Compositions, methods and uses for reprogramming cells into plasmacytoid dendritic cells or type I interferon-producing cells

Abstract

The present disclosure relates to compositions, constructs, and vectors for reprogramming cells into plasmacytoid dendritic cells or type I interferon-producing cells, as well as methods and uses thereof. The present disclosure relates to the development of methods for generating plasmacytoid dendritic cells or type I interferon-producing cells that promote antiviral and antitumor immune responses from differentiated cells, pluripotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthetic transcription factors. More specifically, the present disclosure unexpectedly provides methods for obtaining plasmacytoid dendritic cells or type I interferon-producing cells by direct cell reprogramming using specific combinations of transcription factors.

Description

[Technical Field]

[0001] The present disclosure relates to compositions, methods and uses for reprogramming cells into plasmacytoid dendritic cells or type I interferon-producing cells.

[0002] The present disclosure relates to the development of methods for generating plasmacytoid dendritic cells or type I interferon-producing cells that promote antiviral and antitumor immune responses from differentiated stem cells, pluripotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthetic transcription factors. More specifically, the present disclosure provides methods for obtaining plasmacytoid dendritic cells or type I interferon-producing cells by direct cell reprogramming that unexpectedly and advantageously utilizes specific combinations of transcription factors. [Background technology]

[0003] Cell reprogramming is based on the reconfiguration of epigenetic and transcriptional networks of one cell state into networks of another cell type. Transcription factor (TF) transduction experiments have focused on the plasticity of adult somatic or differentiated cells, providing a novel technique for generating desired cell types. Forced expression of multiple transcription factors can reprogram somatic or differentiated cells into induced pluripotent stem cells (iPSCs), which are surprisingly similar to embryonic stem cells (Takahashi et al., 2006; Takahashi et al., 2007). Alternatively, somatic cells can be converted into other specialized cell types (Pereira et al., 2012). Direct lineage conversion using target-cell-specific transcription factors has been shown to successfully reprogram mouse and human fibroblasts into multiple cell types, including neurons, cardiomyocytes, and hepatocytes (Xu et al., 2015). Lineage conversion has also been demonstrated in the hematopoietic system, where forced expression of transcription factors induced macrophage commitment in B cells and fibroblasts (Xie et al., 2004) and direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors was achieved using the transcription factors Gata2, Gfi1b, cFos, and Etv6 (Pereira et al., 2013). These four transcription factors induce a dynamic, multistep hematopoietic process that progresses through an endothelial-like intermediate cell, recapitulating developmental hematopoiesis in vitro (Pereira et al., 2016).

[0004] Reprogrammed cells hold great promise as therapeutic tools in regenerative medicine, and cells obtained by differentiation of iPSCs are already being tested in clinical trials.

[0005] Cell reprogramming strategies have focused on cell fate flexibility, based on the availability of cell type-specific transcription factors to convert somatic cells into pluripotent cells. Direct lineage conversion from one differentiated cell type to another has been demonstrated and studied to elucidate cell biological mechanisms and for regenerative medicine purposes. Recently, we have demonstrated that a combination of a small number of transcription factors can reprogram unrelated cell types into antigen-presenting dendritic cells (DCs) (Rosa et al., 2018). Classically, functionally distinct dendritic cell subsets—conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs)—are generated from progenitor cells committed to myeloid dendritic cell fate. Conventional dendritic cells are professional antigen-presenting cells (APCs) responsible for antigen-specific immune responses, while plasmacytoid dendritic cells are professional producers of type I interferon during viral infection. They perform both innate and adaptive immune functions and can act as antigen-presenting cells that directly influence T cell responses. The timing and precise mechanisms controlling the branching into different subsets during dendritic cell development remain unclear.

[0006] European Patent Application Publication No. EP3385373 relates to compositions, nucleic acid constructs, methods and kits for inducing or reprogramming cells to a conventional dendritic cell state or an antigen-presenting cell state, which are based in part on the unexpected effects described in this document regarding the novel use of combinations of transcription factors that allow differentiated or undifferentiated cells to be inducible or reprogrammed into DCs or APCs.

[0007] The combination of transcription factors described in European Patent Application Publication No. EP 3385373 induces DCs with a surface phenotype, transcriptional signature, and functional properties characteristic of cDC1, a DC subset, not previously described for its ability to secrete type I interferons (e.g., interferon alpha (IFN-α) and beta (IFN-β)).

[0008] Described herein, induced pDCs or type I interferon-producing cells recapitulate the typical functional properties of pDCs, which detect nucleic acids and respond by secreting type I interferons (Reizis, 2019).

[0009] These facts are disclosed to demonstrate the technical problem that this disclosure addresses. Summary of the Invention

[0010] The subject of the present specification is the identification of multiple isolated transcription factors that unexpectedly reprogram or induce differentiated, pluripotent or multipotent stem cells into pDCs or type I interferon-producing cells in vitro, ex vivo, or in vivo.

[0011] Surprisingly, induced pDCs or type I interferon-producing cells generated by reprogramming as described in this disclosure are capable of constitutively secreting the type I interferons necessary to induce antiviral or antitumor responses.

[0012] Surprisingly, induced pDCs or type I interferon-producing cells generated by reprogramming as described in this disclosure are intrinsically responsive to ligands for toll-like receptor 7 (TLR7) and TLR9, and in response can secrete increased amounts of type I interferons (e.g., interferon alpha (IFN-α) and beta (IFN-β)) required to induce an antiviral response.

[0013] DCs are professional antigen-presenting cells present throughout the body and function at the interface of the innate and adaptive immune systems. They have the ability to capture, process, and present antigens on major histocompatibility complex (MHC) class I and MHC class II molecules to T cells, targeting T cells for different types of immune responses. This ability allows DCs to provide a critical link between the external environment and the adaptive immune system. First, DCs must capture antigen and process it through major histocompatibility complex (MHC) class I and MHC class II molecules. After activation, DCs migrate to local draining lymph nodes, where they can initiate multiple B- and T-cell responses; B- and T-cell responses are key features of adaptive immunity. The effectiveness of initial defense is primarily mediated by the induction of antigen-specific antibodies produced by B lymphocytes. Long-term defense against specific antigens requires the persistence of specific antibodies and the generation of immunological memory, which enables rapid and efficient responses upon subsequent antigen exposure. As professional antigen-presenting cells, DCs have the ability to cross-present antigens, which means that in addition to the classical ability to present exogenous antigens on MHC class II and endogenous antigens on MHC class I, they can also present exogenous antigens on MHC class I, which is an important step in generating cytotoxic T lymphocyte responses (CTLs).

[0014] DCs may express different combinations of surface receptors depending on their ontogeny and / or microenvironment. For example, murine DCs can be classified into distinct subpopulations based on phenotypic criteria. Among these, cDCs from lymphoid tissues are traditionally further divided into two subpopulations: cDC1 and cDC2. Different DC subsets are known to be involved in the specific recognition of specific pathogens and / or to control different immune responses (e.g., Th1 or Th2 (immunity) or regulatory T cell (tolerance)). However, DC phenotype and functional behavior are also significantly conditioned by external activating stimuli, indicating their great plasticity. In vivo, cDC1 and cDC2 subsets differentially initiate Th1 and Th2 responses. Cancer immunotherapy is based on the application of DCs to initiate Th1 or cytotoxic T lymphocyte responses that promote tumor elimination. In parallel with cDCs, pDCs also emerge as a unique subset of DCs specialized for type I interferon (IFN) production after viral infection or self-nucleic acid recognition by TLR7 and TLR9. In addition to contributing to antiviral immunity, pDCs can participate in the initiation of both immunogenic and tolerogenic adaptive immune responses, which are implicated in the pathology of autoimmune diseases and cancer.

[0015] pDCs have the unique characteristic of producing greater amounts of type I interferon than other cell types in response to viruses and / or virus-derived nucleic acids. TLR7 and TLR9 are highly expressed in pDCs in the intracellular endosomal compartment. TLR7 recognizes viral single-stranded RNA, while TLR9 detects viral double-stranded DNA rich in unmethylated CpG oligonucleotides. Activation by viral nucleic acids induces the stimulation and recruitment of the adaptor protein MyD88 and two major intracellular TLR7 / 9 signaling pathways. The first pathway results in type I interferon production, which requires nuclear translocation of IRF7. This nuclear translocation promotes the transcription and secretion of IFN-α and IFN-β. Upon nucleic acid activation, type I interferon secreted by pDCs can bridge the innate and adaptive immune systems, promoting long-term T cell survival and memory, Th1 polarization, CD8 T cell cytotoxic activity, NK cell-mediated cytotoxicity, and B cell proliferation and differentiation. Alternatively, alternative pathway activation leads to the NF-kB-dependent induction of proinflammatory cytokines TNF-α and IL-6, as well as chemokines such as CXCL9 (MIG), CXCL10 (IP-10), CCL3 (MIP-1a), CCL4 (MIP-1b), and CCL5 (RANTES), which can then attract activated CD4 and CD8 T cells to the site of inflammation.

[0016] Currently, DC-based immunotherapy relies on autologous DC precursors (either monocytes, which are associated with less efficient DC generation, or hematopoietic precursors, which can be isolated in very low numbers). Furthermore, in cancer patients, these precursors are usually impaired, resulting in the formation of a heterogeneous mixture of dysfunctional DCs. In contrast, non-hematopoietic cell types, such as fibroblasts, are usually unaffected. Human dermal fibroblasts (HDFs) offer another competitive advantage; they are easily obtained by small skin-punch biopsy and easily expanded by several passages in vitro (15 x 10 cells after 4 weeks). 6 ~20x10 6pDCs) can be cryopreserved and used as needed. Given the fundamental role of DCs functioning at the interface of the innate and adaptive immune systems, there remains a clinical need to find alternative strategies to generate homogeneous populations of functional pDCs that initiate antigen-specific responses.

[0017] In recent years, pDCs have attracted particular attention due to their ability to produce large amounts of IFN and activate T cells. It has been shown that pDC-deficient mice are unable to efficiently clear lymphocytic choriomeningitis virus due to defective CD4+ and CD8+ T cell responses (Cervantes-Barragan et al., 2012). Similarly, null mutations in IRF7 have been reported in patients, negatively affecting the production of type I and type III IFN by pDCs and resulting in severe influenza infection (Ciancanelli et al., 2015). These findings indicate that pDCs are important in fighting viral infections, especially in the early stages where IFN production can limit viral replication (Swiecki and Colonna, 2015). Furthermore, several clinical trials are investigating the potential of pDCs to induce antitumor immune responses (Tel et al., 2013). However, the therapeutic utility of pDCs is likely limited by the low number of these cells circulating in the blood. Therefore, alternative sources of DCs, particularly pDCs, are required to meet therapeutic needs.

[0018] In some embodiments, polypeptide variants or family members having the same or similar activity as a reference polypeptide having a polypeptide sequence (SEQ ID NO:49-SEQ ID NO:96) or a polypeptide encoded by a sequence of SEQ ID NO:1-SEQ ID NO:48 can be utilized in the compositions, methods, and kits described herein. Generally, a particular polypeptide variant encoding a pDC inducer for use in the compositions, vectors, constructs, methods, and kits described herein will have at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to a particular reference polynucleotide or polypeptide sequence, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0019] Methods for aligning sequences for comparison are well known in the art, and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the Needleman and Wunsch algorithm ((1970) J Mol Biol 48:443-453) to obtain a global alignment (across the entire sequences) of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al., (1990) J Mol Biol 215:403-10) calculates the percent sequence identity between two sequences and performs statistical analysis of sequence similarity. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). Global % similarity and identity may also be determined using one of the available methods in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29; MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). As will be apparent to those skilled in the art, minor manual editing may be performed to optimize alignment between conserved motifs. In the present subject matter, sequence identity values, expressed as percentages, were determined by BLAST using default parameters over the entire amino acid or nucleotide sequence.

[0020] In one embodiment for achieving even better results, the transcription factors are IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4), ARID5A (SEQ ID NO: 5, SEQ ID NO: 6), BCL11A (SEQ ID NO: 7, SEQ ID NO: 8), CBFA2T3 (SEQ ID NO: 9, SEQ ID NO: 10), CREB3L2 (SEQ ID NO: 11, SEQ ID NO: 12), ETS1 (SEQ ID NO: 13, SEQ ID NO: 14), HDAC5 (SEQ ID NO: 15, SEQ ID NO: 16), HHEX (SEQ ID NO: 17, SEQ ID NO: 18), HOXA5 (SEQ ID NO: 19, SEQ ID NO: 20), ID3 (SEQ ID NO: 21, SEQ ID NO: 22), IKZF1 (SEQ ID NO: 23, SEQ ID NO: 24), IKZF2 (SEQ ID NO: 25), IKZF3 (SEQ ID NO: 26), IKZF4 (SEQ ID NO: 27), IKZF5 (SEQ ID NO: 28), IKZF6 (SEQ ID NO: 29), IKZF7 (SEQ ID NO: 30), IKZF8 (SEQ ID NO: 31), IKZF9 (SEQ ID NO: 32), IKZF10 (SEQ ID NO: 33), IKZF11 (SEQ ID NO: 34), IKZF12 (SEQ ID NO: 35), IKZF13 (SEQ ID NO: 36), IKZF14 (SEQ ID NO: 37), IKZF15 (SEQ ID NO: 38), IKZF16 (SEQ ID NO: 39), The gene may be encoded by a polynucleotide selected from the group consisting of: IKZF3 (SEQ ID NO: 25, SEQ ID NO: 26), IKZF3 (SEQ ID NO: 27, SEQ ID NO: 28), IKZF5 (SEQ ID NO: 29, SEQ ID NO: 30), IRF7 (SEQ ID NO: 31, SEQ ID NO: 32), MEF2C (SEQ ID NO: 33, SEQ ID NO: 34), MYB (SEQ ID NO: 35, SEQ ID NO: 36), RUNX2 (SEQ ID NO: 37, SEQ ID NO: 38), STAT3 (SEQ ID NO: 39, SEQ ID NO: 40), TCF4 (SEQ ID NO: 41, SEQ ID NO: 42), TCF12 (SEQ ID NO: 43, SEQ ID NO: 44), STAT1 (SEQ ID NO: 45, SEQ ID NO: 46), and TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48).

[0021] In one embodiment of the present disclosure, the transcription factor is IRF8 (SEQ ID NO: 49, SEQ ID NO: 50), SPIB (SEQ ID NO: 51, SEQ ID NO: 52), ARID5A (SEQ ID NO: 53, SEQ ID NO: 54), BCL11A (SEQ ID NO: 55, SEQ ID NO: 56), CBFA2T3 (SEQ ID NO: 57, SEQ ID NO: 58), CREB3L2 (SEQ ID NO: 59, SEQ ID NO: 60), ETS1 (SEQ ID NO: 61, SEQ ID NO: 62), HDAC5 (SEQ ID NO: 63, SEQ ID NO: 64), HHEX (SEQ ID NO: 65, SEQ ID NO: 66), HOXA5 (SEQ ID NO: 67, SEQ ID NO: 68), ID3 (SEQ ID NO: 69, SEQ ID NO: 70), IKZF1 (SEQ ID NO: 71, SEQ ID NO: 72), It may be selected from the group consisting of IKZF2 (SEQ ID NO: 73, SEQ ID NO: 74), IKZF3 (SEQ ID NO: 75, SEQ ID NO: 76), IKZF5 (SEQ ID NO: 77, SEQ ID NO: 78), IRF7 (SEQ ID NO: 79, SEQ ID NO: 80), MEF2C (SEQ ID NO: 81, SEQ ID NO: 82), MYB (SEQ ID NO: 83, SEQ ID NO: 84), RUNX2 (SEQ ID NO: 85, SEQ ID NO: 86), STAT3 (SEQ ID NO: 87, SEQ ID NO: 88), TCF4 (SEQ ID NO: 89, SEQ ID NO: 90), TCF12 (SEQ ID NO: 91, SEQ ID NO: 92), STAT1 (SEQ ID NO: 93, SEQ ID NO: 94), and TSC22D1 (SEQ ID NO: 95, SEQ ID NO: 96).

