Compositions, methods and uses for reprogramming cells into type 2 dendritic cells competent for antigen presentation

Abstract

The present disclosure relates to compositions, methods, and uses for reprogramming cells into conventional dendritic cells (cDCs), particularly cDC2 type (hereinafter referred to as "cDC2" or "CD11b-positive dendritic cells"). The present disclosure relates to the development of methods for generating conventional dendritic cells with antigen-presenting ability from differentiated stem cells, multipotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthetic transcription factors. More particularly, the present disclosure provides methods for obtaining conventional dendritic cells (cDCs), particularly cDC2 type or CD11b-positive dendritic cells, by directly reprogramming cells using a surprising combination of specific transcription factors.

Description

[Technical Field]

[0001] Technical Field The present disclosure relates to compositions, methods and uses thereof for reprogramming cells into conventional dendritic cells (cDCs), in particular, cDC2 type (hereinafter referred to as "cDC2s" or "CD11b-positive dendritic cells").

[0002] The present disclosure relates to the development of a method for generating conventional dendritic cells with antigen-presenting ability from differentiated stem cells, multipotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthetic transcription factors. More particularly, the present disclosure provides a method for obtaining conventional dendritic cells (cDCs), particularly cDC2-type or CD11b-positive dendritic cells, by directly reprogramming cells using a surprising combination of specific transcription factors. [Background technology]

[0003] background Cellular reprogramming relies on rewiring the epigenetic transcriptional network of one cell state to that of a different cell type. Transcription factor (TF) overexpression experiments highlight the plasticity of adult somatic or differentiated cells and provide a new technique for generating any desired cell type. Through forced expression of TFs, somatic or differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which are remarkably similar to embryonic stem cells (ESCs) (Takahashi et al., 2007; Takahashi & Yamanaka, 2006). Alternatively, somatic cells can be directly converted into other specialized cell types (Pereira, Lemischka, & Moore, 2012). Direct lineage conversion has proven successful, using TFs to target cell types, successfully reprogramming mouse and human fibroblasts into several cell types, including neurons, cardiomyocytes, and hepatocytes (Xu, Du, & Deng, 2015). Direct cell conversion has also been demonstrated in the hematopoietic system, where forced expression of TFs induces macrophage fate in B cells and fibroblasts (Xie, Ye, Feng, & Graf, 2004), and direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors was achieved using Gata2, Gfi1b, cFos, and Etv6 (Pereira et al., 2013). These four TFs induce a dynamic, multistep hematopoietic process that progresses through an endothelial-like intermediate, recapitulating hematopoietic development in vitro (Pereira et al., 2016).

[0004] Reprogrammed cells are very promising therapeutic tools for regenerative medicine, and cells obtained by differentiation of iPSCs are already being tested in clinical studies.

[0005] Cellular reprogramming schemes highlight the flexibility of cell fate, with the potential to use cell type-specific TFs to convert somatic cells to pluripotency. Direct lineage conversion from one differentiated cell type to another has also been demonstrated and explored for purposes of elucidating cell biology mechanisms and regenerative medicine. Recently, it has been demonstrated that antigen-presenting dendritic cells can be reprogrammed from unrelated cell types by a small combination of TFs. Classically, myeloid DC-committed progenitors are said to give rise to functionally distinct DC subsets: conventional DCs (cDCs), which are professional antigen-presenting cells (APCs), and plasmacytoid DCs (pDCs). While cDCs promote antigen-specific immune responses, pDCs are specialized producers of type I interferon during viral infection. However, the timing and precise mechanisms that coordinate the divergence of different subsets during DC development have yet to be established.

[0006] DCs are a class of bone marrow-derived cells arising from lymphomyelomyelopoiesis. They scan the organism for pathogens and form an essential boundary between the activation of the innate and adaptive immune systems. DCs act as professional APCs capable of activating T cell responses by presenting peptide antigens complexed with major histocompatibility complexes (MHC) on their surface, along with all necessary soluble and membrane-bound costimulatory molecules. DCs induce primary immune responses, enhance the effector function of previously primed T lymphocytes, and coordinate the communication between innate and adaptive immunity. DCs are found in most tissues, continuously sampling the antigenic environment and monitoring pathogen invasion using several types of receptors. At steady state, and at an increasing rate upon pathogen detection, sentinel DCs in nonlymphoid tissues migrate to lymphoid organs, where they collect and process antigens and present them to T cells. The phenotype acquired by T cells depends on the context of antigen presentation. When antigens are pathogen-derived or self-damaged, DCs receive a danger signal, become activated, and subsequently stimulate T cells, becoming the effectors required to provide protective immunity.

[0007] A key aspect of immune response regulation is the existence of several different types of DCs, each specialized to respond to a particular pathogen and interact with a specific subset of T cells. In this context, three major DC subsets arise: plasmacytoid DCs (pDCs), myeloid / conventional DC1 (cDC1), and myeloid / conventional DC2 (cDC2). This expands the flexibility of the immune system to respond appropriately to a wide range of different pathogens and danger signals.

[0008] cDC2s are characterized by CD11b surface expression and are distinct from naive CD4 + They specialize in MHC-II presentation, which directs T cell polarization toward the Th2 and Th17 helper cytokines (Plantinga et al., 2013). Th2-associated cytokines (IL-4, IL-5, IL-9, and IL-13) mediate responses related to protection against extracellular parasites (Mosmann & Coffman, 1989) and induce allergic and hypersensitivity reactions (Kopf et al., 1993; Zhu & Paul, 2008), while Th17 cytokines are associated with immune responses to extracellular bacteria and fungi and also induce many autoimmune diseases (Weaver, Harrington, Mangan, Gavrieli, & Murphy, 2006). In tumors, cDC2s express CD4+ on MHC-II in tumor-draining lymph nodes. + They are known to complement cDC1 by participating in antigen presentation to T cells (Merad, Sathe, Helft, Miller, & Mortha, 2013). One study demonstrated that antigen presentation by tumor-derived cDC2 promotes the conversion of tumor-associated macrophages to an anti-tumor phenotype on Th17-dependent substrates (Laoui et al., 2016). cDC2 also express autoreactive CD4 + It contributes to the downregulation of effector T cells by priming regulatory T cells (Tregs), which are essential for maintaining self-tolerance by destroying T cells ( Merad et al., 2013 ) and negatively regulating immune responses ( Sakaguchi, 2004 ).

[0009] Additional cDC2 characteristic markers include CD11b, Sirpα, CD4, and ESAM. Due to the inherent heterogeneity of cDC2s, several specific surface markers characterize specific subsets. Recent findings have identified two distinct subsets of cDC2s, defined by distinct transcriptional regulators and distinct immunological functions (Brown et al., 2019). cDC2A is an anti-inflammatory subset defined as Tbet-dependent and characterized by surface expression of ESAM and Clec4a4, whereas cDC2B consists of a significantly pro-inflammatory RORγt-defined subset expressing Clec10a and Clec12a markers.

[0010] The ability of DCs to induce adaptive immunity has spurred research into DC vaccine strategies against bacterial, viral, and parasitic pathogens, as well as immunotherapy in cancer. Indeed, clinical trials utilizing DC-mediated immunotherapy are ongoing for several tumor types, including solid and hematologic tumors (Datta et al., 2014). However, clinical outcomes have been inconsistent, likely associated with the variable efficiency of in vitro-generated DCs. Autologous monocytes can differentiate into DCs in vitro with low efficiency, and very few hematopoietic progenitor cells are isolated. In addition, these progenitor cells are commonly impaired in cancer-bearing patients, resulting in the generation of dysfunctional DCs (Datta et al., 2014; Subklewe et al., 2014). Cancer escape mechanisms may also underlie the lack of consistent therapeutic benefit of DC-based immunotherapy. During tumor progression, cancer cells exploit several immunological processes to escape immune surveillance. These adaptations, together with the heterogeneity of cancer antigens, hinder the recognition of tumor antigens by the immune system, and consequently contribute to the reduced immunogenicity of tumor cells and current immunotherapies.

[0011] The generation of APCs by direct reprogramming opens new opportunities to better understand DC specification and cellular identity, contributing to more efficient control of immune responses using autologous engineered cells.

[0012] Document EP3385373 relates to compositions, nucleic acid constructs, methods and kits for inducing or reprogramming cells to a DC or APC state, which are based in part on the surprising effects of novel uses and combinations of TFs that allow inducing or reprogramming differentiated or undifferentiated cells to DC or APC.

[0013] The reprogrammed cells generated as described in EP3385373 specifically recapitulate the surface marker expression, antigen presentation, cytokine release, and T cell activation characteristics of the cDC1 subset of DCs. The phenotypic characteristics of other DC subsets were not described.

[0014] Antigen-presenting cells (APCs) are a heterogeneous group of immune cells that mediate cellular immune responses by processing and presenting antigens for recognition by certain lymphocytes, such as T cells. Classical APCs include dendritic cells, macrophages, Langerhans cells, and B cells.

[0015] DCs provide a critical link between the external environment and the adaptive immune system through their ability to capture, process, and present antigens to T cells, targeting them to different types of immune responses or inducing tolerance responses.

[0016] Phenotypic criteria allow for the classification of mouse DCs into distinct subpopulations characterized by the expression of distinct surface markers. Conventional DCs (cDCs) in lymphoid tissues are traditionally subdivided into cDC1 and cDC2 subpopulations. Different DC subsets are involved in the specific recognition of certain pathogens and / or regulate different immune responses. cDC1 has been associated with priming Th1 responses, which are important for promoting tumor clearance, whereas the cDC2 subset has been associated with Th1, Th2, Th17 (immune), and Treg (tolerance) responses.

