cells per well on 0.1% gelatin-coated 6-well plates. Cells were incubated overnight with FUW-TetO-TF and FUW-M2rtTA lentiviral particles at a 1:1 ratio. Alternatively, cells were incubated with SFFV-MCS or SFFV-TF lentiviral particles at the same multiplicity of infection (MOI). Lentiviral particles were added to growth medium supplemented with 8 μg / mL polybrene. After overnight incubation, the medium was replaced with fresh growth medium. For FUW-TetO-transduced cells, doxycycline (1 μg / mL) was added to the growth medium. The medium was changed every 2 to 3 days during the culture period. A cytokine cocktail containing stem cell factor (SCF) (10 μg / mL), FMS-like tyrosine kinase 3 ligand (FLT3L) (10 μg / mL), interleukin-3 (IL-3) (5 μg / mL), IL-7 (5 μg / mL), and IL-15 (10 μg / mL) was added to the cultures as indicated. NK reprogramming was assessed by flow cytometry 6 and 12 days after transduction (Figure 15). For flow cytometry, transduced HEFs were dissociated with TrypLE Express, resuspended in PBS 5% FBS, and stained with mouse anti-human CD34 antibody or anti-human CD56 antibody. 1% mouse serum was used to prevent nonspecific antibody binding. Cells were kept at 4°C before analysis on a BD LSRFortessa (BD Biosciences).

[0140] result Combined expression of ETS1, NFIL3, TBET, and EOMES, individually cloned into pSFFV, induced a rare population of human CD34-positive (CD34+) cells on days 6 and 12 of reprogramming (Figure 16). CD34+ cells were not observed in the empty vector (MCS) control. Culture in the presence of a NK differentiation and activation cytokine cocktail increased reprogramming efficiency by 1.5-fold on day 6 and approximately 2-fold on day 12 (Figure 17). Furthermore, combined expression of the four TFs induced human CD56-positive (CD56+) cells 6 days after transduction (Figure 18).

[0141] conclusion These results suggest that four TFs (ETS1, NFIL3, TBET, and EOMES) are sufficient to induce the combination of human CD34+ NK progenitor cells and CD56+ mature NK cells. Furthermore, these data demonstrate that the constitutive lentiviral vector SFFV enables reprogramming from human cells with higher efficiency than the inducible FUW-TetO system. The addition of SCF, FLT3L, IL-3, IL-7, and IL-15 improves the NK reprogramming process.

[0142] Example 7. NK-based immunotherapy can target liquid and solid tumors The cells produced by the methods described herein may be used to treat leukemias, lymphomas such as NCT01807611, chronic myeloid leukemia, multiple myeloma (NCT00720785, NCT02955550), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, B-lymphoid malignancies, acute lymphoblastic leukemia (NCT03019640, NCT03579927, NCT03056339). It can be used to treat or alleviate several cancers and tumors, including blood cancers such as acute myeloid leukemia (NCT02782546, NCT03081780, NCT01787474), childhood acute myeloid leukemia (NCT03068819), plasma cell leukemia (NCT01729091), acute erythroid leukemia, acute megakaryoblastic leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndromes (NCT01823198). The cells produced by the methods described herein can be used to treat or alleviate solid cancers such as neuroblastoma, Ewing's sarcoma, rhabdomyosarcoma (NCT01857934, NCT02100891), pancreatic cancer, colon / rectal cancer, non-small cell lung cancer, gastric cancer, head and neck cancer, squamous cell carcinoma, breast cancer, hepatocellular carcinoma, renal cell carcinoma, melanoma, urothelial carcinoma, cervical cancer, Merkel cell carcinoma (NCT00720785, NCT03319459, NCT03841110), ovarian cancer, fallopian tube cancer, primary peritoneal cancer (NCT03539406), medulloblastoma, ependymoma (NCT02271711), malignant bone neoplasms, lip and oral cancer, endocrine tumors, male reproductive tract tumors, mesothelioma, oral cavity tumors, pharyngeal tumors, bone tumors, thyroid tumors (NCT03420963).