[0022] In one embodiment, the transcription factors of the present disclosure may be used in veterinary or human medicine, particularly in the treatment of infectious diseases, or viral infectious diseases, or virally induced diseases, or cancer, or autoimmune diseases, or neurodegenerative diseases.

[0023] In one embodiment for better results, the cells may be selected from the group consisting of pluripotent stem cells, pluripotent stem cells, hematopoietic stem cells, differentiated cells, tumor cells, cancer cells, cell lines, particularly mesodermally derived cell lines (such as monocyte cell lines), and mixtures thereof, particularly mammalian cells, more particularly murine or human cells.

[0024] In one embodiment for achieving even better results, the transcription factors of the present disclosure may be used as factors for reprogramming or inducing cells selected from the group consisting of pluripotent stem cells, or pluripotent stem cells, or differentiated cells, and mixtures thereof, into dendritic cells, or type I interferon-producing cells, or antigen-presenting cells, preferably into dendritic cells or type I interferon-producing cells.

[0025] In one embodiment for achieving even better results, the transcription factors of the present disclosure may be used as agents for reprogramming or inducing cells selected from the group consisting of pluripotent stem cells, pluripotent stem cells or differentiated cells, and mixtures thereof, into dendritic cells or type I interferon-producing cells.

[0026] In one embodiment for even better results, the transcription factors of the present disclosure may be used as factors to reprogram or induce cells selected from the group consisting of tumor cells, cancer cells, and mixtures thereof, into antigen-presenting cells.

[0027] In one embodiment, the results of the present disclosure demonstrate that labeling pDCs with tdTomato fluorescent protein in Clec9a reporter mice makes this model suitable for screening pDC-inducing factors. IRF8 has been reported to play an important role in cDC1 and pDC specification and is highly expressed in both DC subsets. Therefore, in the present invention, we performed incremental screening in Clec9a reporter mouse embryonic fibroblasts (MEFs) by combining IRF8 with each of the 24 candidate candidates.

[0028] In one embodiment, unexpectedly, IRF8 in combination with SPIB is sufficient to induce reporter activation. Furthermore, combining IRF8 and SPIB with BCL11A, CBFA2T3, CREB3L2, ETS1, STAT1, TCF4, TCF12, or TSC22D1 unexpectedly increases the efficiency of reporter activation. Expression of major histocompatibility complex (MHC) class II molecules, which is important for DC functionality, is also increased by combinations of IRF8-based pDC-inducing transcription factors and IRF8 plus SPIB-based pDC-inducing transcription factors.

[0029] In one embodiment, expression of IRF8 and SPIB is sufficient to induce surface expression of the hematopoietic marker CD45. Notably, when IRF8 and SPIB were combined with IKZF1 or IKZF2, surface expression of CD45 was unexpectedly increased.

[0030] In one embodiment, IRF8 and SPIB can induce the secretion of type I interferons (i.e., IFN-α and IFN-β) after stimulation with TLR7 and TLR9 ligands, a phenotypic characteristic of pDCs, but do not induce the secretion of anti-inflammatory IL-10. When IRF8 and SPIB are combined with BCL11A, CBFA2T3, CREB3L2, ETS1, STAT1, TCF4, TCF12, or TSC22D1, interferon secretion is further increased. Induced DCs generated using the transcription factors PU.1, IRF8, and BATF3 do not exhibit the ability to secrete type I interferons.

[0031] In one embodiment, IRF8 and SPIB can induce the ability to secrete proinflammatory cytokines (particularly IL-6 and TNF-α) after stimulation with TLR7 and TLR9 ligands, a phenotypic characteristic of pDCs. Combining IRF8 and SPIB with ETS1, STAT1, TCF12, or TSC22D1 further increases cytokine secretion.

[0032] In one embodiment, IRF8 and SPIB can induce a phenotypic characteristic of pDCs, i.e., the ability to secrete chemokines, i.e., CCL5 and CXCL10, after stimulation with TLR7 and TLR9 ligands. Combining IRF8 and SPIB with HOXA5, STAT1, TCF12, or TSC22D1 further increases chemokine secretion.

[0033] One aspect of the present subject matter relates to compositions comprising a combination of at least two transcription factors, wherein the at least two transcription factors are IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4), ARID5A (SEQ ID NO:5, SEQ ID NO:6), BCL11A (SEQ ID NO:7, SEQ ID NO:8), CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10), CREB3L2 (SEQ ID NO:11, SEQ ID NO:12), ETS1 (SEQ ID NO:13, SEQ ID NO:14), IKZF1 (SEQ ID NO:23, SEQ ID NO:24), STAT1 (SEQ ID NO:45, SEQ ID NO:46), TCF4 (SEQ ID NO:47, SEQ ID NO:48), TCF5 (SEQ ID NO:49, SEQ ID NO:50), TCF6 (SEQ ID NO:51, SEQ ID NO:52), TCF7 (SEQ ID NO:53, SEQ ID NO:54), TCF8 (SEQ ID NO:55, SEQ ID NO:56), TCF9 (SEQ ID NO:57, SEQ ID NO:58), TCF9 (SEQ ID NO:59, SEQ ID NO:60), TCF9 (SEQ ID NO:61, SEQ ID NO:62), TCF9 (SEQ ID NO:63, SEQ ID NO:64), TCF9 (SEQ ID NO:65, SEQ ID NO:66), TCF9 (SEQ ID NO:67, SEQ ID NO:68), TCF9 (SEQ ID NO:69, SEQ ID NO:70), TCF9 (SEQ ID NO:71, SEQ ID NO:72), TCF9 (SEQ ID NO:73, SEQ ID NO:74), TCF9 (SEQ ID NO:75, SEQ ID NO:76), T 2), TCF12 (SEQ ID NO:43, SEQ ID NO:44), TSC22D1 (SEQ ID NO:47, SEQ ID NO:48), and mixtures thereof; the at least two transcription factors are encoded by isolated or synthetic sequences that show at least 90% identity to any one sequence of a transcription factor selected from the list consisting of TCF12 (SEQ ID NO:43, SEQ ID NO:44), TSC22D1 (SEQ ID NO:47, SEQ ID NO:48), and mixtures thereof; the use of the at least two transcription factors is to reprogram stem cells or differentiated cells, or mixtures thereof, into plasmacytoid dendritic cells or interferon-producing cells or antigen-presenting cells, preferably into plasmacytoid dendritic cells or type I interferon-producing cells in a subject.

[0034] One aspect of the present subject matter relates to a composition comprising at least two transcription factors, IRF8 and SPIB, for use in reprogramming stem cells or differentiated cells, or a mixture thereof, into plasmacytoid dendritic cells or type I interferon-producing cells in a subject.

[0035] In one embodiment, the composition of the present disclosure comprises at least two transcription factors as described herein, which may be isolated or synthetic transcription factors, provided that the combination of at least two transcription factors is not TCF4 (SEQ ID NO:41, SEQ ID NO:42) and IRF8 (SEQ ID NO:1, SEQ ID NO:2).

[0036] In one embodiment, the composition of the present disclosure further comprises one or more transcription factors selected from the group consisting of ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, STAT1, TCF4, TCF12 and TSC22D1, preferably BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, TCF4, TCF12 and TSC22D1.

[0037] In one embodiment, a composition of the present disclosure may comprise a combination of at least two transcription factors, wherein the at least two transcription factors are: IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4), ARID5A (SEQ ID NO:5, SEQ ID NO:6), BCL11A (SEQ ID NO:7, SEQ ID NO:8), CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10), CREB3L2 (SEQ ID NO:11, SEQ ID NO:12), ETS1 (SEQ ID NO:13, SEQ ID NO:14), IKZF1 (SEQ ID NO:23), IRF8 (SEQ ID NO:14), SPIB (SEQ ID NO:15), ARID5A (SEQ ID NO:16), BCL11A (SEQ ID NO:17), CBFA2T3 (SEQ ID NO:18), CREB3L2 (SEQ ID NO:19), CREB3L3 (SEQ ID NO:24), CREB3L4 (SEQ ID NO:25), CREB3L5 (SEQ ID NO:26), CREB3L6 (SEQ ID NO:27), CREB3L7 (SEQ ID NO:28), CREB3L8 (SEQ ID NO:29), CREB3L9 (SEQ ID NO:30), CREB3L10 (SEQ ID NO:31), CREB3L11 (SEQ ID NO:32), CREB3L12 (SEQ ID NO:33), CREB3L13 (SEQ ID NO:34), CREB3L14 (SEQ ID NO:35), CREB3L15 (SEQ ID NO:36), CREB3L16 (SEQ ID NO:37), CREB3L17 (SEQ ID NO:38), CREB3L18 (SEQ ID NO: , SEQ ID NO: 24), STAT1 (SEQ ID NO: 45, SEQ ID NO: 46), TCF4 (SEQ ID NO: 41, SEQ ID NO: 42), TCF12 (SEQ ID NO: 43, SEQ ID NO: 44), TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48), and mixtures thereof, and is encoded by an isolated or synthetic sequence that exhibits at least 95% identity, preferably 96%, 97%, 98% identity; 99% identity, or even complete identity, to the sequence of any one of the following transcription factors selected from the group consisting of: TCF4 (SEQ ID NO: 41, SEQ ID NO: 42), TCF12 (SEQ ID NO: 43, SEQ ID NO: 44), TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48), and mixtures thereof.

[0038] In one embodiment of the present disclosure, the transcription factors are: IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4), ARID5A (SEQ ID NO:5, SEQ ID NO:6), BCL11A (SEQ ID NO:7, SEQ ID NO:8), CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10), CREB3L2 (SEQ ID NO:11, SEQ ID NO:12), ETS1 (SEQ ID NO:13, SEQ ID NO:14), IKZF1 (SEQ ID NO:23, SEQ ID NO:24), ), STAT1 (SEQ ID NO:45, SEQ ID NO:46), TCF4 (SEQ ID NO:41, SEQ ID NO:42), TCF12 (SEQ ID NO:X, SEQ ID NO:X), TSC22D1 (SEQ ID NO:47, SEQ ID NO:48), and mixtures thereof, and preferably exhibits 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, or complete identity to a sequence selected from the group consisting of: TCF4 (SEQ ID NO:41, SEQ ID NO:42), TCF12 (SEQ ID NO:X, SEQ ID NO:X), TSC22D1 (SEQ ID NO:47, SEQ ID NO:48), and mixtures thereof.

[0039] In one embodiment, the compositions of the present disclosure may include a combination of transcription factors selected from the following combinations: IRF8 (SEQ ID NO:1, SEQ ID NO:2) and SPIB (SEQ ID NO:3, SEQ ID NO:4); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and ARID5A (SEQ ID NO:5, SEQ ID NO:6); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:5, SEQ ID NO:6) and BCL11A (SEQ ID NO:7, SEQ ID NO:8); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and CREB3L2 (SEQ ID NO:11, SEQ ID NO:12); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and ETS1 (SEQ ID NO:13, SEQ ID NO:14); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and IKZF1 (SEQ ID NO:23, SEQ ID NO:24); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and STAT1 (SEQ ID NO:45, SEQ ID NO:46); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and TCF4 (SEQ ID NO:41, SEQ ID NO:42); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and TCF12 (SEQ ID NO:43, SEQ ID NO:44); IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4) and TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48), and mixtures thereof.

[0040] In one embodiment, the compositions of the present disclosure may include a combination of transcription factors selected from the following combinations: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; IRF8, SPIB and TSC22D1; and mixtures thereof.

[0041] In one embodiment, the composition of the present disclosure may comprise a combination of transcription factors, wherein the combination of transcription factors is IRF8 (SEQ ID NO:1, SEQ ID NO:2) and SPIB (SEQ ID NO:3, SEQ ID NO:4) or IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and TCF12 (SEQ ID NO:43, SEQ ID NO:44).

[0042] In one embodiment, a composition of the present disclosure may comprise a combination of transcription factors, wherein the combination of transcription factors is IRF8, SPIB, and TCF12.

[0043] In one embodiment, a composition of the present disclosure may comprise a combination of transcription factors, wherein the combination of transcription factors is IRF8, SPIB, and TSC22D1.

[0044] In one embodiment, the compositions of the present disclosure may comprise pluripotent or multipotent stem cells, differentiated cells, and mixtures thereof.

[0045] In one embodiment, the compositions of the present disclosure may be utilized as reprogramming or inducing factors to convert cells selected from the group consisting of tumor cells, cancer cells, and mixtures thereof, into type I interferon-producing cells.

[0046] In one embodiment, the compositions of the present disclosure may be used as pDC reprogramming or inducers, where the antigen is a cancer antigen, an autoantigen, an allergen, an antigen derived from a pathogen and / or an infectious organism.

[0047] In one embodiment, cells obtained using the disclosed combination of transcription factors have the ability to respond to stimulation by TLR7 and TLR9, and / or the cells have the ability to secrete cytokines that are type I interferons (i.e., IFN-α and IFN-β).

[0048] Another aspect of the present disclosure relates to the use of the compositions of the present disclosure in veterinary or human medicine, particularly in immunotherapy, or in the treatment of neurodegenerative or geriatric diseases, or cancer or infectious diseases, or drug screening.

[0049] Another aspect of the present disclosure relates to the use of the compositions of the present disclosure in the diagnosis, therapy or treatment of cancer, antiviral immune responses and immunopathologies, particularly autoimmune diseases and immunodeficiency conditions.

[0050] In one embodiment, the pluripotent, multipotent or differentiated cells are mammalian pluripotent, multipotent or differentiated cells, in particular murine or human cells.

[0051] In one embodiment, the compositions of the present disclosure may be used to treat, cure or diagnose an infectious, viral or virus-induced disease.

[0052] In one embodiment, the compositions of the present disclosure may be used as antiviral or immunogenic compositions.

[0053] Another aspect of the present disclosure relates to constructs or vectors encoding a combination of at least two isolated transcription factors as described herein, preferably a combination of three encoded transcription factors, and more preferably a combination of four transcription factors.

[0054] In one embodiment, the combination of at least two isolated transcription factors is in the following order from 5' to 3': SPIB (SEQ ID NO:3, SEQ ID NO:4) and IRF8 (SEQ ID NO:1, SEQ ID NO:2); IRF8 (SEQ ID NO:1, SEQ ID NO:2) and SPIB (SEQ ID NO:3, SEQ ID NO:4); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and ARID5A (SEQ ID NO:5, SEQ ID NO:6); SPIB (SEQ ID NO:5, SEQ ID NO:6), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and BCL11A (SEQ ID NO:7, SEQ ID NO:8); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and CREB3L2 (SEQ ID NO:11, SEQ ID NO:12); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and ETS1 (SEQ ID NO:13, SEQ ID NO:14); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and IKZF1 (SEQ ID NO:23, SEQ ID NO:24); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and STAT1 (SEQ ID NO:45, SEQ ID NO:46); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TCF4 (SEQ ID NO:41, SEQ ID NO:42); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TCF12 (SEQ ID NO:43, SEQ ID NO:44); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TSC22D1 (SEQ ID NO:47, SEQ ID NO:48).

[0055] Another aspect of the present disclosure relates to a construct or vector encoding a combination of at least two, but preferably three, transcription factors as described herein, wherein the combination of at least two isolated transcription factors is in the following 5' to 3' order: SPIB (SEQ ID NO:3, SEQ ID NO:4) and IRF8 (SEQ ID NO:1, SEQ ID NO:2); IRF8 (SEQ ID NO:1, SEQ ID NO:2) and SPIB (SEQ ID NO:3, SEQ ID NO:4); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and ARID5A (SEQ ID NO:5, SEQ ID NO:6); SPIB (SEQ ID NO:5, SEQ ID NO:6), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and BCL11A (SEQ ID NO:7, SEQ ID NO:8); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and CREB3L2 (SEQ ID NO:11, SEQ ID NO:12); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and ETS1 (SEQ ID NO:13, SEQ ID NO:14); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and IKZF1 (SEQ ID NO:23, SEQ ID NO:24); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and STAT1 (SEQ ID NO:45, SEQ ID NO:46); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TCF4 (SEQ ID NO:41, SEQ ID NO:42); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TCF12 (SEQ ID NO:43, SEQ ID NO:44); SPIB (SEQ ID NO:3, SEQ ID NO:4), IRF8 (SEQ ID NO:1, SEQ ID NO:2) and TSC22D1 (SEQ ID NO:47, SEQ ID NO:48).