[0017] Document EP3385373 relates to compositions, nucleic acid constructs, methods and kits for inducing or initializing cells to a DC or APC state, which are based in part on the surprising effects of novel uses and combinations of TFs that allow inducing or initializing differentiated or undifferentiated cells to DC or APC, more particularly cDC1. Summary of the Invention [Problem to be solved by the invention]

[0018] Currently, DC-based immunotherapy relies on autologous DC precursors, either monocytes, which are associated with inefficient DC production, or hematopoietic progenitor cells, which are isolated in very low numbers. Furthermore, these precursor cells are commonly impaired in cancer-bearing patients, resulting in the generation of dysfunctional DCs. Meanwhile, nonhematopoietic cell types, such as fibroblasts, are typically unaffected. Given the fundamental role of DCs as APCs linking the innate and adaptive immune systems, there remains a clinical need to find alternative strategies for generating functional DCs to prime antigen-specific immune responses. [Means for solving the problem]

[0019] Inducible DCs or APCs generated by the direct reprogramming of the present disclosure surprisingly recapitulate the phenotype of the cDC2 subset of DCs in terms of surface marker expression, cytokine secretion and antigen presentation in MHC-II molecules.

[0020] These facts are disclosed to explain the technical problem addressed by the present disclosure.

[0021] The present subject matter surprisingly identifies several isolated or synthetic TFs that reprogram or induce differentiated cells, multipotent stem cells or pluripotent stem cells into antigen-presenting dendritic cells, more specifically cDC2s, in vitro, ex vivo or in vivo. [Effects of the Invention]

[0022] In one aspect, the present disclosure provides a method for reprogramming stem cells or differentiated cells, or a mixture thereof, into conventional dendritic cells type 2 (cDC2) or CD11b-positive dendritic cells, using PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11), PRDM1 (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17), IRF2 (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23), POU2F2 (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29), TGIF1 (SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35), and / or TGIF1 (SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39) for reprogramming stem cells or differentiated cells, or a mixture thereof, into conventional dendritic cells type 2 (cDC2) or CD11b-positive dendritic cells. or a combination of at least two isolated or synthetic transcription factors that are at least 90% identical to a sequence selected from the list consisting of PU.1 (SEQ ID NO:3, SEQ ID NO:6), IRF4 (SEQ ID NO:9, SEQ ID NO:12), PRDM1 (SEQ ID NO:15, SEQ ID NO:18), IRF2 (SEQ ID NO:21, SEQ ID NO:24), POU2F2 (SEQ ID NO:27, SEQ ID NO:30), TGIF1 (SEQ ID NO:33, SEQ ID NO:36), and a composition comprising a mixture thereof.

[0023] In one aspect, the disclosure includes a composition comprising a combination of at least three isolated or synthetic transcription factors for use in reprogramming stem cells or differentiated cells, or a mixture thereof, into conventional dendritic cells type 2 (cDC2) or CD11b-positive dendritic cells, wherein the first and second transcription factors are isolated and synthetic PU.1 and IRF4 transcription factors that are at least 90% identical to the sequences of PU.1 (SEQ ID NO:3, SEQ ID NO:6) and IRF4 (SEQ ID NO:9, SEQ ID NO:12), and the third transcription factor is an isolated or synthetic transcription factor that is at least 90% identical to a sequence selected from the group consisting of PRDM1 (SEQ ID NO:15, SEQ ID NO:18), IRF2 (SEQ ID NO:21, SEQ ID NO:24), POU2F2 (SEQ ID NO:27, SEQ ID NO:30), TGIF1 (SEQ ID NO:33, SEQ ID NO:36), RBPJ (SEQ ID NO:45, SEQ ID NO:48), and RELB (SEQ ID NO:39, SEQ ID NO:42).

[0024] Variant, as used herein, refers to a sequence having 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to a DNA coding sequence of the present disclosure.

[0025] In a further embodiment, the present disclosure provides an isolated dendritic cell that is at least 90% identical to, and selected from the group consisting of, PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), for use in reprogramming stem cells or differentiated cells, or a mixture thereof, into conventional dendritic cells type 2 (cDC2). The present invention also includes compositions comprising at least two transcription factors encoded by isolated or synthetic sequences, or at least two isolated or synthetic transcription factors that are at least 90% identical to a sequence selected from the group consisting of PU.1 (SEQ ID NO:3, SEQ ID NO:6), IRF4 (SEQ ID NO:9, SEQ ID NO:12), PRDM1 (SEQ ID NO:15, SEQ ID NO:18), IRF2 (SEQ ID NO:21, SEQ ID NO:24), POU2F2 (SEQ ID NO:27, SEQ ID NO:30), and TGIF1 (SEQ ID NO:33, SEQ ID NO:36), as well as mixtures thereof, except for the combination of at least two isolated or synthetic transcription factors consisting of PU.1 (SEQ ID NO:1 to SEQ ID NO:6) and IRF4 (SEQ ID NO:7 to SEQ ID NO:12).

[0026] In one embodiment, the present disclosure provides antibodies to PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11), PRDM1 (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17), IRF2 (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23), POU2F2 (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29), TGIF1 (SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35), RBPJ (SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47), and RELB (SEQ ID NO: 37, SEQ ID NO: 38), for use in reprogramming stem cells or differentiated cells into conventional dendritic cells type 2 (cDC2) or CD11b-positive dendritic cells. and at least two transcription factors encoded by an isolated or synthetic sequence at least 90% identical to a sequence selected from the group consisting of PU.1 (SEQ ID NO:3, SEQ ID NO:6), IRF4 (SEQ ID NO:9, SEQ ID NO:12), PRDM1 (SEQ ID NO:15, SEQ ID NO:18), IRF2 (SEQ ID NO:21, SEQ ID NO:24), POU2F2 (SEQ ID NO:27, SEQ ID NO:30), TGIF1 (SEQ ID NO:33, SEQ ID NO:36), RBPJ (SEQ ID NO:45, SEQ ID NO:48), and RELB (SEQ ID NO:39, SEQ ID NO:42), and mixtures thereof.

[0027] In one embodiment, the disclosure includes a composition for use as described hereinabove, wherein the transcription factors are encoded by polynucleotides that are at least 90% identical to the following sequences, individually: PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41).

[0028] In one embodiment, the present disclosure provides a gene encoding a nucleotide sequence at least 95% identical to the list consisting of PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), and or a combination of at least two isolated or synthetic transcription factors that are at least 95% identical to a sequence selected from the list consisting of PU.1 (SEQ ID NO:3, SEQ ID NO:6), IRF4 (SEQ ID NO:9, SEQ ID NO:12), PRDM1 (SEQ ID NO:15, SEQ ID NO:18), IRF2 (SEQ ID NO:21, SEQ ID NO:24), POU2F2 (SEQ ID NO:27, SEQ ID NO:30), TGIF1 (SEQ ID NO:33, SEQ ID NO:36), and mixtures thereof.

[0029] In one embodiment, the disclosure includes a composition for use as described hereinabove, wherein the transcription factors are individually selected from the following sequences: PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, Isolated or synthetic transcription factors encoded by polynucleotides at least 95% identical to PU.1 (SEQ ID NO: 3, SEQ ID NO: 6), IRF4 (SEQ ID NO: 9, SEQ ID NO: 12), PRDM1 (SEQ ID NO: 15, SEQ ID NO: 18), IRF2 (SEQ ID NO: 21, SEQ ID NO: 24), POU2F2 (SEQ ID NO: 27, SEQ ID NO: 30), TGIF1 (SEQ ID NO: 33, SEQ ID NO: 36), RBPJ (SEQ ID NO: 45, SEQ ID NO: 48), RELB (SEQ ID NO: 39, SEQ ID NO: 42), individually.

[0030] In further embodiments, the present disclosure provides a method for the production of a protein from an isolated or synthetic encoded combination of: PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF4 (SEQ ID NO:7-SEQ ID NO:12); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); IRF4 (SEQ ID NO:7-SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); IRF4 (SEQ ID NO:7-SEQ ID NO:12) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); IRF4 (SEQ ID NO:7-SEQ ID NO:12) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); IRF4 (SEQ ID NO:7-SEQ ID NO:12) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF4 (SEQ ID NO:7-SEQ ID NO:12); IRF2 (SEQ ID NO:19-SEQ ID NO:24) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); IRF2 (SEQ ID NO:19-SEQ ID NO:24) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12), PRDM1 (SEQ ID NO:13-SEQ ID NO:18), IRF2 (SEQ ID NO:19-SEQ ID NO:24), POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RBPJ (SEQ ID NO:43-SEQ ID NO:48); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RELB (SEQ ID NO:37-SEQ ID NO:42); or a mixture thereof The present invention includes a combination of at least two transcription factors selected from the following:

[0031] In one embodiment, the disclosure includes a composition for use as described above, wherein the combination of transcription factors is a combination of: PU.1, IRF4 and PRDM1; PU.1, IRF4 and IRF2; PU.1, IRF4 and POU2F2; PU.1, IRF4 and TGIF1; PU.1, IRF4 and RBPJ; and PU.1, IRF4 and RELB is selected from.

[0032] In one embodiment, the composition of the present disclosure comprises a protein selected from the group consisting of or comprising the following proteins: The transcription factors may include at least three transcription factors encoded by isolated or synthetic sequences that are at least 90% identical to a sequence selected from PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12), PRDM1 (SEQ ID NO:13-SEQ ID NO:18), IRF2 (SEQ ID NO:19-SEQ ID NO:24), POU2F2 (SEQ ID NO:25-SEQ ID NO:30), TGIF1 (SEQ ID NO:31-SEQ ID NO:36), and mixtures thereof.

[0033] In one embodiment, the present disclosure relates to a composition described herein, wherein the combination of transcription factors is PU.1, IRF4 and PRDM1 or PU.1, IRF4 and IRF2.

[0034] In further embodiments, the compositions of the disclosure are derived from the following isolated or synthetic proteins or encoded combinations of the following isolated or synthetic proteins: PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12), PRDM1 (SEQ ID NO:13-SEQ ID NO:18); IRF2 (SEQ ID NO:19-SEQ ID NO:24), POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12), PRDM1 (SEQ ID NO:13-SEQ ID NO:18), IRF2 (SEQ ID NO:19-SEQ ID NO:24), POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RBPJ (SEQ ID NO:43-SEQ ID NO:48); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RELB (SEQ ID NO:37-SEQ ID NO:42); or a mixture thereof The transcription factor may comprise a combination of transcription factors selected from:

[0035] In one embodiment, the combination of isolated or synthetic transcription factors is PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18).