[0143] Sequence Overview [Table 1] TIFF2023554441000003.tif17162 References Bachanova, V. et al. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 123, 3855-3863 (2014). Barry, K. C. et al. A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments. Nat. Med. 24, 1178-1191 (2018). Bottcher, J. P. et al. NK cells stimulate recruitment of cDC1 into the tumor microenvironment promoting cancer immune control. Cell 172, 1022-1037 (2018). Boudreau JE, et al., Cell-extrinsic MHC class I molecule engagement augments human NK cell education programmed by cell-intrinsic MHC class I. Immunity 45, 280-291 (2016). Bezman NA, et al., Molecular definition of the identity and activation of natural killer cells. Nature Immunology13,1000-1009 (2012). E. Vivier et al., Innate or Adaptive Immunity? The Example of Natural Killer Cells. Science 331, 44-49 (2011). Eckelhart,E.,et al., A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK-cell survival and development. Blood117, 1565-1573 (2011). Firas, J., Polo, J., Gough, J., Hayashizaki, Y., Rackham, O., Cell Reprogramming, Patent WO2019177936A1, 2017 Gomes AM, et al. Cooperative Transcription Factor Induction Mediates Hemogenic Reprogramming. Cell Rep 25, 2821- 2835.e7 (2018) Graf T., Enver T., Forcing Cells to Change Lineages, Nature 462, 587-94 (2009) Lopez-Soto, A., et al., Control of metastasis by NK cells. Cancer Cell 32, 135-154 (2017). Noriko Shimasaki et al. NK cells for cancer immunotherapy. Nat Rev Drug Discov 19, 200-218 (2020). Passweg, J. R. et al. Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation. Leukemia 18, 1835-1838 (2004). Pereira CF, et al., Reprogramming cell fates: Insights from combinatorial approaches. Ann N Y Acad Sci 1266 7-17 (2012). Pereira CF, et al., Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell 13, 205-18 (2013). Renoux, V. M. et al., Identification of a human natural killer cell lineage-restricted progenitor in fetal and adult tissues. Immunity 43, 394-407 (2015). Rosa FF, et al., Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Sci Immunol 3(30):4292 (2018) Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126(4):663-76 (2006). Tam, Y. K., et al., Ex vivo expansion of the highly cytotoxic human natural killer-92 cell-line under current good manufacturing practice conditions for clinical adoptive cellular immunotherapy. Cytotherapy 5, 259-272 (2003). Ye Li et al., Human iPSC-Derived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Antitumor Activity. Cell Stem Cell 23, 181-192 (2018).

Claims

1. At least one polynucleotide encoding a combination of at least three different transcription factors selected from the group consisting of ETS1, TBET, NFIL3 and EOMES.

2. a. The transcription factor ETS1 comprises the amino acid sequence of SEQ ID NO: 1 or a biologically active variant of the amino acid sequence of SEQ ID NO: 1; and / or b. said transcription factor TBET comprises the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2, or a biologically active variant thereof; and / or c. The transcription factor NFIL3 comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3, or a biologically active variant thereof; and / or d. The transcription factor EOMES comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or a biologically active variant thereof; At least one polynucleotide according to claim 1.

3. a. the polynucleotide encoding SEQ ID NO: 1 is SEQ ID NO: 22, or a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 22; and / or b. said polynucleotide encoding SEQ ID NO:2 is SEQ ID NO:23 or a sequence having at least 90% sequence identity to SEQ ID NO:23; and / or c. the polynucleotide encoding SEQ ID NO:3 is SEQ ID NO:24 or a sequence having at least 90% sequence identity to SEQ ID NO:24; and / or d. The polynucleotide encoding SEQ ID NO:4 to SEQ ID NO:6 is selected from the group consisting of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, or sequences having at least 90% sequence identity to the sequences SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27; At least one polynucleotide according to claim 2.

4. The combination of at least three transcription factors is ETS1, TBET, NFIL3 and EOMES, ETS1, TBET and NFIL3, ETS1, TBET and EOMES, ETS1, NFIL3 and EOMES, TBET, NFIL3 and EOMES 4. At least one polynucleotide according to any one of claims 1 to 3, selected from the group consisting of:

5. 5. At least one polynucleotide according to any one of claims 1 to 4, wherein the combination further comprises one or more transcription factors selected from the group consisting of ID2, GATA3, ZFP105, IKZF3, TOX, RUNX3, KLF12, ZEB2, IRF2, STAT5, IKZF1, ELF4, ZBTB16, GATA2 and ELF1.