[0056] In one embodiment, the vector is a viral vector; in particular a retroviral, adenoviral, lentiviral, herpesviral, poxviral, paramyxoviridae, rhabdoviral, alphaviral, flaviviral or adeno-associated viral vector.

[0057] In one embodiment, the vector or construct is a synthetic mRNA, a naked alphavirus RNA replicon, or a naked flavivirus RNA replicon.

[0058] In one aspect of the disclosure, the one or more vectors comprise at least two polynucleotide sequences encoding at least two transcription factors, IRF and SPIB, and are for use in reprogramming stem cells or differentiated cells, or a mixture thereof, into plasmacytoid dendritic cells or type I interferon-producing cells in a subject.

[0059] In one embodiment of the present disclosure, the one or more vectors further encode one or more transcription factors selected from the group consisting of ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, STAT1, TCF4, TCF12 and TSC22D1, preferably BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, TCF4, TCF12 and TSC22D1.

[0060] In one embodiment of the present disclosure, the transcription factors of the one or more vectors each exhibit at least 90% identity to a sequence selected from the group consisting of IRF8 (SEQ ID NO: 49, SEQ ID NO: 50), SPIB (SEQ ID NO: 51, SEQ ID NO: 52), ARID5A (SEQ ID NO: 53, SEQ ID NO: 54), BCL11A (SEQ ID NO: 55, SEQ ID NO: 56), CBFA2T3 (SEQ ID NO: 57, SEQ ID NO: 58), CREB3L2 (SEQ ID NO: 59, SEQ ID NO: 60), ETS1 (SEQ ID NO: 61, SEQ ID NO: 62), IKZF1 (SEQ ID NO: 71, SEQ ID NO: 72), TCF4 (SEQ ID NO: 89, SEQ ID NO: 90), TCF12 (SEQ ID NO: 91, SEQ ID NO: 92), STAT1 (SEQ ID NO: 93, SEQ ID NO: 94), and TSC22D1 (SEQ ID NO: 95, SEQ ID NO: 96).

[0061] In one embodiment of the present disclosure, the combination of transcription factors in the one or more vectors is selected from the following combinations: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; and IRF8, SPIB and TSC22D1.

[0062] In one embodiment of the present disclosure, the combination of transcription factors in the one or more vectors is a combination of IRF8, SPIB, and TCF12.

[0063] In one embodiment of the present disclosure, the combination of transcription factors in the one or more vectors is a combination of IRF8, SPIB, and TSC22D1.

[0064] In one embodiment of the present disclosure, the cells utilized for reprogramming with the one or more vectors are selected from the group consisting of pluripotent stem cells, pluripotent stem cells, differentiated cells, tumor cells, cancer cells, and mixtures thereof.

[0065] In one embodiment of the disclosure, the one or more vectors find use in veterinary or human medicine, particularly in the diagnosis, therapy or treatment of antiviral immune responses and immunopathologies, particularly autoimmune diseases and immunodeficiency conditions, neoplasia, particularly cancer, i.e. solid tumors or hematologic tumors, benign tumors, malignant tumors, early cancers, basal cell carcinoma, cervical dysplasia, soft tissue sarcomas, germ cell tumors, retinoblastoma, age-related macular degeneration, Hodgkin's lymphoma, blood cancers, prostate cancer. , ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, breast cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, hollow organ cancer, esophageal cancer, stomach cancer, bile duct cancer, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gallbladder cancer, spleen cancer, brain tumor, lymphatic system cancer, osteosarcoma, pancreatic cancer, leukemia, skin cancer, or myeloma, infectious diseases, viral diseases or virus-induced diseases, disorders of the central and peripheral nervous system, fungi, viruses, chlamydia, bacteria, nanobacteria or for use in the diagnosis, therapy or treatment of a parasitic infectious disease, or a viral or virus-induced disease; for use as an antiviral agent or as an immunogenic composition, for use in immunotherapy, or for use in the treatment or therapy of a neurodegenerative disease; for use in the treatment or therapy of cancer; or for use in the treatment or therapy of an infectious disease, intradermal and transdermal therapy, HIV, SARS coronavirus infection, Asian influenza virus, herpes simplex, influenza virus, shingles, hepatitis, human papillomavirus, Epstein-Barr virus, human T-lymphotrophic virus or viral hepatitis, amyloid diseases, in particular amyloid A amyloidosis, Alzheimer's disease, light chain (AL) amyloidosis, type 2 diabetes, medullary thyroid carcinoma, Parkinson's disease, polyneuropathy, or spongiform encephalopathy-Creutzfeldt-Jakob disease, or for use in drug screening.

[0066] Another aspect of the present disclosure relates to a method of reprogramming or inducing cells to convert into plasmacytoid dendritic cells or type I interferon-producing cells, the method comprising the steps of: transducing cells selected from the group consisting of stem cells or differentiated cells, and mixtures thereof, with one or more vectors; wherein the one or more vectors are one or more vectors comprising at least two nucleic acid sequences encoding sequences exhibiting at least 90% identity, preferably at least 95% identity, to a sequence selected from the group consisting of IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4), ARID5A (SEQ ID NO: 5, SEQ ID NO: 6), BCL11A (SEQ ID NO: 7, SEQ ID NO: 8), CBFA2T3 (SEQ ID NO: 9, SEQ ID NO: 10), CREB3L2 (SEQ ID NO: 11, SEQ ID NO: 12), ETS1 (SEQ ID NO: 13, SEQ ID NO: 14), IKZF1 (SEQ ID NO: 23, SEQ ID NO: 24), TCF4 (SEQ ID NO: 41, SEQ ID NO: 42), TCF12 (SEQ ID NO: 43, SEQ ID NO: 44), STAT1 (SEQ ID NO: 45, SEQ ID NO: 46), TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48), and mixtures thereof; and Culturing the transduced cells in a cell culture medium that supports the growth of plasmacytoid dendritic cells or type I interferon-producing cells.

[0067] Another aspect of the present disclosure relates to an in vitro method for reprogramming or inducing cells to convert into plasmacytoid dendritic cells or type I interferon-producing cells, the method comprising the steps of: transducing cells selected from the group consisting of stem cells or differentiated cells, and mixtures thereof, with one or more vectors; wherein the one or more vectors comprise at least two transcription factors, IRF8 and SPIB; and Culturing the transduced cells in a cell culture medium that supports the growth of plasmacytoid dendritic cells or type I interferon-producing cells.

[0068] In one embodiment, the sequence is: IRF8 (SEQ ID NO:1, SEQ ID NO:2) and SPIB (SEQ ID NO:3, SEQ ID NO:4); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and ARID5A (SEQ ID NO:5, SEQ ID NO:6); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:5, SEQ ID NO:6) and BCL11A (SEQ ID NO:7, SEQ ID NO:8); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and CREB3L2 (SEQ ID NO:11, SEQ ID NO:12); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and ETS1 (SEQ ID NO:13, SEQ ID NO:14); IRF8 (SEQ ID NO:1, SEQ ID NO:2, SPIB (SEQ ID NO:3, SEQ ID NO:4) and IKZF1 (SEQ ID NO:23, SEQ ID NO:24); IRF8 (SEQ ID NO:1, SEQ ID NO:2, SPIB (SEQ ID NO:3, SEQ ID NO:4) and STAT1 (SEQ ID NO:45, SEQ ID NO:46); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and TCF4 (SEQ ID NO:41, SEQ ID NO:42); IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4) and TCF12 (SEQ ID NO:43, SEQ ID NO:44); The antibody is selected from the group consisting of IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4) and TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48) and combinations thereof.

[0069] In one embodiment, the sequence is: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; The combinations are selected from the group consisting of IRF8, SPIB and TSC22D1, and mixtures thereof.

[0070] In one embodiment, the method may comprise culturing the transduced cells for at least 2 days, preferably at least 5 days, more preferably at least 8 days, and even more preferably at least 9 days.

[0071] In one embodiment, the transduction step further comprises at least one vector selected from the group consisting of a nucleic acid sequence encoding IL-12; a nucleic acid sequence encoding IL-4; a nucleic acid sequence encoding IFN-α; a nucleic acid sequence encoding IFN-β; a nucleic acid sequence encoding IFN-γ; a nucleic acid sequence encoding TNF; a nucleic acid sequence encoding GM-CSF; a nucleic acid sequence encoding an siRNA targeting IL-10 RNA, and mixtures thereof.

[0072] In one embodiment, the transduction step further comprises at least one vector comprising a nucleic acid encoding an immunostimulatory cytokine.

[0073] In one embodiment, the cells are selected from the group consisting of pluripotent stem cells, pluripotent stem cells, or differentiated cells, and mixtures thereof; in particular, mammalian cells.

[0074] In one embodiment, the pluripotent stem cells, multipotent stem cells, or differentiated cells are selected from the group consisting of endoderm-derived cells, mesoderm-derived cells, ectoderm-derived cells, pluripotent stem cells (including mesenchymal stem cells, hematopoietic stem cells, intestinal stem cells), pluripotent stem cells, and cell lines.

[0075] In one embodiment, the cell is human or non-human, hi another embodiment, the cell is a mouse cell.

[0076] Another aspect of the present disclosure relates to induced plasmacytoid dendritic cells or induced type I interferon-producing cells, or antigen-presenting cells, preferably induced plasmacytoid dendritic cells or inducible type I interferon-producing cells, obtained by the in vitro method described in the present disclosure.

[0077] Another aspect of the present disclosure relates to a composition comprising a therapeutically effective amount of plasmacytoid dendritic cells, or type I interferon-producing cells, or antigen-presenting cells, preferably induced plasmacytoid dendritic cells or induced type I interferon-producing cells, as described herein, and a pharmaceutically acceptable excipient.

[0078] In one embodiment, the composition may be for use in veterinary or human medicine.

[0079] In one embodiment, the composition may be for use in immunotherapy, or in the treatment or therapy of a neurodegenerative disease, or in the treatment or therapy of a cancer, or in the treatment or therapy of an infectious disease.

[0080] In one embodiment, the composition may further comprise an analgesic, an anti-inflammatory, a chemotherapeutic, a radiotherapeutic, an antibiotic, a diuretic, or a mixture thereof.

[0081] In one embodiment, the composition may further comprise a filler, binder, disintegrant, or lubricant, or a mixture thereof.

[0082] In one embodiment, the composition may be for use in intradermal and transdermal therapy.

[0083] In one embodiment, the composition may be in an injectable form, preferably an in-situ injection.

[0084] In one embodiment, the compositions may be for use in veterinary or human medicine or in drug screening.

[0085] In one embodiment, the composition may be for use in the treatment, therapy or diagnosis of central and peripheral nervous system disorders.

[0086] In one embodiment, the composition may be for use in the treatment, therapy or diagnosis of neoplasia, particularly cancer (such as a solid tumor or a hematological tumor).

[0087] In one embodiment, the composition, vector, or construct may be for use in the treatment, diagnosis, or therapy of benign tumors, malignant tumors, early stage cancers, basal cell carcinoma, cervical dysplasia, soft tissue sarcoma, germ cell tumors, retinoblastoma, age-related macular degeneration, Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, breast cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, hollow organ cancer, esophageal cancer, gastric cancer, bile duct cancer, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gallbladder cancer, spleen cancer, brain tumor, lymphatic system cancer, osteosarcoma, pancreatic cancer, leukemia, skin cancer, or myeloma.

[0088] In one embodiment, the composition, vector or construct may be for use in the treatment, therapy or diagnosis of a fungal, viral, chlamydial, bacterial, nanobacterial or parasitic infectious disease, or a viral disease, or a virus-induced disease.

[0089] In one embodiment, the composition, vector, or construct may be for use in the treatment, therapy, or diagnosis of HIV, SARS coronavirus infection, Asian influenza virus, herpes simplex, influenza virus, shingles, hepatitis, human papillomavirus, Epstein-Barr virus, human T-lymphotropic virus, or viral hepatitis.

[0090] In one embodiment, the composition, vector, or construct may be for use in the treatment, therapy, or diagnosis of an amyloid disease, in particular amyloid A amyloidosis, Alzheimer's disease, light chain (AL) amyloidosis, type 2 diabetes, medullary thyroid carcinoma, Parkinson's disease, polyneuropathy, or spongiform encephalopathy (such as Creutzfeldt-Jakob disease).

[0091] Another aspect of the present disclosure relates to a vaccine or injectable dosage form comprising a composition, vector, or construct described in the present disclosure, and / or induced plasmacytoid dendritic cells as described or induced type I interferon-producing cells or antigen-presenting cells described in the present disclosure, or a mixture thereof.

[0092] Another aspect of the present disclosure relates to a kit comprising at least one of the following elements: Induced plasmacytoid dendritic cells or inducible type I interferon-producing cells or antigen-presenting cells of the present disclosure; a composition as described herein; One or more vectors or constructs as described in this disclosure; or a mixture thereof.

[0093] Another aspect of the present disclosure relates to a kit comprising at least one of the following elements: Induced plasmacytoid dendritic cells or inducible type I interferon-producing cells of the present disclosure; a composition as described herein; One or more vectors or constructs as described in this disclosure; or a mixture thereof.

[0094] In one embodiment, this disclosure provides evidence that IRF8, when combined with SPIB, initiates the pDC program in fibroblasts. These findings provide valuable insights into pDC specification. In the future, generating pDCs by direct reprogramming will pave the way for the induction of antiviral immune responses by artificial autologous cells.

[0095] The following figures provide preferred embodiments that specifically illustrate the present disclosure, but should not be considered as limiting the scope of the invention. [Brief explanation of the drawings]