[0036] In one embodiment, the combination of isolated or synthetic transcription factors is PU.1, IRF4, and PRDM1.

[0037] In a further embodiment, the combination of isolated or synthetic transcription factors is PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18) or PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and IRF2 (SEQ ID NO:19-SEQ ID NO:24).

[0038] In another embodiment, the combination of isolated or synthetic transcription factors is PU.1, IRF4 and PRDM1 or PU.1, IRF4 and IRF2.

[0039] In one embodiment, the compositions of the present disclosure may comprise stem or differentiated cells selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, fibroblasts, tumor cells, cancer cells, and mixtures thereof.

[0040] In one embodiment, the cells may be selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, fibroblasts, tumor cells, cancer cells, and mixtures thereof.

[0041] In further embodiments, the cells may be selected from the group consisting of tumor cells, cancer cells, and mixtures thereof.

[0042] In one embodiment, the antigen may be a cancer antigen, an autoantigen, an allergen, an antigen from a pathogenic and / or infectious organism.

[0043] In one embodiment, the compositions of the present disclosure may be used in veterinary or human medicine, particularly in immunotherapy, or in autoimmune diseases, immune deficiencies, or in neurodegenerative or aging diseases, in cancer or infectious diseases, or for drug screening.

[0044] In a further embodiment, the pluripotent, multipotent or differentiated stem cells are mammalian pluripotent, multipotent or differentiated cells, in particular murine or human cells.

[0045] One aspect of the present disclosure relates to a construct or vector encoding a combination of at least two isolated or synthetic transcription factors of the present disclosure, preferably an encoded isolated or synthetic combination of three transcription factors.

[0046] Another aspect of the present disclosure relates to constructs or vectors encoding the combinations of transcription factors described herein.

[0047] In further embodiments, the disclosure includes a construct or vector, wherein the combination of three isolated or synthetic transcription factors is in the following 5' to 3' sequence order: PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11) and PRDM1 (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17); PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11) and IRF2 (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23) PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29) and TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47); PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11) and RELB (SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41).

[0048] In further embodiments, the disclosure includes a construct or vector, wherein the encoded combination of transcription factors is in the following 5' to 3' sequence order: PU.1, IRF4 and PRDM1; PU.1, IRF4 and IRF2; PU.1, IRF4 and POU2F2; PU.1, IRF4 and TGIF1; PU.1, IRF4 and RBPJ; or PU.1, IRF4 and RELB.

[0049] In a further embodiment, the present disclosure includes a vector, which is a viral vector, in particular a retroviral, adenoviral, lentiviral, herpesviral, poxviral, or adeno-associated viral vector.

[0050] In one embodiment, the vector or construct is a synthetic mRNA, a naked alphavirus RNA replicon, or a naked flavivirus RNA replicon.

[0051] In one aspect of the present disclosure, the present disclosure relates to one or more vectors comprising at least three polynucleotide sequences encoding at least three transcription factors for use in reprogramming stem cells or differentiated cells into conventional dendritic cells type 2 (cDC2) or CD11b-positive dendritic cells, wherein the first and second transcription factors are PU.1 and IRF4, and the third transcription factor is selected from the group consisting of PRDM1, IRF2, POU2F2, RBPJ, RELB, and TGIF1.

[0052] In one embodiment, the disclosure relates to one or more vectors, wherein the transcription factors are individually encoded by a polynucleotide at least 90% identical to a sequence selected from the group consisting of PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41), and RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47).

[0053] In further embodiments, the disclosure includes one or more vectors, wherein the combination of encoded transcription factors is a combination of: PU.1, IRF4 and PRDM1; PU.1, IRF4 and IRF2; PU.1, IRF4 and POU2F2; PU.1, IRF4 and TGIF1; PU.1, IRF4 and RBPJ; or PU.1, IRF4 and RELB is selected from.

[0054] In one aspect, the disclosure relates to one or more vectors comprising at least three polynucleotide sequences encoding at least three transcription factors, wherein the transcription factors are PU.1, IRF4, and PRDM1.

[0055] In one embodiment, the disclosure comprises one or more vectors, wherein the one or more vectors are viral vectors, in particular retroviral, adenoviral, lentiviral, herpesviral, poxviral, paramyxoviral, rhabdoviral, alphaviral, flaviviral or adeno-associated viral vectors.

[0056] In one embodiment, the disclosure includes one or more vectors, wherein the one or more vectors are synthetic mRNA, naked alphavirus RNA replicon, or naked flavivirus RNA replicon.

[0057] In one embodiment, the disclosure comprises one or more vectors, wherein the cells are selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, tumor cells, cancer cells, and mixtures thereof.

[0058] In one embodiment, the disclosure comprises one or more vectors for use in veterinary or human medicine, particularly in immunotherapy, or in the treatment or therapy of neurodegenerative diseases, or autoimmune diseases, immune deficiencies, or in the treatment or therapy of cancer, or in the treatment or therapy of infectious diseases; in intradermal and transdermal therapy; in immunotherapy, or in neurodegenerative diseases or ageing diseases, or in cancer or infectious diseases, for use as drug screening; or in the treatment, therapy or diagnosis of central and peripheral nervous system disorders, neoplasms, particularly cancer, i.e. solid or hematological tumors, immune diseases, particularly autoimmune diseases, hypersensitivity, or immune deficiencies; of fungal, viral, chlamydial, bacterial, nanobacterial or parasitic infections; infections by HIV, SARS coronavirus, Asian influenza virus, herpes simplex, shingles, hepatitis, or viral hepatitis.

[0059] Another aspect of the present disclosure is a method for reprogramming or inducing stem cells or differentiated cells into conventional dendritic cells type 2, 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 comprising at least two nucleic acid sequences encoding sequences at least 90% identical, preferably at least 95% identical, to a sequence from the group consisting of PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41), and mixtures thereof; Culturing the transduced cells in a cell culture medium that supports the growth of dendritic cells or antigen-presenting cells.

[0060] Another aspect of the present disclosure is a method for reprogramming or inducing stem cells or differentiated cells into conventional dendritic cells type 2, 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 encoding at least three transcription factors, wherein the first and second transcription factors are PU.1 and IRF4, and the third transcription factor is selected from the group consisting of PRDM1, IRF2, POU2F2, TGIF1, RELB, and RBPJ, and mixtures thereof; Culturing the transduced cells in a cell culture medium that supports the growth of dendritic cells or antigen-presenting cells.

[0061] In one embodiment, the present disclosure relates to a method described herein, wherein the transduced cells are cultured for at least 2 days, preferably at least 5 days, more preferably at least 8 days, even more preferably at least 9 days, and even more preferably at least 10 days.

[0062] In a further embodiment, the combination of sequences is: PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35); PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47); PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11) and RELB (SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41) It could be.

[0063] In one embodiment, the disclosure includes a construct or vector of the disclosure, wherein the sequence selected from the group is a combination of PU.1 and IRF4.

[0064] In one aspect, the present disclosure provides a method for reprogramming or inducing stem cells or differentiated cells into conventional dendritic cells type 2, comprising the steps of: injecting into a cell selected from the group consisting of stem cells or differentiated cells, and mixtures thereof, one or more of: PU.1 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5), IRF4 (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11), PRDM1 (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17), IRF2 (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23), POU2F2 (SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29), or a combination thereof. No. 29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41); and mixtures thereof; transducing the transduced cells with one or more vectors comprising at least two nucleic acid sequences encoding sequences that are at least 90% identical, preferably at least 95% identical, to a sequence from the group consisting of: TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41); and mixtures thereof; culturing the transduced cells in a cell culture medium that supports the proliferation of dendritic cells or antigen-presenting cells.

[0065] In one embodiment, the combination of three isolated and synthetic transcription factors is in the following 5' to 3' order: PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35). The method includes, but is not limited to, culturing cells transduced with a plurality of isolated and synthetic transcription factors for at least two days, preferably at least five days, more preferably at least eight days, even more preferably at least nine days, and even more preferably at least ten days.

[0066] In further embodiments, the present disclosure includes methods 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, wherein the nucleic acid sequence encodes 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.

[0067] In one embodiment, the present disclosure includes a method wherein the transduction step further includes at least one vector comprising a nucleic acid encoding an immunostimulatory cytokine.

[0068] In further embodiments, the present disclosure includes methods wherein the cells are selected from the group consisting of pluripotent stem cells, or multipotent stem cells, or differentiated cells, and mixtures thereof.

[0069] In a further embodiment, the disclosure includes a method wherein the cell is a mammalian cell.

[0070] In further embodiments, the present disclosure includes methods wherein the pluripotent stem cells, multipotent stem cells, or differentiated cells are selected from the group consisting of endoderm-, mesoderm-, or ectoderm-derived cells, mesenchymal stem cells, hematopoietic stem cells, intestinal stem cells, multipotent stem cells and cell lines, including pluripotent stem cells.

[0071] In one embodiment, the disclosure includes a method wherein the cell is a non-human cell.

[0072] In a further embodiment, the disclosure includes a method wherein the cell is a mouse cell.

[0073] In one embodiment, the disclosure includes a method wherein the cell is a human cell.

[0074] In further embodiments, the present disclosure includes methods wherein the cells are human or mouse fibroblasts, or mammalian umbilical cord blood stem cells.

[0075] Another aspect of the present disclosure relates to induced dendritic cells obtained by the methods of the present disclosure.

[0076] Another aspect of the present disclosure relates to induced dendritic cells transduced with a construct or vector described herein, or one or more of the vectors described herein.

[0077] In a further embodiment, the present disclosure relates to a therapeutically effective amount of induced dendritic cells obtained by the methods of the present disclosure, and a pharmaceutically acceptable excipient.

[0078] In further embodiments, the present disclosure includes methods of use in veterinary or human medicine.