6. 6. One or more constructs or vectors comprising at least one polynucleotide according to any one of claims 1 to 5.

7. 7. The one or more constructs or vectors of claim 6, wherein the vector is a viral vector.

8. 8. The one or more constructs or vectors of any one of claims 6 to 7, wherein the vector is selected from the group consisting of lentiviral vectors, retroviral vectors, adenoviral vectors, herpes virus vectors, poxvirus vectors, flavivirus vectors, rhabdovirus vectors, paramyxovirus vectors, adeno-associated virus vectors, reovirus vectors, papillomavirus vectors, picornavirus vectors, calicivirus vectors, hepadnavirus vectors, togavirus vectors, coronavirus vectors, hepatitis virus vectors, orthomyxovirus vectors, bunyavirus vectors and filovirus vectors.

9. 9. One or more constructs or vectors according to any one of claims 6 to 8, wherein the vector is an oncolytic vector.

10. 10. One or more constructs or vectors according to any one of claims 6 to 9, wherein the construct or vector is a synthetic mRNA, a naked alphavirus RNA replicon, or a naked flavivirus RNA replicon.

11. 11. One or more constructs or vectors according to any one of claims 6 to 10, wherein the vector further comprises a promoter sequence controlling the transcription of at least one polynucleotide according to any one of claims 1 to 5.

12. 12. The one or more constructs or vectors of any one of claims 6 to 11, wherein the promoter sequence is selected from the group consisting of a spleen focus forming virus (SFFV) promoter, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing the myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1 alpha promoter, or a murine stem cell virus (MSCV) promoter.

13. 7. The one or more constructs or vectors of claim 6, wherein the construct or vector is a plasmid.

14. 14. One or more constructs or vectors according to any one of claims 6 to 13, wherein the construct or vector is a CRISPR / CAS activation system.

15. A cell comprising at least one polynucleotide according to any one of claims 1 to 5 and / or one or more constructs or vectors according to any one of claims 6 to 14.

16. The cell of claim 15 , wherein the cell is selected from the group consisting of a stem cell, a differentiated cell, a cancer cell, or a tumor cell.

17. 17. The cell of claim 16, wherein the stem cell is selected from the group consisting of pluripotent and multipotent stem cells, such as mesenchymal stem cells, and hematopoietic stem cells.

18. A pharmaceutical composition comprising at least one polynucleotide according to any one of claims 1 to 5, one or more constructs or vectors according to any one of claims 6 to 14, and / or a cell according to any one of claims 15 to 17.

19. 1. A method for reprogramming or inducing cells into natural killer cells or progenitor cells, comprising: The method comprises the steps of: a. transducing a cell using at least one polynucleotide according to any one of claims 1 to 5, or one or more constructs or vectors according to any one of claims 6 to 14, or a pharmaceutical composition according to claim 18; b. Cultivating and growing the transduced cells in a cell culture medium; c. Obtaining the reprogrammed cells A method comprising:

20. 20. Induced natural killer cells or progenitor cells obtainable by the method of claim 19.

21. At least one polynucleotide according to any one of claims 1 to 5, one or more constructs or vectors according to any one of claims 6 to 14, a cell according to any one of claims 15 to 17, a pharmaceutical composition according to claim 18, and / or an induced natural killer cell or progenitor cell according to claim 20, for use in medicine.

22. At least one polynucleotide according to any one of claims 1 to 5, one or more constructs or vectors according to any one of claims 6 to 14, a cell according to any one of claims 15 to 17, a pharmaceutical composition according to claim 18, and / or an induced natural killer cell or progenitor cell according to claim 20, for use in cancer treatment.

23. At least one polynucleotide according to any one of claims 1 to 5, one or more constructs or vectors according to any one of claims 6 to 14, a cell according to any one of claims 15 to 17, a pharmaceutical composition according to claim 18, and / or an induced natural killer cell or progenitor cell according to claim 20, for use in immunotherapy.