[0096] [Figure 1] Ontogeny of major dendritic cell subsets. Conventional dendritic cell types 1 (cDC1) and 2 (cDC2) arise from a population of conventional dendritic cell precursors (pre-DCs) derived from common dendritic cell precursors (CDPs), whereas plasmacytoid dendritic cells (pDCs) can be derived from either common lymphoid precursors (CLPs) or CDPs. pDCs emerge as a unique subset of DCs specialized for type I interferon (IFN) production after viral infection. cDC1s are highly capable of cross-presentation, induce cytotoxic T cell responses, and preferentially polarize T helper 1 responses. cDC2s are highly capable of antigen presentation to T helper cells and polarize T helper 2 or 17 responses. [Figure 2] Generation of pDCs or type I interferon (IFN)-producing cells by direct cell reprogramming. Findings on the effectiveness of combinations of transcription factors (TFs) disclosed in the present subject matter to induce pDCs from pluripotent stem cells, pluripotent stem cells, and somatic cells (such as fibroblasts). Because pDCs specialize in producing type I IFN, they will find application in the context of personalized immunotherapy. Induced pDCs or combinations of pDC-inducing transcription factors will be utilized to induce immunity in defined pathological conditions, including viral infections and cancer. [Figure 3]Expression of Clec9a, Irf8, TLR7, and TLR9 is restricted to pDCs. Heatmap showing the expression levels of Clec9a, Irf8, TLR7, and TLR9 in pDCs and other ontogenetically related immune cells, specifically cDC1 and cDC2, macrophages, and monocytes. Expression profiles were obtained from data available in the Immunological Genome Project (www.immgen.com). [Figure 4] Candidate transcription factors inducing pDC. 22 candidate transcription factors are highly expressed in pDC compared to cDC1 and cDC2. Heatmap showing gene expression profiles obtained from data available in the Immunological Genome Project. [Figure 5] Clec9a expression in pDCs is shown. 92% of pDCs (gated as CD11clow BST2+ B220+ Siglec-H+ cells) isolated from the spleen of double transgenic Clec9a-Cre x Rosa26-STOP-tdTomato mice expressed tdTomato. [Figure 6] IRF8 in combination with SPIB induces Clec9a reporter activation. Mouse embryonic fibroblasts (MEFs) isolated from double-transgenic Clec9a-tdTomato mice were transduced with a pool of inducible lentiviral vectors encoding IRF8 alone or in combination with other pDC-inducing transcription factors, and flow cytometry analysis was performed 5 days after Dox addition. Quantification of tdTomato+ cells after transduction with IRF8 alone or in combination with individual pDC-inducing candidate transcription factors. M2rtTA-transduced cells are also included as a control. [Figure 7] Induced pDCs express antigen-presenting molecules on their cell surface. Flow cytometry analysis of major histocompatibility complex class II (MHC-II) expression in tdTomato+ cells generated on day 8 after overexpression of IRF8 and SPIB. [Figure 8] Combining IRF8 and SPIB with other pDC-inducing transcription factors increases the efficiency of Clec9a reporter activation. Double-transgenic Clec9a-tdTomato MEFs were transduced with IRF8 and SPIB alone or in combination with individual pDC-inducing factors, and tdTomato+ cells were quantified. Flow cytometry analysis was performed on day 8. M2rtTa-transduced cells were also included as a control. [Figure 9] Combining IRF8 and SPIB with other pDC-inducing transcription factors increases MHC-II expression on the cell surface. Flow cytometry analysis of MHC-II expression on day 8 of MEFs transduced with M2rtTA, IRF8 + SPIB alone, or IRF8 + SPIB in combination with individual pDC-inducing transcription factors. Quantification of MHC-II levels is shown for tdTomato-negative (tdT-) and -positive (tdT+) populations. [Figure 10] Upon stimulation with TLR7 and TLR9, induced pDCs secrete type I interferons. MEFs were transduced with M2rtTA, PIB (PU.1, IRF8, and BATF3), IRF8 and SPIB alone, or IRF8 and SPIB in combination with ARID5A, BCL11A, CBFA2T3, ETS1, HOXA5, TCF4, and TCF12, and tdTomato+ cells were sorted on day 9 after doxycycline addition. Purified tdTomato+ cells were incubated overnight with TLR7 (R848) and TLR9 (CpG ODN1668), and cytokine secretion in the supernatant was quantified using cytometric bead arrays for IFN-α (A), IFN-β (B), and IL-10 (C). [Figure 11] Additional candidate transcription factors inducing pDC. Expression levels of Stat1 (A) and Tsc22d1 (B) are higher in pDC compared to cDC1 and cDC2. Gene expression profile derived from data available in the Immunological Genome Project. [Figure 12]Combining IRF8 and SPIB with other pDC-inducing transcription factors increases the efficiency of Clec9a reporter activation and MHC-II surface expression. Flow cytometry analysis of (A) Clec9a reporter activation and (B) MHC-II expression was performed on day 5 in MEFs transduced with M2rtTA (M2), IRF8 + SPIB alone, or IRF8 + SPIB in combination with STAT1 or TSC22D1. M2rtTa-transduced cells were also included as a control. [Figure 13] Upon stimulation with TLR7 and TLR9, induced pDCs secrete type I interferons. MEFs were transduced with M2rtTA, IRF8, and SPIB alone, or IRF8 and SPIB in combination with STAT1 and TSC22D1, and tdTomato+ cells were sorted on day 9 after doxycycline addition. Purified tdTomato+ cells were incubated overnight with TLR7 (R848) and TLR9 (CpG ODN1668) as triggers, and cytokine secretion was quantified in the supernatants for IFN-α (A), IFN-β (B), and IL-10 (C) using cytometric bead arrays. [Figure 14] Induced pDCs express CD45 on the cell surface. Flow cytometry analysis of CD45 expression on day 5 in MEFs transduced with M2rtTA, PIB (PU.1, IRF8, and BATF3), IRF8 + SPIB alone, or IRF8 + SPIB in combination with individual pDC-inducing transcription factors. M2rtTa (M2)-transduced cells were also included as a control. [Figure 15]Upon stimulation with TLR7 and TLR9, induced pDCs secrete proinflammatory cytokines and chemokines. MEFs were transduced with M2rtTA, IRF8, and SPIB alone, or IRF8 and SPIB in combination with ARID4A, BCL11A, CBFA2T3, ETS1, HOXA5, STAT1, TCF4, TCF12, and TSC22D1, and tdTomato+ cells were sorted on day 9 after doxycycline treatment. Purified tdTomato+ cells were incubated overnight with TLR7 (R848) and TLR9 (CpG ODN1668), and cytokine secretion in the supernatant was quantified using cytometric bead arrays for IL-6 (A), TNF-α (B), CCL5 (C), and CXCL10 (D). DETAILED DESCRIPTION OF THE INVENTION

[0097] The present disclosure relates to compositions, nucleic acid constructs, vectors, methods, and kits for reprogramming cells into pDCs or type I interferon-producing cells. The objective is to generate pDCs and type I interferon-producing cells capable of inducing antiviral and antitumor responses from differentiated cells, pluripotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthesized transcription factors. More specifically, the present disclosure provides methods for obtaining pDCs or type I interferon-producing cells using direct cell reprogramming through the unexpected use of specific combinations of transcription factors.

[0098] Natural DCs are bone marrow-derived cells that disseminate throughout all tissues. DCs are constantly on the lookout for information about their environment and relay it to cells of the adaptive immune system (T cells and B cells). After phagocytosing antigens, DCs initiate immune responses by presenting processed antigens (in the form of peptide-major histocompatibility complex (MHC) molecule complexes) to naive (i.e., antigen-naive) T cells in lymphoid tissues. DCs can be divided into two major subsets: plasmacytoid dendritic cells (pDCs) and conventional or classical dendritic cell DCs (cDCs). cDCs can be further divided into type 1 cDCs (cDC1) and type 2 cDCs (cDC2) (Figure 1). Typical characteristics of pDCs are their round morphology and low levels of MHC-II expression, which is upregulated after activation. After activation, pDCs secrete various cytokines and chemokines, namely, tumor necrosis factor (TNF)-α, IFN-α, IFN-β, IL-12, IL-6, and chemokines (e.g., CCL5 and CXCL10), which initiate and / or promote multiple T and B lymphocyte responses. In addition, pDCs typically overexpress costimulatory molecules and MHC molecules. pDCs express high levels of the nucleic acid-sensing Toll-like receptors (TLRs), TLR7 and TLR9. Upon TLR7 or TLR9 stimulation, pDCs can detect pathogen-derived nucleic acids and produce large amounts of type I interferons (i.e., IFN-α and IFN-β). Activated pDCs actively produce cytokines and are capable of both antigen presentation and cross-presentation; however, it is currently unclear whether these functional characteristics are the result of the same cell or distinct, functionally specialized pDC subsets. cDCs are generally identified based on their high expression of major histocompatibility complex class II molecules (MHC-II), costimulatory molecules (such as CD80 / 86 and CD40), and the integrin CD11c, as well as their superior ability to migrate from non-lymphoid to lymphoid organs and stimulate naive T cells.DNGR-1 (also known as CLEC9A) is a receptor for necrotic cells that supports cross-priming of cytotoxic T lymphocyte responses (CTLs) against dead cell-associated antigens in mice. DNGR-1 is selectively and highly expressed in mouse cDC1, cDC2, and pDC, where it recognizes intracellular ligands that are released after cell death. Recently, it has been shown that DC precursors (CDPs) destined for conventional or plasmacytoid DC lineages and their progeny in lymphoid tissues can be distinguished based on Clec9a expression (Schraml et al., 2015).

[0099] As described herein, the successful identification of pDC-inducing factors capable of reprogramming differentiated cells into induced pDCs would advance our fundamental understanding of pDC biology in many ways. This work would provide detailed insight into the minimal transcriptional network of pDCs. Furthermore, identifying pDC-inducing factors would provide an unprecedented opportunity to understand how the pDC state is established and how key regulatory mechanisms are realized.

[0100] Transcription factors (TFs) play a critical role in the specification of any developing cell type. The success of direct reprogramming strategies using TF-mediated reprogramming suggests that it is equally reasonable to use such factors to induce the differentiation of pluripotent ES / iPS cells or pluripotent stem cells toward specific fate. Thus, using the pDC inducer factors identified herein, it is possible to induce the differentiation of ES / iPS cells toward specific DC fates by expressing transcription factors that are highly present in DCs. Furthermore, using the pDC inducer factors identified herein, it is possible to induce the differentiation of pluripotent hematopoietic stem and progenitor cells toward specific DC fates by expressing transcription factors that are highly present in DCs.

[0101] Typically, nucleic acids (e.g., DNA or RNA) encoding pDC-inducing factors or constructs thereof are introduced into cells by single or repeated transduction or transformation, with or without the use of viral vectors, and as described herein, cells that resemble pDCs morphologically, biochemically, and functionally are obtained through expression of the gene product and / or translation of the RNA molecule. After priming with an appropriate antigen, these induced pDCs are able to capture, process, and present the antigen to effector cells of the immune system (macrophages, T cells, B cells, and NK cells) and secrete type I IFNs to elicit antigen-specific immune responses against viral infections and cancers (Figure 2).

[0102] One aspect of the present disclosure is the use of transcription factors, or combinations of transcription factors, in cancer cells (in situ or ex vivo) to force cancer cells to secrete type I IFN, present cancer cell antigens to immune cells, and secrete other inflammatory cytokines and chemoattractants within the tumor microenvironment. This method circumvents cancer escape mechanisms and enhances tumor immunogenicity, making it a viable strategy for improving clinical outcomes of anti-cancer immunotherapy.

[0103] Compared with cDC1 and cDC2 and other related monocytes, plasmacytoid DCs specifically express TLR7 and TLR9 (Figure 3). The TLR7 and TLR9 receptors enable pDCs to sense viral DNA and respond by secreting type I IFN. Plasmacytoid DCs and cDC1s express Clec9a and Irf8.

[0104] In one embodiment, 22 candidate transcription factors were selected based on the higher expression levels of specific genes in plasmacytoid DCs compared to cDC1 and cDC2 (Figure 4). Each of the 22 candidate transcription factors was individually cloned into a doxycycline (Dox)-inducible lentiviral vector that demonstrated reprogramming.

[0105] In one embodiment, to screen for the effects of pDC-inducing transcription factors and combinations of pDC-inducing transcription factors on cell reprogramming, we used mouse embryonic fibroblasts (MEFs) containing a DC-specific reporter (Clec9a-Cre x R26-stop-tdTomato) to screen for the ability of pDC-inducing transcription factors to activate the DC-specific Clec9a-tomato reporter (Figure 4). In Clec9a-tomato reporter mice, tdTomato fluorescent protein was expressed exclusively in CDPs, pre-DCs, cDCs, and pDCs (Schraml et al., 2015). Macrophages, other immune lineages, or monocyte-derived DCs in culture do not express Clec9a and therefore do not activate reporter or tdTomato protein expression (Figure 3). Splenocytes isolated from Clec9a reporter mice were analyzed to determine the expression of pDC cells (CD11c). low BST2 + B220 + SiglecH + We confirmed that 92.4% of the cells (gated on the 3D cells) expressed tdTomato fluorescent protein (Figure 5).

[0106] Double transgenic Clec9a-tdTomato reporter MEFs were isolated from E13.5 embryos and subjected to fluorescence-activated cell sorting to remove contaminating tdTomato+ or CD45+ cells that may already be committed to the hematopoietic lineage.

[0107] In one embodiment, SPIB and IRF8 are sufficient for Clec9a activation.

[0108] In one experiment, Clec9a reporter MEFs were transduced with IRF8 alone or a combination of two factors, IRF8 and one of 21 other candidate transcription factors, and tdTomato expression was assessed. After transduction of 21 combinations of two candidate transcription factors or IRF8 alone, tdTomato+ cells emerged 5 days after Dox addition (Figure 6). The combination of IRF8 and SPIB generated 5% tdTomato+ cells, suggesting that IRF8 and SPIB are the minimal combination of factors required to induce reporter activation. TdTomato+ cells were not detected after transduction with the control M2rtTA vector or IRF8 alone.

[0109] In one embodiment, MHC-II, a marker of antigen-presenting cells, was expressed in approximately 60% of MEFs transduced with IRF8 and SPIB (FIG. 7).

[0110] In one experiment, we evaluated the effects of expressing the remaining 20 candidate transcription factors together with IRF8 and SPIB (Figure 8). Of the 20 transcription factors tested, HHEX, ID3, MEF2C, and RUNX2 were observed to have a negative effect on the number of tdTomato+ cells generated. Addition of ARID5A, HDAC5, HOXA5, IKZF1, IKZF2, IKZF3, IKZF5, IRF7, MYB, and STAT3 did not affect the number of tdTomato+ cells generated. Addition of BCL11A, CBFA2T3, CREB3L2, ETS1, TCF12, and TCF4 positively affected the number of tdTomato+ cells generated, thereby increasing the efficiency of reporter activation.

[0111] In one experiment, we determined whether activation of the Clec9a-tdTomato reporter was reflected in the surface expression of MHC-II, a key component of the antigen presentation machinery. Notably, we observed that tdTomato+ cells expressed MHC-II on their surface at day 8 (Figure 9).

[0112] In one embodiment, in addition to MHC-II surface molecules, pDCs secrete cytokines with inflammatory functions important for initiating T cell responses. These responses can be initiated by triggering at least 11 different Toll-like receptors (TLRs), which allow for the specific recognition of different conserved microbial or viral structures. Within the dendritic cell category, pDCs specifically respond to triggering by TLR7 and TLR9 (Figure 3). We investigated whether induced pDCs secrete cytokines into the culture medium upon TLR7 stimulation (with Resiquimod or R848) and TLR9 stimulation (with ODN1668) (Figure 10). Upon TLR challenge of induced pDCs, increased IFN-α secretion was observed in tdTomato+ cells reprogrammed with the following combinations: IRF8 and SPIB (16.6-fold); IRF8, SPIB, and ARID5A (3.9-fold); IRF8, SPIB, and BCL11A (14.8-fold); IRF8, SPIB, and CBFA2T3 (3.5-fold); IRF8, SPIB, and ETS1 (3.7-fold); IRF8, SPIB, and TCF4 (3.6-fold); IRF8, SPIB, and TCF12 (8.3-fold); and IRF8, SPIB, and HOXA5 (7.7-fold). These are the fold increases observed when cells reprogrammed with each TF combination were stimulated versus unstimulated. TLR challenge of induced pDCs also increased IFN-β secretion by tdTomato+ cells reprogrammed with the following combinations: IRF8 and SPIB (1.5-fold); IRF8, SPIB, and ARID5A (2.3-fold); IRF8, SPIB, and BCL11A (2.3-fold); IRF8, SPIB, and CBFA2T3 (1.9-fold); IRF8, SPIB, and ETS1 (1.6-fold); IRF8, SPIB, and TCF4 (1.3-fold); IRF8, SPIB, and TCF12 (1.9-fold); and IRF8, SPIB, and HOXA5 (2.6-fold). TLR challenge of induced pDCs did not increase secretion of the anti-inflammatory cytokine IL-10.Importantly, tdTomato+ cells obtained by transduction with a combination of PU.1, IRF8, and BATF3 (PIB) transcription factors did not respond to TLR7 or TLR9 and did not secrete IFN-α or IFN-β. Collectively, these data demonstrate that by combining reporter activation with interferon secretion, we have successfully identified a combination of transcription factors capable of inducing pDCs or type I interferon-secreting cells by direct cell reprogramming.

[0113] In one embodiment, two additional candidate transcription factors were selected based on their high expression levels specifically in plasmacytoid DCs compared with cDC1 and cDC2: STAT1 and TSC22D1 (Figure 11). These two transcription factors were cloned into the same doxycycline (Dox)-inducible lentiviral vectors that had been shown to induce reprogramming, and then tested in combination with IRF8 and SPIB to assess their ability to induce Clec9a-tdTomato reporter activation (Figure 12). Interestingly, the addition of STAT1 or TSC22D1 to the combination of IRF8 and SPIB enhanced Clec9a reporter activation compared with IRF8 and SPIB alone (Figure 12A). Induced pDCs generated by IRF8, SPIB, and STAT1, as well as those generated by IRF8, SPIB, and TSC22D1, also expressed MHC-II molecules on their cell surface (Figure 12B), indicating the acquisition of pDC fate.