[0079] In further embodiments, the present disclosure includes methods of use in immunotherapy, or in the treatment or therapy of neurodegenerative diseases, or autoimmune diseases, immune deficiencies, or in the treatment or therapy of cancer, or in the treatment or therapy of infectious diseases.

[0080] In one embodiment, the present disclosure includes a method further comprising administering to the patient an antiviral agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, an antibiotic, a diuretic, or a mixture thereof.

[0081] In further embodiments, the present disclosure includes compositions further comprising a filler, binder, disintegrant, or lubricant, or mixtures thereof.

[0082] In further embodiments, the present disclosure includes compositions for use in intradermal and transdermal therapy.

[0083] In further embodiments, the present disclosure includes injectable formulations, particularly in situ injections.

[0084] In one embodiment, the present disclosure includes compositions for use as drug screens in veterinary or human medicine, particularly in immunotherapy, or in neurodegenerative or aging diseases, or in cancer or infectious diseases.

[0085] In further embodiments, the present disclosure includes compositions for use in the treatment, therapy, or diagnosis of disorders of the central and peripheral nervous system.

[0086] In a further embodiment, the present disclosure includes compositions for use in the treatment, therapy or diagnosis of neoplasia, particularly cancer, i.e., solid tumors or hematological tumors.

[0087] In a further embodiment, the present disclosure includes compositions for use in the treatment, diagnosis, or therapy of cancer or immune disorders, i.e., autoimmune diseases, hypersensitivity disorders, or immunodeficiency disorders.

[0088] In one embodiment, the present disclosure includes a composition for use in the treatment, therapy, or diagnosis of a fungal, viral, chlamydial, bacterial, nanobacterial, or parasitic infection.

[0089] In further embodiments, the present disclosure includes compositions for use in the treatment, therapy, or diagnosis of infection by HIV, SARS coronavirus, Asian influenza virus, herpes simplex, shingles, hepatitis, or viral hepatitis.

[0090] In a further embodiment, the present disclosure includes a vaccine for cancer comprising a composition according to any one of the preceding claims, or an induced dendritic cell of the present disclosure, or a mixture thereof.

[0091] In one aspect, the present disclosure relates to a vaccine or injectable formulation, particularly an in situ injection, against cancer comprising a composition as described herein, or an induced dendritic cell as described herein, or a mixture thereof.

[0092] In a further embodiment, the present disclosure provides a composition comprising the following components: Induced dendritic cells of the present disclosure; a composition described herein; a vector or construct of the present disclosure; or a mixture thereof The kit includes at least one of:

[0093] Surprisingly, induced DCs generated by the reprogramming described in this disclosure exhibit the intrinsic surface marker phenotype of conventional dendritic cell type 2 (CD11b), as well as cytokine secretion and antigen presentation on MHC-II molecules.

[0094] The present disclosure relates to a composition comprising a combination of at least two isolated transcription factors encoded by a sequence 90% identical to a sequence from the group consisting of PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41) as a reprogramming or induction factor for cells selected from the group consisting of stem cells or differentiated cells, or a mixture thereof.

[0095] Polypeptide variants or family members having the same or similar activity as the reference polypeptides encoded by the reference sequences (SEQ ID NOS: 1-36) can be used in the compositions, methods, and kits described herein. Generally, a particular polypeptide variant encoding a DC inducer for use in the compositions, 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 that particular reference polynucleotide or polypeptide, as determined by the sequence alignment algorithms and parameters described herein and known to those of skill in the art.

[0096] Methods of sequence alignment for comparison include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find a global (sequence-wide) alignment 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 percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). Global percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: An application for generating similarity / identity matrices using protein or DNA sequences). A small amount of manual editing can be performed to optimize alignment between conserved motifs, as will be apparent to those skilled in the art. The sequence identity values ​​expressed as percentages in this subject matter were determined across the entire amino acid sequence using BLAST with default parameters.

[0097] In one embodiment, any of the DNA coding sequences of the present disclosure can be modified, substituted, or altered to include one or more, preferably 0, 1, 2, 3, 4, 5, 6, different deoxyribonucleotide bases.

[0098] In one embodiment, the present disclosure verified that the majority of cDC2s in Clec9a reporter mice were labeled with tdTomato fluorescent protein, making this model suitable for screening of cDC2 inducers. PU.1 has been described to play an important role in DC development, and IRF4 has been described to ensure cDC2 specification. Furthermore, both PU.1 and IRF4 are highly expressed on cDC2 subsets. Therefore, the present disclosure combined PU.1 and IRF4 with an additional 33 cDC2 inducer candidates and performed additional screening in Clec9a reporter mouse embryonic fibroblasts (MEFs).

[0099] In one embodiment, PU.1 in combination with IRF4 and PRDM1 is sufficient to induce Clec9a reporter activation and surface expression of the cDC2 surface marker CD11b. Furthermore, expression of major histocompatibility complex (MHC) class II molecules, which are important for DC function, is induced by PU.1 in combination with IRF4 and PRDM1.

[0100] A polycistronic construct encoding PU.1 followed by IRF4 and PRDM1 increases the efficiency of Clec9a reporter activation. The resulting tdTomato + Upon TLR stimulation, cells secrete proinflammatory TNF-α and target antigens loaded on MHC-II to CD4 + It exhibits the ability to present to T cells and induce their proliferation and activation.

[0101] In one embodiment, PU.1 in combination with IRF4 and IRF2 induces significant reporter activation. Furthermore, PU.1 in combination with IRF4 and IRF2 results in an expansion of the tdT+CD11b+ double positive cell population.

[0102] In one embodiment, PU.1 in combination with IRF4 and POU2F2 or PU.1 in combination with IRF4 and TGIF1 results in increased expression of CD11b, a cDC2-specific surface marker.

[0103] In one embodiment, PU.1 in combination with IRF4 and RBPJ or PU.1 in combination with IRF4 and RELB results in increased expression of CD11b, a cDC2-specific surface marker.

[0104] In summary, we demonstrate that combining PU.1 and IRF4 with PRDM1, IRF2, RBPJ, RELB, POU2F2, or TGIF1 induces a cDC2 phenotype in fibroblasts. These findings provide insight into the heterogeneity of cDC2 specification. Direct reprogramming of next-generation cDC2s opens the possibility of inducing immunostimulatory and tolerogenic responses using autologous engineered cells.

[0105] In one embodiment, the combination of isolated transcription factors comprises: PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); or PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RBPJ (SEQ ID NO:43-SEQ ID NO:48); Or it may be PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12) and RELB (SEQ ID NO:37-SEQ ID NO:42).

[0106] Another aspect of the present disclosure is the use of a combination of at least two sequences from the group consisting of PU.1 (SEQ ID NO:1-SEQ ID NO:6), IRF4 (SEQ ID NO:7-SEQ ID NO:12), PRDM1 (SEQ ID NO:13-SEQ ID NO:18), IRF2 (SEQ ID NO:19-SEQ ID NO:24), POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36). The isolated transcription factors are the following combinations: PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF4 (SEQ ID NO:7-SEQ ID NO:12); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); or IRF4 (SEQ ID NO:7-SEQ ID NO:12) and PRDM1 (SEQ ID NO:13-SEQ ID NO:18); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or IRF4 (SEQ ID NO:7-SEQ ID NO:12) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); or IRF4 (SEQ ID NO:7-SEQ ID NO:12) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or IRF4 (SEQ ID NO:7-SEQ ID NO:12) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); or PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or PRDM1 (SEQ ID NO:13-SEQ ID NO:18) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and IRF2 (SEQ ID NO:19-SEQ ID NO:24); or IRF2 (SEQ ID NO:19-SEQ ID NO:24) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or IRF2 (SEQ ID NO:19-SEQ ID NO:24) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or PU.1 (SEQ ID NO:1-SEQ ID NO:6) and POU2F2 (SEQ ID NO:25-SEQ ID NO:30); or POU2F2 (SEQ ID NO:25-SEQ ID NO:30) and TGIF1 (SEQ ID NO:31-SEQ ID NO:36); or PU.1 (SEQ ID NO: 1-6) and TGIF1 (SEQ ID NO: 31-36) may include:

[0107] In one embodiment for better results, the cells may be selected from the group consisting of pluripotent stem cells, multipotent stem cells, differentiated cells, tumor cells, cancer cells, and mixtures thereof, particularly mammalian cells, more particularly murine or human cells.

[0108] In one embodiment, the isolated transcription factors of the present disclosure may be used for veterinary or human medical applications, particularly in infectious diseases, or viral diseases, or virally induced diseases, or neurodegenerative diseases, or in cancer, or in diabetes, or in immunotherapy, or in autoimmune diseases, or in hypersensitivity.

[0109] In one embodiment for better results, the isolated transcription factor of the present disclosure may be used as a reprogramming factor or inducer of cells selected from the group consisting of pluripotent stem cells, or multipotent stem cells, or differentiated cells, and mixtures thereof, into dendritic cells or interferon-producing cells.

[0110] The following figures provide preferred embodiments to illustrate the present disclosure and should not be considered as limiting the scope of the present invention. [Brief explanation of the drawings]

[0111] [Figure 1]Figure 1 - Ontogeny of the three major DC subsets. DCs emerge from common DC precursors (CDPs) in the bone marrow and can develop into different DC subsets: cDC1s, which primarily perform antigen cross-presentation and promote Th1 and cytotoxic T cell responses; pDCs, which act as interferon type I producers during viral infection; and cDC2s, which primarily perform MHC-II antigen presentation and promote Th2, Th17, and Treg responses. [Figure 2] Figure 2 - Schematic diagram of applications of directly reprogrammed cDC2s. Fibroblasts obtained from patients are reprogrammed into cDC2 cells that can be applied in personalized immunotherapy. The induced cDC2s can be used to induce immunity against parasites, extracellular pathogens, promote antitumor responses, or induce immune tolerance to self-antigens in autoimmune or hypersensitivity situations. [Figure 3] Figure 3 - Splenic cDC2s express high levels of tdTomato protein driven by the Clec9a-tdTomato reporter. (A) Flow cytometry analysis of tdTomato expression in splenic cDC1 (MHC-II+ CD11c+ CD8a+) and cDC2 (MHC-II+ CD11c+ CD11b+) populations isolated from Clec9a-tdTomato mice. (B) Quantification of tdTomato+ cells in cDC1 (CD8α+) ​​and cDC2 (CD8α-CD11b+). [Figure 4]Figure 4 - Clec9a-reporter activation and gene expression patterns are surprisingly suitable for identifying factors for cDC2 specification. (A) Experimental design to screen for cDC2-inducing transcription factors (TFs). The combination of PU.1, IRF8, and BATF3 (PIB) induces the reprogramming of Clec9a-tdTomato (Clec9a-tdT) mouse embryonic fibroblasts (MEFs) into cDC1-like derived cells. This combination retains the essential TFs of cDCs and is modified to identify combinations for cDC2 reprogramming. (B) Comparison of Spi1, Irf8, and Batf3 expression in cDC1 and cDC2 (GSE15907). Fold changes in gene expression are indicated in parentheses. (C and D) Quantification of tdTomato+ cells 6 days after transduction with the combination of PU.1 + BATF3 and different members of the IRF family (IRF1–IRF9) or PU.1 + IRF4. (E) Expression of the Irf gene family in cDC1 and cDC2 (GSE15907). Fold changes are indicated in parentheses. *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001, unpaired t-test and one-way ANOVA. [Figure 5] Figure 5 - Strategy for identifying cDC2-inducible transcription factors. (A) Schematic of the combinatorial screening strategy for cDC2 reprogramming. PU.1 and IRF4 were overexpressed along with additional individual candidate TFs. Clec9a reporter activation and expression of the cDC2 surface marker CD11b were assessed on day 6 of reprogramming. (B) Candidate TFs are highly enriched in cDC2 compared to cDC1 and pDC populations. Heatmap of expression of PU.1, IRF4, and 33 candidates in cDC1, cDC2, and pDC populations (GSE15907). [Figure 6]Figure 6 - Clec9a reporter-based screen for cDC2-inducible TFs identifies novel regulators of cDC2 reprogramming. Representative plots of flow cytometry analysis (A) and quantification of tdTomato-positive (tdTomato+) (B) cells at day 6 after transduction of Clec9a reporter MEFs with PU.1+IRF4 in combination with individual additional candidates (mean ± SD; screening data and statistics for 2-7 replicates per condition). MEFs transduced with M2rtTA, PU.1+IRF8+BATF3, and PU.1+IRF4+BATF3 were included as controls. *P<0.05, **P<0.01, one-way ANOVA. [Figure 7] Figure 7 - CD11b expression-based screen for cDC2-inducible TFs identifies novel regulators of cDC2 reprogramming. Representative plots of flow cytometry analysis (A) and quantification of CD11b+ (B) cells at day 6 after transduction of Clec9a reporter MEFs with PU.1+IRF4 in combination with individual additional candidates (mean ± SD; screening data and statistics for 2-7 replicates per condition). MEFs transduced with M2rtTA, PU.1+IRF8+BATF3, and PU.1+IRF4+BATF3 were included as controls. *P<0.05, **P<0.01, one-way ANOVA. [Figure 8] Figure 8 - Induction of cDC2-like cells from mouse fibroblasts by a combination of three transcription factors. Representative plots of flow cytometry analysis (A) and quantification of double-positive tdTomato+CD11b+ (B) cells 6 days after transduction with PU.1+IRF4 in combination with individual additional candidates (mean ± SD, screening data consisting of two replicates per condition). M2rtTA-transduced MEFs were included as a control. [Figure 9] Figure 9 - PU.1, IRF4, and PRDM1 are sufficient and necessary for cDC2 reprogramming. Quantification of tdT+ cells (A) and CD11b+ cells (B) gated within the tdT+ population after transduction of PU.1+IRF4+PRDM1 and individual removal of TFs from the 3 TF pool or expression of individual TFs on day 6 (n=2, mean ± SD). M2rtTA-transduced MEFs were included as a control. [Figure 10] Figure 10 - PU.1, IRF4, and PRDM1 are enriched in cDC2 cells. (A) Gene expression of Spi1, Irf4, and Prdm1 in DC populations (pDC, cDC1, and cDC2). Spi1, Irf4, and Prdm1 are more highly expressed in cDC2, with Prdm1 specifically more highly expressed in cDC2. (B) The combination of Spi1, Irf4, and Prdm1 is predominantly enriched in CD8a-DCs among 96 mouse tissues and cell types. Gene expression data (GeneAtlas MOE430) were log-transformed and normalized to a range of 0–1 for each gene, after which the highest mean expression was determined for Spi1, Irf4, and Prdm1. [Figure 11] Figure 11 - PU.1, IRF4, and PRDM1 induce CD45 and MHC-II surface expression. (A) Representative flow cytometry plots and (B) quantification of MHC-II+ cells (n=2, mean ± SD) of M2rtTA, PU.1+IRF8+BATF3, and PU.1+IRF4+PRDM1 transduced MEFs at day 6. (C) Quantification of MHC-II+ cells within tdT+ in M2rtTA, PU.1+IRF8+BATF3, and PU.1+IRF4+PRDM1 transduced MEFs at day 6 (n=2, mean ± SD). (D) CD45 and MHC-II expression within tdT- and tdT+ populations in PU.1+IRF4+PRDM1 transduced MEFs at day 9. [Figure 12] Figure 12 - The combination of PU.1 and IRF4 is sufficient for Clec9a reporter activation. Quantification of tdTomato-positive (tdTomato+) cells at day 6 after transduction of Clec9a reporter MEFs with PU.1 in combination with each additional candidate (mean ± SD; data from screening duplicates per condition). M2rtTA-transduced MEFs were included as a control. [Figure 13]Figure 13 - DC2-inducible TF combinations induce progressive Clec9a reporter activation. Kinetics of Clec9a-tdTomato reporter activation for the combinations PU.1 + IRF4 + PRDM1 (P + I4 + P) and PU.1 + IRF4 + IRF2 (P + I4 + I2). M2rtTA-transduced MEFs were included as a control. [Figure 14] Figure 14 - PU.1, IRF4, and IRF2 are minimal and sufficient combinations to induce a DC phenotype independent of PRDM1. (A) Quantification of tdTomato+ cells after transduction with PU.1+IRF4+IRF2 and individual removal of TFs from the 3-TF pool or individual TF expression on day 6 (n=2, mean ± SD). (B) The combination of Spi1, Irf4, and Irf2 is predominantly enriched in CD8a-DCs among 96 mouse tissues and cell types. Gene expression data (GeneAtlas MOE430) were log-transformed and normalized to a range of 0-1 for each gene, and then the highest mean expression was determined for Spi1+Irf4+Irf2. (C) Quantification of TdTomato+ cells after transduction with PU.1+IRF4 in combination with PRDM1, IRF2, or PRDM1+IRF2 on day 6 of reprogramming. (D) Schematic of the polycistronic construct encoding Spi1 followed by Irf4 and Irf2 separated by the self-cleaving peptides P2A and T2A within the pFUW-TetO plasmid (PI4I2 poly). (E) Quantification of TdTomato+ cells after transduction with M2rtTA, individual PU.1, IRF4 and IRF2 factors (P+I4+I2), and the PI4I2 poly construct on day 6 of reprogramming. [Figure 15]Figure 15 - Polycistronic PI4P vectors (PI4P poly) improve reprogramming efficiency. (A) Schematic diagram of a polycistronic construct encoding Spi1 followed by Irf4 and Prdm1, separated by the self-cleaving peptides P2A and T2A inserted into the pFUW-TetO plasmid (PI4P poly). (B) Representative flow cytometry plots and (C) quantification (mean ± SD, n = 2) of TdT+ cells after transduction with M2rtTA, PU.1, IRF4, and PRDM1 encoded by individual vectors (P+I4+P), the polycistronic PU.1, IRF8, and BATF3 combination (PI8B poly), and the polycistronic PU.1, IRF4, and PRDM1 (PI4P poly) construct on day 6 of reprogramming. (D) Fluorescence microscopy comparison of MEFs transduced with M2rtTA, PI8B poly, and PI4P poly on day 9 of reprogramming, showing tdT+ cell morphology (white arrow). [Figure 16] Figure 16 - PIP-induced cells secrete the TNF-α proinflammatory cytokine. Quantification of TNF-α and IL-10 concentrations in the supernatant of FACS-sorted tdTomato+ cells at day 9 of reprogramming induced by the PU.1, IRF4, and PRDM1 polycistronic vector (PI4Ppoly) before (-) or after overnight TLR stimulation with LPS, poly I:C (PiC), R848, and CpG ODN1585. [Figure 17] Figure 17 - PIP expression induces the ability to present antigens in MHC-II molecules to CD4+ T cells in an antigen-specific manner. Quantification of CTV dilution of OVA-specific OT-II Rag2KO CD4+ T cells (CD4+ TCRb+) after co-culture with different stimulation conditions: no stimulation (-), LPS, PiC, R848, and CpG ODN1585, of MEFs, sorted PIP-TdT+ cells (day 9), and bone marrow DCs (BM-DCs) pre-loaded or not with OVA peptide (323-339). DETAILED DESCRIPTION OF THE INVENTION

[0112] Detailed Description The present disclosure relates to the development of compositions, nucleic acid constructs, methods, and kits for reprogramming cells into conventional dendritic cells, particularly conventional dendritic cell type 2 (cDC2), particularly methods for generating cDCs, particularly cDC2s, from differentiated stem cells, multipotent stem cells, or pluripotent stem cells by introducing and expressing isolated / synthesized transcription factors. More particularly, the present disclosure provides methods for obtaining cDCs, particularly cDC2s, by direct cell reprogramming involving the surprisingly beneficial use of specific transcription factor combinations. Such compositions, nucleic acid constructs, methods, and kits can be used to induce dendritic cells in vitro, ex vivo, or in vivo, and these induced DCs or APCs can be used for immunotherapy applications.