[0114] In one embodiment, induced pDCs were stimulated with TLR7 / 9, and increased IFN-α secretion was observed in tdTomato+ cells reprogrammed with IRF8, SPIB, and STAT1 (1.3-fold) and in tdTomato+ cells reprogrammed with IRF8, SPIB, and TSC22D1 (4.8-fold) compared to induced pDCs generated with IRF8 and SPIB (Figure 13A). When ISTAT1 or TSC22D1 was added, increased IFN-β secretion was also observed in tdTomato+ cells reprogrammed with IRF8, SPIB, and STAT1 (1.3-fold) and in tdTomato+ cells reprogrammed with IRF8, SPIB, and TSC22D1 (4.1-fold) compared to cells induced with IRF8 and SPIB (Figure 13B). Induced pDCs generated by IRF8, SPIB, and STAT1 and those generated by IRF8, SPIB, and TSC22D1 failed to secrete the anti-inflammatory cytokine IL-10. These data indicate that IRF8, SPIB, and STAT1, as well as IRF8, SPIB, and TSC22D1, can induce fate-directed reprogramming of induced pDCs and induce type I interferon secretion.

[0115] In one embodiment, surface expression of the hematopoietic marker CD45 is also a hallmark of pDCs. We investigated whether a combination of pDC-inducing factors can induce surface expression of CD45. CD45 is expressed on 1.79±0.33% of induced pDCs generated with IRF8 and SPIB. Surprisingly, when IKZF1 is combined with IRF8 and SPIB, the CD45+ cell population increases to a maximum of 10.16±0.91% (a 5.6-fold increase).

[0116] In one embodiment, when pDCs recognize viral or self-nucleic acids via TLR7 and TLR9, two distinct pathways are induced, depending on the compartment in which TLR7 and TLR9 encounter their ligands. The MYD88-IRF7 pathway leads to the secretion of type I interferons, whereas the MYD88-NF-κB pathway leads to the production of inflammatory cytokines and chemokines. We next investigated whether TLR7 / 9 stimulation induces the secretion of other cytokines from induced pDCs (Figure 15). Indeed, purified tdTomato+ cells generated by overexpressing IRF8 and SPIB express the inflammatory cytokines IL-6 and TNF-α and the chemokines CCL5 and CXCL10. Importantly, the addition of STAT1, TCF12, or TSC22D1 to the combination of IRF8 and SPIB increases the secretion of IL-6, TNF-α, CCL5, and CXCL10 in a TLR-dependent manner. TLR-stimulated induced pDCs secreted increased IL-6 (5.6-fold), TdTomato (7.5-fold), and TSC22D1-reprogrammed tdTomato cells (3.5-fold), with similar increases in TNF-α (1.8-fold, 3.1-fold, and 1.3-fold, respectively). TLR-stimulated chemokine CCL5 secretion was also increased (2.5-fold, 3.1-fold, and 2.4-fold) in tdTomato (2.5-fold, 3.1-fold, and 2.4-fold) reprogrammed tdTomato cells (3.1-fold, 3.1-fold, and 2.4-fold, respectively) in tdTomato (2.5-fold, 3.1-fold, and 2.4-fold) reprogrammed tdTomato cells (3.1-fold, 3.1-fold, and 2.4-fold) reprogrammed tdTomato cells (3.1-fold, 3.1-fold, and 2.3-fold, respectively) in tdTomato (2.8-fold, 3.1-fold, and 2.3-fold) reprogrammed tdTomato cells (3.1-fold, 3.1-fold, and 2.4-fold ...3-fold) in tdTomato (2.8-fold, 3.1-fold, and 2.3-fold) reprogrammed tdTomato cells). Regarding CXCL10, its expression was increased by 2.8-fold and 1.7-fold by TLR stimulation in induced pDCs generated by overexpression of IRF8, SPIB, and TCF12, and in induced pDCs generated by overexpression of IRF8, SPIB, and TSC22D1, respectively.Collectively, these data demonstrate that we have successfully identified a combination of transcription factors sufficient to induce pDCs from unrelated cell types that are capable of responding to TLR7 and TLR9 stimulation through two intracellular pathways that elicit type I interferon secretion and the secretion of inflammatory cytokines and chemokines.

[0117] In one experiment, the coding region for each candidate TF was individually cloned into the inducible lentiviral pFUW-TetO vector, in which transcription factor expression is controlled by a tetracycline operator and a minimal CMV promoter. This was combined with the lentiviral vector described above (FUW-M2rtTA), which contains the reverse tetracycline-controlled transactivator M2rtTA under the control of the constitutively active human ubiquitin C promoter. Human embryonic kidney (HEK) 293T cells were transfected with a mixture of TF-encoding plasmids, packaging constructs, and VSV-G envelope protein. After 36, 48, and 60 hours, viral supernatants were harvested and filtered (0.45 μm, Corning) and then used either immediately or after 40-fold concentration using Amicon ultracentrifugal filters (Millipore).

[0118] In some embodiments, polypeptide variants or family members having the same or similar activity as the reference polypeptides encoded by the sequences presented in the sequence listing can be utilized in the compositions, methods, and kits described herein. Generally, variants of particular polypeptides encoding pDC inducers utilized in the compositions, methods, and kits described herein have at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0119] In one embodiment, Homo sapiens interferon regulatory factor 8 (IRF8), mRNA (SEQ ID NO: 1), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0120] In one embodiment, Homo sapiens Spi-B (SPIB), mRNA (SEQ ID NO: 3), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0121] In one embodiment, Homo sapiens AT-rich interacting domain 5A (ARID5A), mRNA (SEQ ID NO: 5), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0122] In one embodiment, it is understood that the Homo sapiens BAF chromatin remodeling complex subunit (BCL11A), mRNA (SEQ ID NO: 7) and optimized or different codons encoding the same amino acids are also encompassed by the nucleic acids as set forth herein.

[0123] In one embodiment, Homo sapiens CBFA2 / RUNX1 partner transcriptional corepressor 3 (CBFA2T3), mRNA (SEQ ID NO: 9), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0124] In one embodiment, it is understood that Homo sapiens cAMP response element binding protein 3-like protein 2 (CREB3L2), mRNA (SEQ ID NO: 11), and optimized or different codons encoding the same amino acids are also encompassed by the nucleic acids as set forth herein.

[0125] In one embodiment, Homo sapiens ETS proto-oncogene 1 (ETS1), mRNA (SEQ ID NO: 13), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0126] In one embodiment, Homo sapiens homeobox A5 (HOXA5), mRNA (SEQ ID NO: 19), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0127] In one embodiment, it is understood that Homo sapiens IKAROS family zinc finger 1 (IKZF1), mRNA (SEQ ID NO: 23), and optimized or different codons encoding the same amino acids are also encompassed by the nucleic acids as set forth herein.

[0128] In one embodiment, Homo sapiens signal transducer and activator of transcription 1 (STAT1), mRNA (SEQ ID NO: 45), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0129] In one embodiment, Homo sapiens transcription factor 4 (TCF4), mRNA (SEQ ID NO: 41), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0130] In one embodiment, Homo sapiens transcription factor 12 (TCF12), mRNA (SEQ ID NO: 43), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0131] In one embodiment, Homo sapiens TSC22 domain family protein 1 (TSC22D1), mRNA (SEQ ID NO: 47), and optimized or different codons encoding the same amino acids are understood to be encompassed by the nucleic acids as set forth herein.

[0132] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the number of pDC-inducing factors used or selected to generate pDCs from starting somatic cells (such as fibroblasts or cells of the hematopoietic system, pluripotent stem cells, induced pluripotent stem cells, cancer cells, or tumor cells) is at least 2. In some embodiments, the number of pDC-inducing factors used or selected is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 33, at least 35, at least 40, or more.

[0133] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, a nucleic acid sequence or construct encoding a pDC inducer (such as IRF8, SPIB, ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, HOXA5, IZKF1, TCF4, TCF12, and TSC22D1) is inserted into or operably linked to an expression vector suitable for cell transformation using standard molecular biology techniques. As used herein, the term "vector" refers to a nucleic acid molecule, such as a dsDNA molecule, that confers useful biological or biochemical properties to an inserted nucleotide sequence (such as a nucleic acid construct or replacement cassette described herein). Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences capable of replicating or causing replication in vitro or in a host cell, or transporting a desired nucleic acid segment to a desired site within a host cell. A vector may have one or more restriction endonuclease recognition sites (whether Type I, Type II, Type IIs, Type III, or Type IV) that allow for unambiguous cleavage of sequences without impairing the essential biological function of the vector, and into which nucleic acid fragments may be ligated or inserted for replication and cloning. A vector may also contain one or more recombination sites that allow for the exchange of nucleic acid sequences between two nucleic acid molecules. A vector may further contain, for example, primer sites for PCR, transcription and / or translation initiation and / or control sites, recombination signals, replicons, additional selectable markers, etc. A vector may also contain one or more selectable markers suitable for use in identifying cells transformed with the vector.

[0134] In some embodiments of compositions, vectors, constructs, methods and kits described herein, this expression vector is virus vector.Some virus-mediated expression methods utilize retrovirus vector, adenovirus vector, lentivirus vector, paramyxovirus family virus vector, rhabdovirus vector, alphavirus vector, flavivirus vector, herpesvirus vector, poxvirus vector and adeno-associated virus (AAV) vector, and this expression method has been used for gene transfer for a long time, and is well known in the art.

[0135] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the viral vector is a retrovirus. Retroviruses provide a convenient platform for gene transfer. A selected gene can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and introduced into target cells of interest either in vivo or ex vivo. Several retroviral systems have been reported. See, e.g., U.S. Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-90; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-52; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop. 3:102-09. In some embodiments of the compositions, vectors, methods, and kits described herein, the retrovirus is replication-deficient. Retroviral vector systems take advantage of the fact that a minimal vector containing the 5' and 3' LTRs and a packaging signal is sufficient for vector packaging, infection, and integration into target cells, provided that viral structural proteins are supplied in trans in the packaging cell line.The fundamental advantages of retroviral vectors for gene transfer include high efficiency of infection and gene expression in most cell types, precise integration of single-copy vectors into target cell chromosomal DNA, and ease of manipulation of the retroviral genome.

[0136] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the viral vector is an adenovirus-based expression vector. Unlike retroviruses, which integrate into the host genome, adenoviruses remain extrachromosomal, thereby minimizing the risk of insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-74; Bett et al. (1993) J. Virol. 67:5911-21; Mittereder et al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994) J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, KL (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human Gene Therapy 4:461-76). Adenoviral vectors infect a wide variety of cell types and have a broad host range. They have high infection efficiency, direct high-level expression of heterologous genes, and achieve long-term in vivo expression of those genes. The virus is fully infectious in the cell-free viral particle form, eliminating the need for injection of producer cell lines. Regarding safety, adenoviruses have not been associated with serious pathologies in humans, and recombinant vectors derived from them can be rendered replication-impaired by deleting the early region 1 ("E1") of the viral genome. Adenoviruses are also relatively easy to produce in large quantities. The adenoviral vectors used in the compositions, vectors, methods, and kits described herein can be derived from any of a variety of adenovirus serotypes, including, but not limited to, more than 40 serotype strains of adenovirus (e.g., serotypes 2, 5, 12, 40, and 41). Adenoviral vectors for use herein are preferably replication-deficient and contain a pDC inducer of interest operably linked to a suitable promoter.

[0137] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, nucleic acid sequences encoding pDC inducers (such as IRF8, SPIB, ARID5A, BCL11A, CBFA2T3, ETS1, HOXA5, IKZF1, STAT1, TCF4, TCF12, and TSC22D1) are introduced or delivered using one or more inducible lentiviral vectors. Control of expression of pDC inducers delivered using one or more inducible lentiviral vectors can, in some embodiments, be achieved by contacting cells containing at least one pDC inducer under the control of, or operably linked to, an inducible promoter in an expression vector with a regulator (e.g., doxycycline) or other inducer. When using certain inducible lentiviral vectors, contacting such cells with an inducer induces expression of the pDC inducer, whereas withdrawal of the regulator inhibits expression. When using other inducible lentiviral vectors, the presence of a regulator inhibits expression, whereas removal of the regulator allows expression. As used herein, the term "induction of expression" refers to the expression of a gene (such as a pDC-inducing factor encoded by an inducible viral vector) in the presence of an inducer, e.g., or in the presence of one or more substances or factors that cause endogenous expression of the gene in a cell.

[0138] Some embodiments of the aspects described herein utilize a doxycycline (Dox)-inducible lentiviral system. Unlike retroviruses, lentiviruses can transduce quiescent cells, allowing for easier transduction of a broader range of hematopoietic cell types. For example, the pFUW-tetO lentiviral system has been shown to transduce primary hematopoietic progenitor cells with high efficiency.

[0139] In some embodiments of the methods described herein, pDC inducer factors (IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4), ARID5A (SEQ ID NO: 5, SEQ ID NO: 6), BCL11A (SEQ ID NO: 7, SEQ ID NO: 8), CBFA2T3 (SEQ ID NO: 9, SEQ ID NO: 10), CREB3L2 (SEQ ID NO: 11, SEQ ID NO: 12), ETS1 (SEQ ID NO: 13, SEQ ID NO: 14), HDAC5 (SEQ ID NO: 15, SEQ ID NO: 16), HHEX (SEQ ID NO: 17, SEQ ID NO: 18), HOXA5 (SEQ ID NO: 19, SEQ ID NO: 20), ID3 (SEQ ID NO: 21, SEQ ID NO: 22), IKZF1 (SEQ ID NO: 23, SEQ ID NO: 24), IKZF2 (SEQ ID NO: 25), IKZF3 (SEQ ID NO: 26), IKZF4 (SEQ ID NO: 27), IKZF5 (SEQ ID NO: 28), IKZF6 (SEQ ID NO: 29), IKZF7 (SEQ ID NO: 30), IKZF8 (SEQ ID NO: 31), IKZF9 (SEQ ID NO: 32), IKZF10 (SEQ ID NO: 33), IKZF11 (SEQ ID NO: 34), IKZF12 (SEQ ID NO: 35), IKZF13 (SEQ ID NO: 36), IKZF14 (SEQ ID NO: 37), IKZF15 (SEQ ID NO: 38), IKZF16 (SEQ ID NO: 3 Nucleic acid sequences encoding IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-30, IL-31, IL-32, IL-33, IL-34, IL-45, IL-46, IL-47, IL-48, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-79, IL-80, IL-81, IL-82, IL-83, IL-84, IL-85, IL-86, IL-87, IL-89, IL-90, IL-91, IL-92, IL-93, IL-94, IL-95, IL-96, IL-97, IL-98, IL-99, IL-99, IL-99, IL-99, IL-99, IL-99, IL-99, IL-99, IL-91, IL-92, IL-93, IL-94, IL-95, IL-96, IL-97, IL-99, IL-99, IL-99, IL-99, IL-99, IL-9 Because non-integrating vectors do not become part of the host genome, they often transiently express nucleic acids in a cell population. This is in part because many non-integrating vectors are replication-impairing. Thus, non-integrating vectors offer several advantages over retroviral vectors, including, but not limited to: (1) no disruption of the host genome, (2) transient expression, and (3) no residual viral integration products.Some non-limiting examples of non-integrating vectors for use in the methods described herein include adenovirus, baculovirus, alphavirus, picornavirus, and vaccinia virus. In some embodiments of the methods described herein, the non-integrating viral vector is adenovirus. Other advantages of non-integrating viral vectors include the ability to produce high titers, stability in vivo, and efficient infection of host cells.

[0140] In some embodiments, the nucleic acid constructs and vectors used to generate induced pDCs, which are included in the compositions, vectors, constructs, methods, and kits described herein, can further comprise one or more sequences encoding selectable markers for positive and negative selection of cells. Such selectable marker sequences typically confer resistance or sensitivity to antibiotics that are not normally present in cells in the absence of the nucleic acid construct. A selectable marker can be used in conjunction with a selection agent (such as an antibiotic) to select cells expressing the introduced nucleic acid construct in culture. A sequence encoding a positive selectable marker typically confers resistance to an antibiotic; i.e., the presence of a positive selectable marker sequence in the genome of a cell renders the cell sensitive to the antibiotic or drug. A sequence encoding a negative selectable marker typically confers sensitivity to an antibiotic or drug; i.e., the presence of a negative selectable marker in the genome of a cell renders the cell sensitive to the antibiotic or drug.