[0113] Natural DCs are bone marrow-derived cells that disseminate throughout all tissues. DCs are poised to sample the environment and transmit the collected information to cells of the adaptive immune system (T cells and B cells). Once antigens are engulfed, 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. After activation, DCs typically overexpress costimulatory and MHC molecules in addition to secreting various cytokines involved in the initiation and / or enhancement of many T and B lymphocyte responses, namely, type I interferons, tumor necrosis factor (TNF)-α, IFN-γ, IL-12, and IL-6. Therefore, DCs are generally distinguished by 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 remarkable ability to secrete proinflammatory cytokines, migrate from non-lymphoid to lymphoid organs, and stimulate naive T cells. In mice and humans, distinct subsets of DCs can be variably defined by phenotype, ontogeny, and function (Figure 1). They include conventional DC subset 1 (cDC1, CD8α), found in lymphoid organs, which exhibit the ability for cross-presentation on MHC class I and induce CTL responses against infectious pathogens or tumors. +pDCs act by producing large amounts of type I interferon in response to viral infection. On the other hand, cDC2s are skilled at MHC-II presentation, leading to Th2 and Th17 T cell responses. In addition to T cell priming, cDC2s are involved in establishing self-tolerance to antigens by priming Tregs or by contributing to the negative selection of autoreactive T cells in the thymus. DNGR-1, also known as CLEC9A, is a necrotic cell receptor that supports the cross-priming of CTLs against dead cell-associated antigens in mice. DNGR-1 is selectively expressed at high levels by mouse cDC1 DCs, cDC2 DCs, and pDCs. Clec9a expression has recently been shown to enable the differentiation of conventional or plasmacytoid DC lineages and DC precursors (CDPs) that give rise to multiple lineages and their progeny in lymphoid tissues.

[0114] The successful identification of DC inducers capable of reprogramming differentiated cells described herein into inducible DCs, specifically cDC2s, advances our fundamental understanding of cDC2 biology and heterogeneity in many ways. This study provides in-depth insight into the cDC2 transcriptional network. Additionally, identifying DC inducers offers unprecedented opportunities to understand how the DC state is established and the critical regulatory mechanisms in place.

[0115] Transcription factors play a critical role in the specification of all cell types during development. The success of direct reprogramming strategies using transcription factor-mediated reprogramming indicates that it is equally feasible to use such factors to direct the differentiation of pluripotent ES / iPS cells or multipotent stem cells toward specific fates. Thus, the DC inducers identified herein can be used to achieve directed differentiation of ES / iPS cells toward a definitive DC fate through expression of DC-enriched transcription factors. Furthermore, the DC inducers identified herein can be used to achieve directed differentiation of multipotent hematopoietic stem and progenitor cells toward a definitive DC fate through expression of DC-enriched transcription factors.

[0116] An aspect of the present disclosure is the use of TFs or combinations of TFs to generate cells that can present autoantigens and generate a tolerogenic response. This method represents a viable strategy for tolerogenic immunotherapy in the setting of autoimmune and hypersensitivity disorders.

[0117] Fibroblasts can be obtained from human sources and then reprogrammed into cDC2s for immunomodulatory purposes (Figure 2). Given the known immune roles of cDC2s, these generated cDC2s can also be applied to promote antiparasitic immunity, immunity to extracellular pathogens, immune tolerance to self-antigens in the context of autoimmunity or hypersensitivity, or, when combined with cDC1s, to promote antitumor immunity.

[0118] A nucleic acid, e.g., DNA or RNA, or a construct thereof, encoding a DC inducer is introduced into cells by single or repeated transfection with or without a viral vector, resulting in expression of the gene product and / or translation of the RNA molecule in cells morphologically, biochemically, and functionally similar to the cDC2s described herein. These inducible cDC2s express the cDC2 surface marker CD11b.

[0119] In one embodiment, to screen the effects of cDC2-inducing TFs and cDC2-inducing TF combinations on cell reprogramming, mouse embryonic fibroblasts (MEFs) carrying a DC-specific reporter (Clec9a-Cre x R26-stop-tdTomato) were used, where reporter activation was used to reveal DC2-inducing TFs. In Clec9a-tomato reporter mice, tdTomato fluorescent protein is exclusively expressed by CDPs, pre-DCs, cDCs, and pDCs. Macrophages, other immune lineages, or monocyte-derived DCs in culture do not express Clec9a and therefore do not express tdTomato protein. Splenocytes isolated from Clec9a reporter mice were analyzed to identify cDC2 cells (CD11c + MHC-II + CD8a- CD11b + We confirmed that 78.9% of the cells (gated by ) expressed tdTomato fluorescent protein (Figure 3).

[0120] Double transgenic Clec9a-tdTomato reporter MEFs were isolated from E13.5 embryos and, by use of fluorescence-activated cell sorting (FACS), removed any contaminating tdTomato that may already be committed to the hematopoietic lineage. + or CD45 + was excluded from the cells.

[0121] Reprogramming of fibroblasts into cDC1-like cells was recently demonstrated through the combinatorial overexpression of PU.1, IRF8, and BATF3 (PIB). This combination was identified by screening Clec9a-Cre x R26-stop-tdT (Clec9a-tdT) mice, which express PU.1, IRF8, and BATF3 in cDC1, cDC2, and pDC (Rosa et al., 2018). Using this same DC reporter, we identified cDC2 transcription factors that direct the cDC2 lineage by altering the reported TF combination (Figure 4A).

[0122] Comparing the expression of Spi1, Irf8, and Batf3 across cDC populations showed that Spi1 was highly expressed in cDC2, whereas Irf8 and Batf3 were less expressed compared to cDC1 (Figure 4B). Furthermore, loss-of-function studies showed that PU.1 deletion impaired specification of the entire DC lineage, suggesting that it is required for continued DC development. Collectively, these data support the maintenance of PU.1 for cDC2 reprogramming and suggest that IRF8 and BATF3 can be replaced by other TFs.

[0123] Because the IRF family of TFs is known to be important for DC development, maturation, and functional roles (Gabriele & Ozato, 2007), we tested the replacement of IRF8 with other IRF proteins in combination with PU.1 and BATF3 for Clec9a reporter activation. IRF4 resulted in significant tdT expression (Figure 4C), suggesting that IRF4 can replace IRF8 in DC reprogramming. IRF4 is thought to be required for cDC2 development. On the other hand, IRF8 and BATF3 are thought to be important only for cDC1 development, suggesting that they are negligible for cDC2 reprogramming. However, combined overexpression of PU.1 and IRF4 did not result in significant Clec9a reporter activation (0.21%, Figure 4C and D), suggesting that these two TFs may be necessary, but not sufficient, to induce cDC2 reprogramming. Furthermore, when comparing the expression of all Irf gene family members between cDC1 and cDC2 populations, Irf4 was significantly more expressed in cDC2 (Figure 4E), further demonstrating its importance for cDC2 reprogramming. Indeed, from the entire IRF family, only Irf4 (2.6-fold) and, to a lesser extent, Irf2 (1.3-fold) were overrepresented in cDC2 cells.

[0124] In one embodiment, to screen for cDC2 reprogramming TF combinations, candidate TFs were individually combined with PU.1 and IRF4 and assessed for Clec9a reporter activation and expression of the cDC2 surface marker CD11b (Figure 5A).

[0125] In one embodiment, 33 candidate cDC2-inducing TFs were selected due to their differentially enriched gene expression in cDC2s compared with cDC1s and pDCs (Figure 5B). These 33 candidate TFs, along with PU.1 and IRF4, were individually cloned into a reprogramming-proven doxycycline (Dox)-inducible lentiviral vector.

[0126] In one embodiment, to screen for cDC2 reprogramming TF combinations, candidate TFs were first individually combined with PU.1 and assessed for Clec9a reporter activation (FIG. 12). From this screen, only the PU.1+IRF4 combination was found to induce significant rates of TdT + Cells were obtained and this combination was established as a baseline for further cDC2-inducing combinations.

[0127] In one embodiment, screening of candidate TFs demonstrated that combining PRDM1 or IRF2 with PU.1 and IRF4 resulted in significant Clec9a reporter activation (Figure 6) and tdT + CD11b + We identified an increase in the double population (Figure 8). Combining PRDM1 with PU.1 and IRF4 also resulted in increased expression of the cDC2 surface marker CD11b.

[0128] In one embodiment, combining PRDM1, RBPJ, RELB, POU2F2, or TGIF1 with PU.1 and IRF4 results in increased expression of the cDC2 surface marker CD11b (Figure 7). Collectively, these data identify PRDM1, RBPJ, RELB, POU2F2, and TGIF1 as additional cDC2-indicating TFs that may be responsible for the induction of distinct cDC2 cell states that reflect the inherent diversity within cDC2 subsets.

[0129] In one embodiment, to assess whether PU.1 + IRF4 + PRDM1 represents a minimal network for reprogramming, each of the factors was individually removed from the three TF pools. Removal of each of these individual TFs reduced Clec9a reporter activation and CD11b expression, and individual expression of each TF resulted in low expression of tdT and CD11b (Figures 9A and 9B). Collectively, this data implicates PU.1 + IRF4 + PRDM1 as a TF combination sufficient for Clec9a-tdT reporter activation and CD11b surface expression.

[0130] Comparison of the individual expression of Spi1, Irf4, and Prdm1 revealed enrichment of all three TFs in cDC2s, with PRDM1 showing the highest expression in cDC2s compared with other DC populations (Figure 10A). The combined expression of Spi1, Irf4, and Prdm1 also significantly increased CD8a - highly associated with DCs (Fig. 10B).