[0141] The nucleic acid constructs and vectors for generating induced pDCs in the compositions, vectors, constructs, methods, and kits described herein can, in some embodiments, further comprise other nucleic acid elements for the regulation, expression, or stabilization of the construct or other vector genetic elements, such as promoters, enhancers, TATA boxes, ribosome binding sites, IRESs, and 2A-like self-cleaving sequences, as known to those skilled in the art.

[0142] The nucleic acid constructs and vectors for generating induced pDCs in the compositions, vectors, constructs, methods, and kits described herein can further comprise a 2A-like self-cleaving sequence. Self-cleaving 2A peptides, 18-22 amino acids in length, mediate "ribosomal skipping" between proline and glycine residues, inhibiting peptide bond formation without affecting downstream translation. These peptides encode multiple proteins as a polyprotein, which dissociates into its component proteins during translation. Examples of 2A-like self-cleaving sequences that can be used in accordance with the present invention include, but are not limited to, self-cleaving sequences from the Picornaviridae family of viruses (including aphthoviruses such as foot-and-mouth disease virus (FMDV), equine rhinitis virus type A (ERAV), Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1)) and cardioviruses (e.g., Theiler's murine encephalitis virus) and encephalomyocarditis virus).

[0143] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the vectors selected from the group consisting of IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4), ARID5A (SEQ ID NO: 5, SEQ ID NO: 6), BCL11A (SEQ ID NO: 7, SEQ ID NO: 8), CBFA2T3 (SEQ ID NO: 9, SEQ ID NO: 10), CREB3L2 (SEQ ID NO: 11, SEQ ID NO: 12), ETS1 (SEQ ID NO: 13, SEQ ID NO: 14), HOXA5 (SEQ ID NO: 19, SEQ ID NO: 20), IKZF1 (SEQ ID NO: 23, SEQ ID NO: 24), IKZF2 (SEQ ID NO: 25, SEQ ID NO: 26), IKZF3 (SEQ ID NO: 27, SEQ ID NO: 28), IKZF4 (SEQ ID NO: 29, SEQ ID NO: 30), IKZF5 (SEQ ID NO: 31, SEQ ID NO: 32), IKZF6 (SEQ ID NO: 33, SEQ ID NO: 34), IKZF7 (SEQ ID NO: 35, SEQ ID NO: 36), IKZF8 (SEQ ID NO: 37, SEQ ID NO: 38), IKZF9 (SEQ ID NO: 39, SEQ ID NO: 40), IKZF10 (SEQ ID NO: 41, SEQ ID NO: 42), IKZF11 (SEQ ID NO: 43, SEQ ID NO: 44), IKZF12 (SEQ ID NO: 45, SEQ ID NO: 46), IKZF13 (SEQ ID NO: pDC-inducing factors, such as TCF4 (SEQ ID NO:24), TCF4 (SEQ ID NO:41, SEQ ID NO:42), TCF12 (SEQ ID NO:43, SEQ ID NO:44), STAT1 (SEQ ID NO:45, SEQ ID NO:46), and TSC22D1 (SEQ ID NO:47, SEQ ID NO:48), are provided as synthetic, modified RNA or are introduced or delivered to cells as synthetic, modified RNA in a manner as described in U.S. Patent Application No. 2012-0046346-A1, the contents of which are incorporated herein by reference in their entirety. In those embodiments in which synthetic, modified RNA is used to reprogram cells into induced pDCs according to the methods described herein, the methods can include repeatedly contacting the cells with or repeatedly transforming the cells with synthetic, modified RNA encoding a pDC inducer (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 or more transformations, etc.).

[0144] In addition to one or more modified nucleosides, the modified mRNAs used in the compositions, vectors, constructs, methods, and kits described herein can include any additional modifications known to those of skill in the art and described in U.S. Patent Applications 2012-0046346-A1 and 20120251618A1, and International Patent Application 2012 / 019168. Such additional elements include, for example, a 5' cap (e.g., an anti-reverse cap analog containing a 5'-5'-triphosphate guanine-guanine linkage in which one guanine has an N7 methyl group and a 3'-O-methyl group). caps made using recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme (which can introduce a standard 5'-5'-triphosphate linkage between the 5'-most nucleotide of an mRNA and a guanine nucleotide, where the guanine contains an N7 methylation and the final 5'-nucleotide contains a 2'-O-methyl forming a Cap1 structure); caps made using a terminal poly(A) strand (e.g., a terminal poly(A) strand greater than 30 nucleotides in length, a terminal poly(A) strand greater than 35 nucleotides in length, a terminal poly(A) strand greater than 40 nucleotides, at least 45 nucleotides, at least 55 nucleotides, at least 60 nucleotides in length, a terminal poly(A) tail of at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides or more; a Kozak sequence; a 3' untranslated region (3'UTR); a 5' untranslated region (5'UTR); one or more intron nucleotide sequences that are capable of being removed from the nucleic acid, or a combination thereof.

[0145] The modified mRNA used in the compositions, vectors, constructs, methods, and kits described herein may further comprise an internal ribosome entry site (IRES). A single IRES can act as the only ribosome binding site, or it can serve as one of multiple ribosome binding sites in an mRNA. An mRNA containing multiple functional ribosome binding sites can encode multiple peptides or polypeptides (such as the pDC inducers described herein) and be independently translated by ribosomes ("polycistronic mRNA"). When a nucleic acid has an IRES, a second translatable region can optionally be added. Examples of IRES sequences that can be utilized in accordance with the present invention include, but are not limited to, IRES sequences derived from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SW), or cricket paralysis viruses (CrPV).

[0146] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least one modified nucleoside. In some embodiments of the compositions, vectors, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least two modified nucleosides.

[0147] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the modified nucleoside is 5-methylcytosine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'dU), 4-thiouridine (s4U), 5-methyluridine ( In some embodiments, the modified nucleoside is selected from the group consisting of 5-methylcytosine (5mC), pseudouracil, or a combination thereof.

[0148] Modified mRNA need not be uniformly modified throughout the entire length of the molecule. Different nucleotide modifications and / or different backbone structures can be present at various positions in the nucleic acid. Those skilled in the art will understand that nucleotide analogs or other modifications can be present at any position in the nucleic acid as long as the function of the nucleic acid is not substantially impaired. Modifications can also be at the 5' or 3' end. The nucleic acid can contain a minimum of one modified nucleotide, a maximum of 100% modified nucleotides, or any percentage in between (such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides).

[0149] In some embodiments of the present disclosure, the vector or construct is a replicating viral vector or viral particle, such as a Paramyxoviridae viral vector, a Rhabdoviridae vector, an alphavirus and / or flavivirus particle, or a non-viral vector or particle, such as a naked alphavirus or flavivirus RNA replicon.

[0150] In some embodiments, it is preferred, but not absolutely necessary, that each specific nucleoside in the molecule be modified (e.g., each cytosine is a modified cytosine, e.g., 5-methylcytosine, each uracil is a modified uracil, e.g., pseudouracil, etc.). For example, the modified mRNA can also include modified pyrimidines (e.g., uracil or cytosine). In some embodiments, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in a nucleic acid are replaced with modified uracils. It is also understood that nucleosides at different positions within the same type of nucleoside in a particular synthetic, modified RNA molecule can be modified in different ways. The modified uracils can be replaced with a single compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some embodiments, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in a nucleic acid may be replaced with modified cytosines. The modified cytosines may be replaced with a single compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures) (e.g., some cytosine modifications are 5mC and others are 2'-O-methylcytosine or other cytosine analogs). Such multiply modified synthetic RNA molecules may be generated using a ribonucleoside mixture or mixture containing all the desired modified nucleosides during RNA molecule synthesis, so that only the desired modified nucleosides are incorporated into the resulting RNA molecule encoding a pDC inducer.

[0151] In certain embodiments, modified nucleic acids introduced into cells need to be degraded within the cell (e.g., when precisely timed protein production is required). Thus, in some embodiments of the compositions, vectors, methods, and kits described herein, the present disclosure provides modified nucleic acids that include a degradation domain that can act in a directed manner within the cell.

[0152] It is understood that induced pDCs can be generated by introducing pDC-inducing factors in the form of nucleic acids (DNA or RNA) or amino acid sequences; however, in some embodiments of the compositions, vectors, methods, and kits described herein, induced pDCs can also be induced using other methods (e.g., by treating cells with a substance (such as a small molecule or small molecule cocktail) that induces expression of one or more of the pDC-inducing factors).

[0153] Expression of pDC inducers introduced into cells or induced in cell populations using the compositions, vectors, methods, and kits described herein can be detected by any of several techniques known to those skilled in the art, including, for example, Western blot analysis, immunochemistry, and fluorescence-mediated detection.

[0154] To determine whether a particular combination of pDC-inducing factors generates induced pDCs, one or more DC activities or parameters can be measured (such as, in some embodiments, differential expression of surface antigens). Generation of induced DCs using the compositions, vectors, methods, and kits described herein preferably results in the appearance of cell surface phenotypes characteristic of endogenous pDCs (e.g., CD45 and MHC-II).

[0155] DCs are most reliably distinguished from other immune cells by their functional behavior. Functional aspects of DC phenotype or DC activity (such as the ability of induced pDCs to secrete cytokines) can be readily determined by those skilled in the art using common methods known in the art. In some embodiments of the aspects described herein, functional assays to identify reprogramming factors can be used. For example, in some embodiments, cytokine secretion can be used to confirm the immunomodulatory properties of induced pDCs generated using the compositions, vectors, methods, and kits described herein. In particular, for the purpose of characterizing the functionality of pDCs, it is highly relevant to examine their ability to respond to intracellular DNA via the TLR7 and TLR9 pathways and to secrete type I interferon.

[0156] As used herein, the terms "cellular parameter," "DC parameter," or "cytokine secretion" refer to a measurable element or attribute of endogenous or natural DCs, particularly elements that can be accurately measured. A cellular parameter can be a measurable parameter related to cellular phenotype, function, or behavior. Such cellular parameters include changes in characteristics and markers of DCs or DC populations, including, but not limited to, changes in viability, changes in cell proliferation, and changes in expression of one or more markers or combinations of markers; such markers can also include cell surface determinants (e.g., receptors), proteins (including their conformational or post-translational modifications), lipids, carbohydrates, organic or inorganic molecules, nucleic acids (e.g., mRNA, DNA), and global gene expression patterns. Such cellular parameters can be measured using a variety of assays known to those skilled in the art. For example, viability and cell proliferation can be measured by assays such as trypan blue exclusion, CFSE dilution, and 3H-thymidine incorporation. Expression of protein or polypeptide markers can be measured, for example, using flow cytometry, Western blot techniques, or microscopy. Gene expression profiles can be measured, for example, using RNA sequencing technology and quantitative or semi-quantitative real-time PCR assays. Cellular parameters can also refer to functional parameters or functional activity. Many cellular parameters provide quantitative measurement values, but semi-quantitative or qualitative results may also be acceptable in some cases. Measurement values ​​can include a single determination value, or can include the mean, median, or variance. Characteristically, a range of parameter measurements can be obtained from each parameter in multiple identical assays. Because variation is expected, standard statistical methods, including common statistical methods used to obtain a single value, are used to obtain a range of values ​​for each test parameter set.

[0157] In some embodiments of the compositions, vectors, methods, and kits described herein, additional factors and agents can be used to enhance induced pDC reprogramming, for example, factors and agents that modify epigenetic pathways can be used to enhance reprogramming to induced pDC.

[0158] Essentially any primary somatic cell type can be used to generate induced pDCs or to reprogram somatic cells into induced pDCs using the compositions, vectors, methods, and kits described herein. These primary somatic cell types also include other stem cell types, including pluripotent stem cells, such as induced pluripotent stem cells (iPS cells); other pluripotent stem cells; oligopotent stem cells; and unipotent stem cells. 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, and pancreatic cells, as well as the stem cells from which these cells are derived. The cells may be any isolated primary cells derived from somatic tissues, including, but not limited to, spleen, bone marrow, blood, brain, liver, lung, gastrointestinal tract, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc. In some embodiments, the term "somatic cell" further includes primary cells grown in culture if the somatic cells are not immortalized. When cells are maintained in vitro, standard 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 readily available to those skilled in the art.

[0159] In some embodiments of these and all similar aspects described herein, the somatic cells are fibroblasts.

[0160] In some embodiments of these and all similar aspects described herein, the somatic cell may be a cell of the hematopoietic system.

[0161] In some embodiments of these and all similar aspects described herein, the somatic cell may be a cancer cell or a tumor cell.

[0162] In some embodiments of the compositions, vectors, constructs, methods, and kits described herein, the somatic cells that are reprogrammed into induced pDC cells are cells of hematopoietic origin. As used herein, the terms "hematopoietic-derived cells," "hematopoietic-derived differentiated cells," "hematopoietic cells," and "cells of hematopoietic origin" refer to cells derived from or differentiated from pluripotent hematopoietic stem cells (HSCs). Thus, cells of the hematopoietic system for use with the compositions, vectors, constructs, methods, and kits described herein include pluripotent, oligopotent, and 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 that have 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 cell" refers to pluripotent, oligopotent, and lineage-restricted hematopoietic cells that have the potential to differentiate into two or more hematopoietic cell types, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, and lymphocytes, including B cells and T cells. Hematopoietic progenitor cells include multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), and pre-megakaryocyte-erythroid common precursor progenitors. Lineage-restricted hematopoietic progenitor cells include common 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, pre-granulocyte / macrophage precursor cells (GMPs), and pro-mast cells (ProMCs).

[0163] Cells of hematopoietic origin used in the compositions, vectors, methods, and kits described herein can be obtained from sources known to contain these cells (such as fetal tissue, umbilical cord blood, bone marrow, peripheral blood, mobilized peripheral blood, spleen, liver, thymus, lymph nodes, etc.). Cells obtained from these sources can be expanded ex vivo using methods acceptable to those skilled in the art before use in the compositions, vectors, constructs, methods, and kits for generating induced pDCs described herein. For example, cells can be sorted, fractionated, treated to remove specific cell types, or otherwise manipulated to obtain cell populations for use in the methods described herein using procedures acceptable to those skilled in the art. Mononuclear lymphocytes may be collected by repeated lymphopheresis using a continuous flow cell separator, as described, for example, in U.S. Pat. No. 4,690,915, or isolated using an affinity purification step in a method used for CLP (common lymphoid progenitor) cell lineages (CLP); such methods include flow cytometry using a cytometer, magnetic separation using antibody- or protein-coated beads, affinity chromatography, or solid-phase support affinity separation (cells are retained on a substrate depending on the expression or lack of expression of a particular protein or type of protein), or batch purification using one or more antibodies against one or more surface antigens specifically expressed by the cell type of interest. Cells of hematopoietic origin can also be obtained from peripheral blood. Prior to collection of cells from peripheral blood, subjects can be treated with cytokines (e.g., granulocyte colony-stimulating factor) to promote cell migration from the bone marrow to the blood compartment and / or to promote activation and / or proliferation of the desired population. For example, any method suitable for identifying surface proteins can be used to isolate cells of hematopoietic origin from a heterogeneous population. In some embodiments, a clonal population of cells of hematopoietic origin (such as lymphocytes) is obtained. In some embodiments, the cells of hematopoietic origin are not a clonal population.

[0164] Furthermore, for various aspects and embodiments of the compositions, vectors, constructs, methods, and kits described herein, the somatic cells can be obtained from any mammalian species, including, but not limited to, murine, bovine, simian, porcine, equine, ovine, or human cells. In some embodiments, the somatic cells are human cells. In some embodiments, the cells are derived from a non-human organism (such as a non-human mammal).

[0165] Generally, the methods for generating induced pDCs described herein involve culturing or expanding somatic cells (e.g., cells of hematopoietic origin) in any culture medium available and known to those skilled in the art. Such media include, but are not limited to, Dulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle's Minimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco's Medium®, RPMI-1640 Medium®, or serum-free media for culturing and expanding DCs. Many media are also available as sodium-containing or sodium-free, low-glucose preparations. In some embodiments, media used with the methods described herein can include one or more immunostimulatory cytokines. Commonly used growth factors include, but are not limited to, G-CSF, GM-CSF, TNF-α, IL-4, IL-3, Flt-3 ligand, and kit ligand. Furthermore, in preferred embodiments, the immunostimulatory cytokine is selected from the group consisting of interleukins (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-9, IL-10, IL-12, IL-18, IL-19, IL-20); interferons (e.g., IFN-α, IFN-β, IFN-γ), tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt-3 ligand, and kit ligand.