[0131] Further analysis of cell surface marker expression showed that 13.62% of PU.1+IRF4+PRDM1-transduced cells expressed MHC-II, compared with 14.43% in PU.1+IRF8+BATF3-generated DCs (Figures 11A and B). + Within the compartment, 35.58% of PU.1+IRF4+PRDM1-induced cells expressed surface MHC-II (Figure 11C). + While 20.10% of cells co-expressed surface MHC-II and CD45, only 1.36% of cells expressed tdT - MHC-II in the compartment + CD45 + (FIG. 11D). These data further support that PU.1+IRF4+PRDM1 induces hematopoiesis and the acquisition of an APC phenotype and antigen-presenting machinery.

[0132] The combination of PU.1, IRF4, and IRF2 was also further evaluated for its role in DC reprogramming. Removal of each individual TF abolished Clec9a reporter activation, and individual expression of each TF resulted in low TdT expression (Figure 14A). Combined expression of Spi1, Irf4, and Irf2 also highly associated with CD8a-DCs (Figure 14B). Given that adding PRDM1 and IRF2 individually to PU.1 and IRF4 resulted in productive Clec9a reporter activation, we investigated coexpression of these four TFs to address potential synergistic effects. However, overexpression of PU.1, IRF4, IRF2, and PRDM1 abolished Clec9a reporter activation, suggesting a cross-inhibitory role of PRDM1 and IRF2 in DC reprogramming (Figure 14C). Collectively, this data implicates PU.1+IRF4+IRF2 as a minimal and sufficient combination to induce a DC phenotype independent of PRDM1 and suggests the induction of distinct subsets of cDC2s.

[0133] Upon transduction with PU.1, IRF4, and PRDM1 or PU.1, IRF4, and IRF2, activation of the Clec9a reporter is detectable for both combinations starting on day 2. TdT expression peaks between days 9 (PU.1 + IRF4 + IRF2) and 10 (PU.1, IRF4, and PRDM1) (Figure 13).

[0134] Polycistronic constructs encoding combinations of transcription factors have been used to enhance reprogramming efficiency. Transduction of MEFs with a polycistronic construct (PI4I2 poly) encoding Spi1 followed by Irf4 and Irf2 (Figure 14D) resulted in increased reprogramming efficiency when compared to individual expression (P+I4+I2) (Figure 14E). Furthermore, a polycistronic construct (PI4P) encoding Spi1 followed by Irf4 and Prdm1, separated by the self-cleaving peptides P2A and T2A, resulted in increased reprogramming efficiency. ポリ ) was produced (Figure 15A). Compared with the individual expression (P + I4 + P), PI4P ポリThe polycistronic construct PU.1+IRF8+BATF3 (PI8B) for cDC1-like reprogramming ポリ ) resulted in an increase in reprogramming efficiency reaching 12.20%, a percentage comparable to that of PI8B (Figures 15B and 15C). ポリ and PI4P ポリ tdT resulting from the combination of both + The dendritic morphology of the cells is highlighted (Figure 15C).

[0135] The typical immunoregulatory characteristic of DCs is their ability to secrete cytokines. Proinflammatory cDC2s have been described to secrete TNF-α in response to TLR stimulation. On the other hand, anti-inflammatory cDC2s are characterized by the secretion of IL-10, which further mediates their immunoregulatory functions. ポリ Generate TdT + Cytokine secretion of cells was measured upon stimulation with the toll-like receptors TLR3 (PiC-polyinosinic:polycytidylic acid), TLR4 (LPS-lipopolysaccharide), TLR7 / TLR8 (R848-resiquimod), and TLR9 (CpG ODN1585). Overexpression of PU.1, IRF8, and PRDM1 induced the ability to secrete proinflammatory tumor necrosis factor-α (TNF-α), which increased 2.2-fold after LPS challenge (Figure 16). In contrast, the anti-inflammatory cytokine IL-10 was not detected. These results suggest that PI4P induces proinflammatory DC2 cells.

[0136] CD4 + PI4P sorted on day 9 to characterize the functional ability of generated cells to promote antigen-specific proliferation of T cells ポリ Generate TdT + Cells, MEFs, and bone marrow-derived DCs (BM-DCs) were cultured using OT-II CD4 T cells expressing T cell receptors specific for the ovalbumin (OVA) peptide 323-329 presented in the context of MHC-II molecules. + When pre-loaded with OVA peptide 323-339, PIP-induced cells increased by 10.67±1.16% OT-II CD4 +In the presence of LPS and R848, PIP-induced cells acquired the ability to induce T cell proliferation (CTVlow). + These data indicate that CD4 T cells are more likely to be involved in the proliferation of CD4 T cells than CD4 T cells, respectively. + This supports the ability of PI4P-induced cells to load and present antigens on MHC-II molecules, promoting T cell responses (Fig. 17).

[0137] A recent update on DC heterogeneity identified two distinct subsets of cDC2s, defined by distinct transcriptional regulators and distinct anti- and pro-inflammatory functions (Brown et al., 2019). Coincidentally, in this report, analysis of the top-defining TF genes for each of these newly identified subsets highlights PRDM1 as a top TF associated with cDC2B, a subset characterized by its pro-inflammatory phenotype. These data therefore further support the pro-inflammatory phenotype of the PU.1+IRF4+PRDM1-generated DCs reported in this disclosure, which are therefore similar to the cDC2B phenotype.

[0138] In some embodiments, polypeptide variants or family members having the same or similar activity as the reference polypeptides encoded by the sequences provided in the sequence listing may be used in the compositions, methods, and kits described herein. Generally, a particular polypeptide variant encoding a cDC2 inducer for use in the compositions, methods, and kits described herein will 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 with that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0139] In one embodiment, human PU.1 transcription factor (PU.1), mRNA (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5) and codon-optimized or different codons encoding the same amino acids are also intended to be encompassed by reference to the nucleic acids described herein.

[0140] In one embodiment, human interferon regulatory factor 4 (IRF4), mRNA (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11) and codon-optimized or different codons encoding the same amino acids are also intended to be encompassed by reference to the nucleic acids described herein.

[0141] In one embodiment, human PR domain zinc finger protein 1 (PRDM1), mRNA (SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17) and codon-optimized or different codons encoding the same amino acids are also intended to be encompassed by reference to the nucleic acids described herein.

[0142] In one embodiment, human interferon regulatory factor 2 (IRF2), mRNA (SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 23) and codon-optimized or different codons encoding the same amino acids are also contemplated as being encompassed by reference to the nucleic acids described herein.

[0143] In one embodiment, human POU class 2 homeobox 2 (POU2F2), mRNA (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29) and codon-optimized or different codons encoding the same amino acids are also intended to be encompassed by reference to the nucleic acids described herein.

[0144] In one embodiment, it is intended that reference to the nucleic acids described herein encompasses the human homeobox protein TGIF1 (TGIF1), mRNA (SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35) and codon-optimized or different codons encoding the same amino acids.

[0145] In one embodiment, human recombinant binding protein Suppressor of Hairless (RBPJ), mRNA (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47) and codon-optimized or different codons encoding the same amino acids are also contemplated as being encompassed by reference to the nucleic acids described herein.

[0146] In one embodiment, human transcription factor RelB (RELB), mRNA (SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41) and codon-optimized or different codons encoding the same amino acids are also intended to be encompassed by reference to the nucleic acids described herein.

[0147] In some embodiments of the compositions, constructs, vectors, methods, and kits provided herein, the number of cDC2 inducers used or selected to generate cDC2s derived from starting somatic cells, such as fibroblasts or hematopoietic lineage cells, multipotent stem cells, induced pluripotent stem cells, cancer or tumor cells, is at least 2. In some embodiments, the number of cDC2 inducers 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.

[0148] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, a nucleic acid sequence or construct encoding a cDC2 inducer(s), such as PU.1, IRF4, PRDM1, IRF2, RBPJ, RELB, POU2F2, and TGIF1, is inserted into or operably linked to an expression vector suitable for transfection of cells using standard molecular biology techniques. As used herein, "vector" refers to a nucleic acid molecule, such as a dsDNA molecule, that provides 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 (ARSs), centromeres, and other sequences that can replicate or be replicated in vitro or within a host cell, or that can deliver a desired nucleic acid segment to a desired location within a host cell. A vector can have one or more restriction endonuclease recognition sites (whether type I, II, or IIs) that allow sequences to be cleaved in a determinable manner without losing the essential biological function of the vector, and into which nucleic acid fragments can be spliced ​​or inserted to effect 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 provide, for example, primer sites for PCR, transcription and / or translation initiation and / or control sites, recombination signals, replicons, additional selection markers, etc. A vector may further contain one or more selection markers suitable for use in identifying cells transformed with the vector.

[0149] In some embodiments of the compositions, methods, constructs, vectors, and kits described herein, the expression vector is a viral vector. Several viral-mediated expression methods employ retroviral, adenoviral, lentiviral, herpesviral, poxviral, and adeno-associated viral (AAV) vectors, and such expression methods are used in gene delivery and are well known in the art.

[0150] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, the viral vector is a retrovirus. Retroviruses provide a convenient platform for gene delivery. 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 delivered to target cells of the subject either in vivo or ex vivo. Numerous retroviral systems have been described. 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, methods, and kits described herein, the retrovirus is replication-deficient. Retroviral vector systems take advantage of the fact that a minimal vector containing 5' and 3' LTRs and a packaging signal is sufficient to enable 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 efficient infection and gene expression in most cell types, precise single-copy vector integration into target cell chromosomal DNA, and ease of manipulation of the retroviral genome.

[0151] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, the viral vector is an adenovirus-based expression vector. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thereby minimizing the risk associated with 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 cells, have a broad host range, exhibit highly efficient infectivity, directly express heterologous genes at high levels, and achieve long-term expression of those genes in vivo. Because the virus is fully infectious as a cell-free virion, injection of producer cell lines is not necessary. Regarding safety, adenoviruses are not associated with serious human pathologies, and recombinant vectors derived from the virus can be made replication-deficient by deleting the early region 1 ("E1") of the viral genome. Adenoviruses can also be produced relatively easily in large quantities. Adenoviral vectors for use in the compositions, methods, and kits described herein can be derived from any of a variety of adenovirus serotypes, including, but not limited to, any of the more than 40 serotype strains of adenovirus, such as serotypes 2, 5, 12, 40, and 41. The adenoviral vectors used herein are preferably replication-deficient and contain a cDC2 inducer of interest operably linked to an appropriate promoter.