[0166] The cultured cells can be maintained, for example, in suspension or attached to a solid support (e.g., seeded on extracellular matrix components or feeder cells). In some embodiments, the cells used in the methods described herein may require additional factors to aid cell attachment to the solid support (e.g., type I and type II collagen, chondroitin sulfate, fibronectin, "superfibronectin," and fibronectin-like polymers, gelatin, poly-D and poly-L-lysine, thrombospondin, and vitronectin). In some embodiments, the cells are suitable for growth in suspension culture. Suspension-competent host cells are generally monodisperse or grow in a loose, aggregated form that is not substantially aggregated. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., cells of hematopoietic origin (e.g., lymphoid cells)) and cells that have become suspension-competent through modification or adaptation of adhesion-dependent cells (e.g., epithelial cells, fibroblasts).

[0167] In some embodiments of these and all similar aspects described herein, the isolated induced pDCs further comprise a pharmaceutically acceptable carrier for administration to a subject in need thereof.

[0168] Also provided herein in some aspects are methods for treating a subject in need of treatment to induce an antigen-specific immune response to eliminate cancer cells or infectious agents, the methods utilizing pDC-inducing compositions and vectors and the methods for preparing induced pDCs described herein, or the methods utilizing isolated induced pDCs and their cell clones prepared using a combination of pDC-inducing factors, pDC-inducing compositions and vectors, or the methods for preparing induced pDCs described herein. In such treatment methods, somatic cells (such as fibroblasts or hematopoietic lineage cells) are first isolated from the subject, and the isolated cells can be transduced or transformed with a pDC-inducing composition containing an expression vector or synthetic mRNA, respectively, as described herein. Isolated induced pDCs prepared using the combination of pDC-inducing factors, pDC-inducing compositions, or methods for preparing induced pDCs described herein can then be administered to the subject, such as by systemic injection of the induced pDCs.

[0169] Also provided herein in some aspects are methods for treating a subject in need of treatment to induce an antigen-specific immune response to eliminate cancer cells or infectious agents, using a combination of the pDC-inducing compositions and vectors described herein and pDC-inducing factors. In such treatment methods, cancer cells are transduced with a pDC-inducing composition comprising an expression vector, as described herein. Cancer cells can be first isolated from a subject, transduced with a pDC-inducing composition comprising an expression vector, and then administered to the subject by systemic injection, for example. Alternatively, cancer cells can be transduced in situ or in vivo with a pDC-inducing composition comprising an expression viral vector.

[0170] Reprogrammed induced pDCs generated using the compositions, vectors, constructs, methods, and kits described herein can be used directly or administered to a subject in need of immunotherapy in some embodiments of the therapeutic methods described herein. Accordingly, various embodiments of the methods described herein comprise administering to an individual or subject in need of cell therapy an effective amount of induced pDCs or a population of induced pDCs generated using any of the compositions, vectors, methods, and kits described herein. The administered cells or cell populations can be autologous or derived from one or more non-autologous sources. Furthermore, such induced pDCs can be administered in a manner that allows them to migrate to lymph nodes and activate effector T cells.

[0171] Various methods for administering cells to a subject are known to those skilled in the art. Such methods may include systemic injection, e.g., intravenous injection, or implanting cells at a target site in a subject. Cells may be introduced into a delivery device that facilitates introduction into a subject by injection or implantation. Such a delivery device may include a tube, e.g., a catheter, through which cells and fluids are injected into the body of the recipient subject. In a preferred embodiment, the tube further comprises a needle, e.g., through which cells can be introduced to a desired site in a subject. Cells can be prepared in a variety of different forms for delivery. For example, cells can be suspended in a solution or gel, or, when placed in such a delivery device, the cells can be embedded in a support matrix. Cells can also be mixed with a pharmaceutically acceptable carrier or diluent that allows the cells to survive when mixed with the carrier or diluent.

[0172] Thus, the cells generated by the methods described herein can be used to prepare cells for treating or alleviating a number of cancers and tumors, including breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain tumor, primary brain tumor, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian carcinoma, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder carcinoma, pancreatic carcinoma, stomach cancer, colon cancer, prostate cancer, and the like. carcinoma), genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, uterine endothelial carcinoma, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid tumor, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical dysplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, sarcoma of the soft tissue, osteosarcoma, primary macroglobulinemia, and retinoblastoma.

[0173] In addition, the methods of the present invention can be used to prevent or eliminate infection with pathogens known to predispose individuals to certain cancers. Pathogens of particular interest for use in the cancer vaccines described herein include hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (liver cancer), Epstein-Barr virus (EBV) (Burkitt's lymphoma, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease (PTLD) in immunosuppressed individuals), HTLV-1 (adult T-cell leukemia), oncogenic human papillomavirus types 16, 18, 33, and 45 (adult cervical cancer), and the bacterium Helicobacter pylori (B-cell gastric lymphoma). Other medically relevant microorganisms that can serve as antigens in mammals, and more specifically in humans, have been extensively described in the literature (e.g., C.G. A. Thomas, Medical Microbiology, Bailliere Tindall, (1983)).

[0174] In addition to the above, the methods of the present invention can be used to treat viral infections. Examples of viral pathogens include, but are not limited to, infectious viruses that infect mammals, and more specifically, humans.Examples of infectious viruses include Retroviridae (e.g., human immunodeficiency virus (HIV-I (also called HTLV-III, LAV, or HTLV-III / LAV) or HIV-III; and other isolates, such as HIV-LP)); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Viviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronaviruses such as SARS coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantaan virus, Bunyaviridae, Phleboviruses, and Nairoviruses; Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis B virus (HBV). Rupesviruses; Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), agents of non-A, non-B hepatitis (Class 1 = orally transmitted; Class 2 = parenterally transmitted (i.e., hepatitis C); Norwalk virus and related viruses, and astroviruses).

[0175] In addition to the above, the method of the present invention may be used to target Gram-negative and Gram-positive bacteria in vertebrates. Such Gram-positive bacteria include, but are not limited to, Pasteurella sp., Staphylococcus sp., and Streptococcus sp. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas sp., and Salmonella sp. Specific examples of infectious bacteria include Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacterium sp. (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium gordonii), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus (viridans streptococci), Streptococcus faecalis, Streptococcus bovis, and Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhodopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii.

[0176] In addition to the above, the methods of the present invention may be used to target pathogens, including but not limited to infectious fungi and parasites that infect mammals, and more specifically, humans. Examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0177] In addition, the method of the present invention may be used to target parasites (such as intracellular parasites and obligate intracellular parasites), including, but not limited to, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax, Plasmodium simian, Babesia murine, Babesia polymorpha, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timorii, Ascaris lumbricoides, Onchocerca volvulus, and Schistosoma mekonnensis.

[0178] Once induced pDCs are modified, they can be used to induce a tolerogenic response against one or more target antigens, including suppression of future or current immune responses. Thus, induced pDCs are useful for treating or preventing undesirable immune responses, including, for example, transplant rejection, graft-versus-host disease, allergies, parasitic diseases, inflammatory diseases, and autoimmune diseases. Examples of transplant rejection that can be treated or prevented according to the present invention include rejection associated with bone marrow transplants and rejection associated with organ transplants such as heart, liver, pancreas, kidney, lung, eye, and skin. Examples of allergies include seasonal respiratory allergies; allergies to aeroallergens, such as hay fever; allergies treatable by reducing serum IgE and eosinophilia; asthma; eczema; animal allergies; food allergies; latex allergies; dermatitis; or allergies treatable by allergic desensitization. Examples of autoimmune diseases that can be treated or prevented by the present invention include psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff-man syndrome, thyroiditis, Sydenham's chorea, rheumatoid arthritis, diabetes, and multiple sclerosis. Examples of inflammatory diseases include Crohn's disease, chronic inflammatory eye disease, chronic inflammatory lung disease, and chronic inflammatory liver disease, autoimmune hemolytic anemia, idiopathic leukopenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, irritable bowel syndrome, systemic lupus erythematosus (SLE), multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome (antiphospholipid syndrome), idiopathic myxedema, hyperthyroidism, pernicious anemia, autoimmune atrophic gastritis, and adipocyte myelopathy. These include: Crohn's disease, insulin-dependent diabetes mellitus (IDDM), Goodpasture's syndrome, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, sympathetic ophthalmia, Hashimoto's disease / chronic thyroiditis, celiac disease / dermatitis herpetiformis, and demyelinating diseases, primary biliary cirrhosis, mixed connective tissue disease, chronic active hepatitis, Graves' disease / hyperthyroidism, scleroderma, chronic idiopathic thrombocytopenic purpura, diabetic neuropathy, and septic shock.

[0179] Pharmaceutically acceptable carrier and diluent include saline solution, aqueous buffer solution, solvent and / or dispersion medium.The use of such carrier and diluent is well known in the art.This solution is preferably sterile and liquid.Preferably, this solution before introducing into cell is stable under the conditions of manufacture and storage, and is protected from the contaminating action of microorganisms such as bacteria and fungi by using, for example, paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0180] Preferably, the cell administration method is relatively non-invasive, for example, by intravenous injection, pulmonary delivery by inhalation, topical administration, or intranasal administration.However, the cell administration route depends on the tissue to be treated, and may include transplantation.Cell delivery methods are known to those skilled in the art, and those skilled in the medical field who utilize the methods and compositions described herein can conceive of delivery methods.

[0181] In some aspects, the present specification also provides kits for generating induced pDCs, which kits include pDC-inducing compositions that include one or more of the expression vector elements described herein.

[0182] Also in some aspects, the present specification provides kits that include one or more of the pDC induction factors described herein as components for the methods of generating induced pDCs described herein.

[0183] Thus, in some aspects, the present disclosure provides a kit for preparing induced dendritic cells, the kit comprising the following elements: (a) one or more expression vectors encoding at least one, two, three, four, five, six, seven, eight, or more pDC inducer factors, the pDC inducer factors being: IRF8 (SEQ ID NO: 1, SEQ ID NO: 2), SPIB (SEQ ID NO: 3, SEQ ID NO: 4), ARID5A (SEQ ID NO: 5, SEQ ID NO: 6), BCL11A (SEQ ID NO: 7, SEQ ID NO: 8), CBFA2T3 (SEQ ID NO: 9, SEQ ID NO: 10), CREB3L2 (SEQ ID NO: 11, SEQ ID NO: 12), ETS1 (SEQ ID NO: 13, SEQ ID NO: 14), HDAC5 (SEQ ID NO: 15, SEQ ID NO: 16), HHEX (SEQ ID NO: 17, SEQ ID NO: 18), HOXA5 (SEQ ID NO: 19, SEQ ID NO: 20), ID3 (SEQ ID NO: 21, SEQ ID NO: 22), IKZF1 (SEQ ID NO: 23, SEQ ID NO: 24), IKZF2 (SEQ ID NO: 25), IKZF3 (SEQ ID NO: 26), IKZF4 (SEQ ID NO: 27), IKZF5 (SEQ ID NO: 28), IKZF6 (SEQ ID NO: 29), IKZF7 (SEQ ID NO: 30), IKZF8 (SEQ ID NO: 31), IKZF9 (SEQ ID NO: 32), IKZF10 (SEQ ID NO: 33), IKZF11 (SEQ ID NO: 34), IKZF12 (SEQ ID NO: 35), IKZF13 (SEQ ID NO: 36), IKZF14 (SEQ ID NO: 37), IKZF15 (SEQ ID NO: 38), IKZF16 (SEQ ID NO: 39), IKZF17 (SEQ ID NO: an expression vector selected from among: IKZF3 (SEQ ID NO: 25, SEQ ID NO: 26), IKZF3 (SEQ ID NO: 27, SEQ ID NO: 28), IKZF5 (SEQ ID NO: 29, SEQ ID NO: 30), IRF7 (SEQ ID NO: 31, SEQ ID NO: 32), MEF2C (SEQ ID NO: 33, SEQ ID NO: 34), MYB (SEQ ID NO: 35, SEQ ID NO: 36), RUNX2 (SEQ ID NO: 37, SEQ ID NO: 38), STAT3 (SEQ ID NO: 39, SEQ ID NO: 40), TCF4 (SEQ ID NO: 41, SEQ ID NO: 42), TCF12 (SEQ ID NO: 43, SEQ ID NO: 44), STAT1 (SEQ ID NO: 45, SEQ ID NO: 46), and TSC22D1 (SEQ ID NO: 47, SEQ ID NO: 48); and (b) Packaging and its instructions.

[0184] In some embodiments, the kits described herein can further provide the synthetic mRNA or one or more expression vectors encoding pDC-inducing factors, which can be provided in the form of a mixture or in individual aliquots.

[0185] In some embodiments, the kits can further comprise agents that increase the efficiency of reprogramming, hi some embodiments, the kits can further comprise one or more antibodies or primer reagents for detecting cell type-specific markers that identify cells induced to the pDC state.

[0186] In some embodiments, the kit can further comprise a buffer. In some such embodiments, the buffer is ribonuclease-free TE buffer (pH 7.0). In some embodiments, the kit further comprises a container containing cell culture medium.

[0187] Any of the kits described herein further include buffers, cell culture media, transduction or transformation media, and / or media additives. In preferred embodiments, the buffers, cell culture media, transformation media, and / or media additives are deoxyribonuclease-free and ribonuclease-free. In some embodiments, the synthetic, modified RNA provided in the kit may be in a specific amount or mass of non-solution form, such as 20 μg of lyophilized form, such that the end user can add an appropriate amount of buffer or medium to achieve a desired concentration of the components, such as 100 ng / μl.

[0188] Any of the kits described herein can further include a device that facilitates single administration or repeated or multiple infusions of cells generated using the kit components described herein, such as a non-implantable delivery device (e.g., a needle, syringe, pen device) or an implantable delivery device (e.g., a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intracapsular), or a reservoir). In some such embodiments, the delivery device can include a mechanism for dispensing a unit dose of a pharmaceutical composition comprising induced pDCs. In some embodiments, the device releases the composition continuously, e.g., by diffusion. In some embodiments, the device can include a sensor in the subject that monitors a parameter. For example, the device can include a pump, and, e.g., optionally, associated electronics.

[0189] In one embodiment, induced pDCs are artificially generated, for example, by altering gene expression in somatic, pluripotent, progenitor, or stem cells of at least one of the factors disclosed herein, or by exposing any one of these cell types to at least one protein or RNA that produces at least one protein, as disclosed herein. Additionally, the cells may be further generated by exposure to a small molecule that activates at least one of the factors disclosed herein. In some aspects, at least two, three, four, five, six, seven, or eight factors are used to generate induced pDCs.

[0190] In one embodiment, data on the expression of Clec9a, Tlr7, Tlr9, and candidate transcription factors in DC subsets and other myeloid cells (monocytes, macrophages, and microglia) are obtained from the Immgen database (www.immgen.org). Expression data were analyzed using cluster 3.0 (log-transformed data; centered genes; centered, clustered, and normalized cell types) and displayed in Treeview. Alternatively, expression values ​​were displayed using GraphPad Prism.

[0191] In one experiment, mouse embryonic fibroblasts (MEFs) were isolated and purified as follows: Clec9aCre / Cre animals (10) were crossed with Rosa26-stopflox-tdTomato reporter mice (Jackson Laboratory) to generate double homozygous Clec9aCre / Cre RosatdTomato / tdTomato (Clec9A-tdTomato) mice. All animals were housed at controlled temperature (23±2°C) under a fixed 12-h light / dark cycle with free access to food and water.

[0192] In one experiment, primary cultures of MEFs were isolated from E13.5 embryos of Clec9A-tdTomato or C57BL / 6 mice (6, 10). The head, fetal liver, and all other organs were removed, and the remaining tissues were mechanically dissociated. Dissected tissues were enzymatically digested with 0.12% trypsin / 0.1 mM ethylenediaminetetraacetic acid (EDTA) solution (3 mL per embryo). Incubation was performed at 37°C for 15 minutes. An additional 3 mL of the same solution was added per embryo, followed by another 15-minute incubation. A single-cell suspension was obtained and plated in growth medium onto 10 cm tissue culture dishes coated with 0.1% gelatin. Cells were grown for 2–3 days until the dish surface was densely covered, after which they were dissociated using Tryple Express and frozen in fetal bovine serum (FBS) / 10% dimethyl sulfoxide (DMSO). Prior to seeding for lentiviral transduction, MEFs were sorted to remove any remaining CD45+ and tdTomato+ cells (potentially hematopoietic cells).

[0193] In one embodiment, HEK293T cells and 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. All tissue culture reagents were from Thermo Fisher Scientific unless otherwise stated.

[0194] In one experiment, viral transduction and reprogramming experiments were performed as follows: Clec9A-tdTomato MEFs were seeded at a density of 40,000 cells per well in 6-well plates coated with 0.1% gelatin. Cells were incubated overnight with FUW-TetO-transcription factor and FUW-M2rtTA lentiviral particles at a 1:1 ratio in growth medium supplemented with 8 μg / mL polybrene. For transcription factor combination tests, the same MOI was used for each viral particle. Cells were transduced twice on consecutive days, and after overnight incubation, the medium was replaced with fresh growth medium. After the second transduction, doxycycline (1 μg / mL) was added to the growth medium (day 0). The medium was replaced every 2–3 days during the culture period. The appearance of tdTomato+ cells was analyzed 5–8 days after transduction.

[0195] In one embodiment, flow cytometry analysis was performed as follows: transduced Clec9A-tdTomato MEFs were dissociated with TrypLE Express, resuspended in 200 μL of PBS (containing 5% FBS), and stored at 4°C before analysis on a BD FACS LSR II (BD Biosciences). For analysis of MHC-II cell surface marker expression, dissociated cells were incubated with APC-conjugated rat anti-mouse IA / IE antibody (Biolegend) or APC-Cy7-conjugated anti-CD45 antibody (Biolegend) diluted in PBS (containing 5% FBS) for 30 minutes at 4°C; this incubation was performed in the presence of rat serum (1 / 100, GeneTex) to block nonspecific binding. Cells were washed with PBS (containing 5% FBS) and resuspended in PBS (containing 5% FBS) before analysis on a BD FACS LSR II. Flow cytometry data were analyzed using FlowJo software (FLOWJO, LLC, version 7.6).

[0196] In one embodiment, fluorescence-activated cell sorting (FACS) was performed as follows: To purify Clec9A-tdTomato MEFs, cells were incubated with APC-Cy7-conjugated anti-CD45 antibody (Biolegend) diluted in PBS (containing 5% FBS) for 30 minutes at 4°C. MEFs were then washed with PBS (containing 5% FBS) and resuspended in PBS (containing 5% FBS), after which tdTomato-CD45- MEFs were purified using a BD FACSAria III. When tdTomato+ cells are described, the cells were purified using a BD FACSAria III. FACS data were processed using FlowJo software.

[0197] In one experiment, fresh spleens from Clec9a-tdTomato mice were ground between two sterile slides with frosted edges. Cells were collected in PBS containing 5% FBS and filtered through a 70 μm cell strainer (BD Biosciences). Red blood cells were lysed at room temperature for 8 minutes using BD Pharm Lyse (BD Biosciences). The hemolysis reaction was stopped by adding at least 5 volumes of PBS (containing 5% FBS). Spleen cells were washed once to obtain a single-cell suspension. These suspensions were incubated with a mixture of FITC-conjugated anti-CD11c antibody (Biolegend), APC-conjugated anti-Bst2 antibody (Biolegend), APC-Cy7-conjugated anti-B220 antibody (Biolegend), and BV605-conjugated anti-Siglec-H antibody (Biolegend) diluted in PBS (containing 5% FBS) in the presence of rat serum at 4°C for 30 minutes. Single staining and negative fluorescence controls were also included for proper compensation and gating. After 30 minutes at 4°C, cells were washed and resuspended in PBS (containing 3% FBS) and analyzed on a BD FACSAria III (BDiosciences). Data from this analysis were processed using FlowJo software. Splenic pDCs were analyzed as single live cells, CD11clow, Bst2+, B220+, Siglec-H. + was selected as.

[0198] In one experiment, inflammatory cytokine assays were performed as follows: tdTomato+ cells were sorted by FACS on day 9 and cultured overnight with 1 μg / mL R848 (Invivogen) and 0.5 μM CpG ODN1668 (Invivogen) for TLR7 and TLR9 stimulation, respectively. The levels of cytokines interferon-α (IFN-α), interferon-β (IFN-β), interleukin-10 (IL-10), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), CC motif chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 10 (CXCL10) were assessed on day 10. 50 μL of culture supernatant was collected from wells of a 96-well plate and analyzed using the LEGENDplex Mouse Antiviral Response Panel (13-plex) (Biolegend) in a V-plate according to the manufacturer's instructions. Data acquisition was performed using a BD FACS LSR II, and data analysis was performed using LEGENDplex Data Analysis, version 8.0 (Biolegend).

[0199] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the above description, but rather is set forth in the appended claims.

[0200] In the specification of a patent claim, when the singular form of an element or feature is used, it also includes the plural form, and vice versa, unless specifically excluded. For example, the term "transcription factor" or "transcription factor" includes the plural form "transcription factors" or "transcription factors," and vice versa. In the patent claims, unless otherwise indicated or the context clearly indicates otherwise, articles such as "a," "an," and "the" may mean one or more than one. Unless otherwise indicated or the context clearly indicates otherwise, claims or statements including "or" between one or more members of a group are deemed to be valid regardless of whether a single member, multiple members, or all members of the group are present in, used in, or related to a particular product or process. The invention includes embodiments in which only a single member of the group is present in, used in, or associated with a particular product or process, as well as embodiments in which more than one or all of the members of the group are present in, used in, or associated with a particular product or process.

[0201] Furthermore, it should be understood that any variation, combination, or modification that adds to another claim one or more limitations, elements, clauses, descriptive language, etc., contained in one or more claims or in the relevant portion of the description is encompassed by the present invention. For example, any claim that is dependent on another claim can be modified to include one or more limitations that are present in any of the other claims that are dependent on the same base claim.

[0202] Furthermore, when a claim recites a composition, it should be understood that it encompasses methods of using that composition for any purpose disclosed herein, and methods of making that composition, either according to the methods disclosed herein or other methods known in the art, unless a potential conflict or inconsistency is specifically indicated or would be apparent to one of ordinary skill in the art.

[0203] When ranges are presented, they are inclusive of their endpoints. Furthermore, unless otherwise expressly stated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to mean that any particular value falling within the stated range can be considered to be up to one-tenth of the unit of the lower limit of that range, unless the context clearly indicates otherwise in different embodiments of the invention. Also, unless otherwise expressly stated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to mean that any particular value falling within that range can be considered to be up to one-tenth of the unit of the lower limit of that range, if the endpoint of that subrange within that particular range is expressed to the same degree of accuracy as one-tenth of the unit of the lower limit of that range.

[0204] The present disclosure should not be understood as being limited to the described embodiments, as those skilled in the art will envision numerous possibilities for modifying them.

[0205] The above embodiments can be combined.

[0206] The table below provides a summary of the sequences included in this disclosure.

[0207] [Table 1] TIFF0007728755000002.tif63159

[0208] The following claims further define and define particular embodiments of the present disclosure.

Claims

1. A composition comprising at least two transcription factors, IRF8 and SPIB, for use in reprogramming stem cells or differentiated cells or a mixture thereof to convert them into plasmacytoid dendritic cells in a subject.

2. 2. The composition of claim 1, further comprising one or more other transcription factors selected from the group consisting of ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, IKZF2, STAT1, TCF4, TCF12 and TSC22D1.

3. 3. The composition of claim 1 or 2, wherein the transcription factors are individually at least 90% identical to a sequence selected from the group consisting of IRF8 (SEQ ID NO:49, SEQ ID NO:50), SPIB (SEQ ID NO:51, SEQ ID NO:52), ARID5A (SEQ ID NO:53, SEQ ID NO:54), BCL11A (SEQ ID NO:55, SEQ ID NO:56), CBFA2T3 (SEQ ID NO:57, SEQ ID NO:58), CREB3L2 (SEQ ID NO:59, SEQ ID NO:60), ETS1 (SEQ ID NO:61, SEQ ID NO:62), IKZF1 (SEQ ID NO:71, SEQ ID NO:72), IKZF2 (SEQ ID NO:73, SEQ ID NO:74), TCF4 (SEQ ID NO:89, SEQ ID NO:90), TCF12 (SEQ ID NO:91, SEQ ID NO:92), STAT1 (SEQ ID NO:93, SEQ ID NO:94), and TSC22D1 (SEQ ID NO:95, SEQ ID NO:96).

4. The composition according to any one of claims 1 to 3, wherein the composition comprises: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and IKZF2; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; IRF8, SPIB and TSC22D1, or mixtures thereof, A composition comprising a combination of transcription factors selected from the combinations of:

5. 5. The composition of claim 4, wherein the combination of transcription factors is a combination of IRF8, SPIB and TCF12.

6. 5. The composition of claim 4, wherein the combination of transcription factors is a combination of IRF8, SPIB, and TSC22D1.

7. 5. The composition of claim 4, wherein the combination of transcription factors is a combination of IRF8, SPIB, and IKZF2.

8. 8. The composition of any one of claims 1 to 7, wherein the stem cells or differentiated cells or mixtures thereof are selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, tumor cells, cancer cells, and mixtures thereof.

9. A composition according to any one of claims 1 to 8 for use in veterinary or human medicine.

10. The composition according to any one of claims 1 to 9, for use in the treatment of an autoimmune disease, cancer or an infectious disease.

11. 1. One or more constructs or vectors encoding at least two transcription factors, said two transcription factors being SPIB and IRF8.

12. 12. One or more constructs or vectors according to claim 11, wherein said vectors are viral vectors.

13. 13. One or more constructs or vectors according to any one of claims 11 to 12, wherein said one or more constructs or vectors are synthetic mRNA, naked alphavirus RNA replicon or naked flavivirus RNA replicon.

14. A composition comprising one or more vectors, wherein said vectors comprise at least two polynucleotide sequences, said vectors encoding at least two transcription factors IRF8 and SPIB in combination, said composition being for use in reprogramming stem cells or differentiated cells or a mixture thereof into plasmacytoid dendritic cells in a subject.

15. 15. The composition of claim 14, wherein the vector further encodes one or more other transcription factors selected from the group consisting of ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, IKZF2, STAT1, TCF4, TCF12 and TSC22D1.

16. 16. The composition of any one of claims 14-15, wherein the transcription factors are individually at least 90% identical to a sequence selected from the group consisting of IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4), ARID5A (SEQ ID NO:5, SEQ ID NO:6), BCL11A (SEQ ID NO:7, SEQ ID NO:8), CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10), CREB3L2 (SEQ ID NO:11, SEQ ID NO:12), ETS1 (SEQ ID NO:13, SEQ ID NO:14), IKZF1 (SEQ ID NO:23, SEQ ID NO:24), IKZF2 (SEQ ID NO:25, SEQ ID NO:26), TCF4 (SEQ ID NO:41, SEQ ID NO:42), TCF12 (SEQ ID NO:43, SEQ ID NO:44), STAT1 (SEQ ID NO:45, SEQ ID NO:46), and TSC22D1 (SEQ ID NO:47, SEQ ID NO:48).

17. The composition according to any one of claims 14 to 16, wherein the one or more vectors are: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and IKZF2; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; IRF8, SPIB and TSC22D1, or mixtures thereof, A composition encoding a combination of transcription factors selected from the combinations of:

18. The composition according to any one of claims 14 to 17, wherein the combination of transcription factors is a combination of IRF8, SPIB and TCF12.

19. 18. The composition of any one of claims 14 to 17, wherein the combination of transcription factors is a combination of IRF8, SPIB, and TSC22D1.

20. 18. The composition of any one of claims 14 to 17, wherein the combination of transcription factors is a combination of IRF8, SPIB, and IKZF2.

21. The composition of any one of claims 14 to 20, wherein the vector is a viral vector.

22. The composition of any one of claims 14 to 21, wherein the vector is a synthetic mRNA, a naked alphavirus RNA replicon, or a naked flavivirus RNA replicon.

23. 23. The composition of any one of claims 14 to 22, wherein the stem cells or differentiated cells or mixtures thereof are selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, tumor cells, cancer cells, and mixtures thereof.

24. A composition according to any one of claims 14 to 23, for use in veterinary or human medicine.

25. The composition according to any one of claims 14 to 24, for use in the treatment of an autoimmune disease, cancer or an infectious disease.

26. An in vitro method for reprogramming or inducing cells into plasmacytoid dendritic cells, the method comprising transducing cells selected from the group consisting of stem cells, differentiated cells, and mixtures thereof with one or more vectors comprising at least two transcription factors selected from the group consisting of IRF8 and SPIB.

27. 27. The method of claim 26, wherein the one or more vectors further comprise one or more transcription factors selected from the group consisting of ARID5A, BCL11A, CBFA2T3, CREB3L2, ETS1, IKZF1, IKZF2, STAT1, TCF4, TCF12 and TSC22D1.

28. 28. The method of any one of claims 26-27, wherein the transcription factors are individually at least 90% identical to a sequence selected from the group consisting of IRF8 (SEQ ID NO:1, SEQ ID NO:2), SPIB (SEQ ID NO:3, SEQ ID NO:4), ARID5A (SEQ ID NO:5, SEQ ID NO:6), BCL11A (SEQ ID NO:7, SEQ ID NO:8), CBFA2T3 (SEQ ID NO:9, SEQ ID NO:10), CREB3L2 (SEQ ID NO:11, SEQ ID NO:12), ETS1 (SEQ ID NO:13, SEQ ID NO:14), IKZF1 (SEQ ID NO:23, SEQ ID NO:24), IKZF2 (SEQ ID NO:25, SEQ ID NO:26), TCF4 (SEQ ID NO:41, SEQ ID NO:42), TCF12 (SEQ ID NO:43, SEQ ID NO:44), STAT1 (SEQ ID NO:45, SEQ ID NO:46), and TSC22D1 (SEQ ID NO:47, SEQ ID NO:48).

29. 29. The method of any one of claims 26 to 28, wherein the one or more vectors comprise: IRF8 and SPIB; IRF8, SPIB and ARID5A; IRF8, SPIB and BCL11A; IRF8, SPIB and CBFA2T3; IRF8, SPIB and CREB3L2; IRF8, SPIB and ETS1; IRF8, SPIB and IKZF1; IRF8, SPIB and IKZF2; IRF8, SPIB and STAT1; IRF8, SPIB and TCF4; IRF8, SPIB and TCF12; IRF8, SPIB and TSC22D1, or mixtures thereof, The method comprises a combination of transcription factors selected from the combinations:

30. The method according to any one of claims 26 to 29, wherein the transduced cells are cultured for 2 to 9 days.

31. The method of any one of claims 26 to 30, wherein the transduction step further comprises at least one vector selected from the group consisting of a nucleic acid sequence encoding IL-12; a nucleic acid sequence encoding IL-4; a nucleic acid sequence encoding IFN-α; a nucleic acid sequence encoding IFN-β; a nucleic acid sequence encoding IFN-γ; a nucleic acid sequence encoding TNF; a nucleic acid sequence encoding GM-CSF; a nucleic acid sequence encoding siRNA targeting IL-10 RNA, and mixtures thereof.

32. The method according to any one of claims 26 to 31, wherein the stem cells or differentiated cells or mixtures thereof are mammalian, human, non-human or murine cells selected from the group consisting of pluripotent stem cells, multipotent stem cells, and differentiated cells; wherein the pluripotent stem cells, multipotent stem cells, or differentiated cells are selected from the group consisting of endoderm-derived cells, mesoderm-derived cells, ectoderm-derived cells, and pluripotent stem cells, including mesenchymal stem cells, hematopoietic stem cells, intestinal stem cells, pluripotent stem cells, and cell lines.

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