[0152] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, nucleic acid sequences encoding cDC2 inducer(s), such as PU.1, IRF4, PRDM1, IRF2, RBPJ, RELB, POU2F2, and TGIF1, are introduced or delivered using one or more inducible lentiviral vectors. Control of expression of cDC2 inducers delivered using one or more inducible lentiviral vectors can, in some embodiments, be achieved by contacting cells harboring at least one DC inducer under the control of an inducible promoter or in an expression vector operably linked to an inducible promoter with a regulating agent (e.g., doxycycline) or other inducer. When using some types of inducible lentiviral vectors, contacting such cells with an inducer induces expression of the cDC2 inducer, while withdrawal of the regulating agent inhibits expression. When using other types of inducible lentiviral vectors, the presence of a regulating agent inhibits expression, while removal of the regulating agent permits expression. As used herein, the term "induction of expression" refers to the expression of a gene, such as a cDC2-inducing factor, encoded by an inducible viral vector in the presence of, for example, an inducer, or in the presence of one or more agents or factors that cause endogenous expression of the gene in a cell.

[0153] In some embodiments of the aspects described herein, a doxycycline (Dox)-inducible lentivirus system is used. Unlike retroviruses, lentiviruses can transduce quiescent cells, making them suitable for transducing a wider variety of hematopoietic cell types. For example, the pFUW-tetO lentivirus system has been shown to transduce primary hematopoietic progenitor cells with high efficiency.

[0154] In some embodiments of the methods described herein, nucleic acid sequences encoding cDC2 inducer(s), such as PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41), are introduced or delivered using a non-integrating vector (e.g., adenovirus). While integrating vectors, such as retroviral vectors, can integrate into the host cell genome and potentially disrupt normal gene function, non-integrating vectors control expression of the gene product by extrachromosomal transcription. Because non-integrating vectors do not become part of the host genome, they tend to transiently express nucleic acids within a cell population. This is due, in part, to the fact that non-integrating vectors are often replication-defective. As such, non-integrating vectors have 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 with 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 an adenovirus. Other advantages of non-integrating viral vectors include their ability to be produced at high titers, their stability in vivo, and their efficient infection of host cells.

[0155] Nucleic acid constructs and vectors for use in generating inducible cDC2s in the compositions, methods, and kits described herein can further include, in some embodiments, one or more sequences encoding selectable markers for positive and negative selection of cells. Such selectable marker sequences can typically provide a characteristic of resistance or sensitivity to antibiotics not normally found in cells in the absence of introduction of the nucleic acid construct. The selectable marker can be used in combination with a selection agent, such as an antibiotic, to select in culture for cells expressing the inserted nucleic acid construct. A sequence encoding a positive selectable marker typically confers antibiotic resistance, i.e., when the positive selectable marker sequence is present in the genome of a cell, the cell is sensitive to the antibiotic or drug. A sequence encoding a negative selectable marker typically confers sensitivity to an antibiotic or drug, i.e., when the negative selectable marker is present in the genome of a cell, the cell is sensitive to the antibiotic or drug.

[0156] The nucleic acid constructs and vectors for use in generating inducible cDC2s in the compositions, methods, and kits described herein can, in some embodiments, further comprise other nucleic acid elements for regulation, expression, or stabilization of the genetic elements of the construct or other vector, such as promoters, enhancers, TATA boxes, ribosome binding sites, IRESs, and the like, known to those of skill in the art.

[0157] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, the DC inducer(s) such as PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), and RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41) are provided as or introduced or delivered to cells as synthetic modified RNAs as described in U.S. Patent Publication 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 inducible cDC2s according to the methods described herein, the methods can involve repeated contacting of cells or repeated transfections, such as, for example, at least 2, 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 25, at least 30, or more transfections, of synthetic modified RNA encoding a DC inducer.

[0158] In addition to one or more modified nucleosides, modified mRNA for use in the compositions, constructs, vectors, methods, and kits described herein can include any additional modifications known to those of skill in the art and described in U.S. Patent Publication Nos. 2012 / 0046346A1 and 2012 / 0251618A1, and PCT Publication WO2012 / 019168. Such other components include, for example, a 5' cap (e.g., an anti-reverse cap analog (ARCA) cap comprising a 5'-5'-triphosphate guanine-guanine linkage in which one guanine comprises an N7 methyl group and a 3'-O-methyl group; caps made using recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzymes that can create a standard 5'-5'-triphosphate linkage between the 5'-most nucleotide of an mRNA and a guanine nucleotide, where the guanine comprises an N7 methylation and the final 5'-nucleotide comprises a 2'-O-methyl generating a Cap1 structure); a poly(A) tail (e.g., greater than 30 nucleotides in length, greater than 35 nucleotides in length, at least 40 nucleotides in length, at least poly-A tail of 45 nucleotides, at least 55 nucleotides, at least 60 nucleotides, 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 intronic nucleotide sequences that can be excised from the nucleic acid, or any combination thereof.

[0159] In one embodiment, the modified mRNA for use in the compositions, constructs, vectors, methods, and kits described herein can further comprise an internal ribosome entry site (IRES). The IRES can act as the only ribosome binding site or can function as one of multiple ribosome binding sites in the mRNA. An mRNA containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by ribosomes ("multicistronic mRNA"), such as the cDC2 inducers described herein. When a nucleic acid is provided with an IRES, a second translatable region is optionally further provided. Examples of IRES sequences that can be used in accordance with the present disclosure include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SW), or cricket paralysis viruses (CrPV).

[0160] In some embodiments of the compositions, constructs, vectors, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least one modified nucleoside. In some embodiments of the compositions, methods, and kits described herein, the synthetic, modified RNA molecule comprises at least two modified nucleosides.

[0161] In some embodiments of the compositions, constructs, vectors, 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 (m In some embodiments, the modified nucleoside is selected from the group consisting of 5-methylcytosine (5mC), pseudouracil, or a combination thereof.

[0162] Modified mRNA need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can be present at various positions in the nucleic acid. Those skilled in the art will understand that nucleotide analogs or other modification(s) can be placed at any position(s) in the nucleic acid so as not to substantially reduce the function of the nucleic acid. Modifications can also be 5' or 3' terminal modifications. Nucleic acids can contain a minimum of one and a maximum of 100% modified nucleotides, or any percentage therebetween, such as at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

[0163] In some embodiments, it is preferred, but not absolutely necessary, that each occurrence of a given nucleoside in a 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, a modified mRNA can include a modified pyrimidine such as 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 substituted with modified uracil. It is also contemplated that different occurrences of the same nucleoside can be modified in different ways in a given synthetic, modified RNA molecule. Modified uracils can be substituted with a 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 can be substituted with modified cytosines. The modified cytosines can be substituted with a 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 cytosines modified as 5mC and others modified as 2'-O-methylcytosine or other cytosine analogs). Such multiply modified synthetic RNA molecules can be produced using a ribonucleoside blend or mixture containing all of the desired modified nucleosides, such that when the RNA molecule is synthesized, only the desired modified nucleosides are incorporated into the resulting RNA molecule encoding the cDC2 inducer.

[0164] In certain embodiments, it may be desirable to degrade modified nucleic acids introduced into cells within the cell, for example, when precise timing of protein production is desired. Accordingly, in some embodiments of the compositions, methods, and kits described herein, provided herein are modified nucleic acids that include a degradation domain that can act in a directed manner within the cell.

[0165] It is understood that inducible cDC2s can be generated by delivery of cDC2-inducing factors in the form of nucleic acids (DNA or RNA) or amino acid sequences; however, in some embodiments of the compositions, constructs, vectors, methods, and kits described herein, inducible cDC2s can be induced using other methods, such as, for example, by treatment of cells with an agent, such as a small molecule or cocktail of small molecules, that induces expression of one or more cDC2-inducing factors.

[0166] Detection of expression of cDC2-inducing factors introduced into cells or induced in cell populations using the compositions, constructs, vectors, methods, and kits described herein can be accomplished by any of several techniques known to those of skill in the art, including, for example, Western blot analysis, immunocytochemistry, and fluorescence-mediated detection.

[0167] To distinguish whether a given combination of DC inducers has generated inducible cDC2s, in some embodiments, one or more DC activities or parameters, such as differential expression of surface antigens, can be measured. Generation of inducible DCs using the compositions, methods, and kits described herein preferably results in the appearance of cell surface phenotypes characteristic of endogenous cDC2s, such as, for example, CD45, MHC-II, CD11b, Sirpa, CD4, ESAM, Clec4a4, Clec10a, Clec12a, and Mgl2.

[0168] DCs are most reliably distinguished from other immune cells by their functional behavior. Functional aspects of the cDC2 phenotype or cDC2 activity, such as the ability of inducible cDC2s to secrete cytokines, can be readily determined by those skilled in the art using routine methods known in the art. In some embodiments of the aspects described herein, functional assays can be used to identify reprogramming factors. For example, in some embodiments, cytokine secretion can be used to confirm the immunomodulatory properties of inducible cDC2s generated using the compositions, constructs, vectors, methods, and kits described herein.

[0169] As used herein, the terms "cellular parameter," "DC parameter," or "cytokine secretion" refer to a measurable component or quality of endogenous or natural DCs, particularly a component that can be accurately measured. A cellular parameter can be any measurable parameter related to cellular phenotype, function, or behavior. Such cellular parameters include changes in DC or DC population characteristics and markers, including, but not limited to, changes in viability, cell proliferation, expression of one or more markers or combinations of markers, such as cell surface determinants, such as receptors, proteins, including conformational or post-translational modifications, lipids, carbohydrates, organic or inorganic molecules, nucleic acids, such as mRNA, DNA, and global gene expression patterns. Such cellular parameters can be measured using any of 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 assays, Western blot techniques, or microscopy. Gene expression profiles can be assayed, for example, using RNA sequencing methods and quantitative or semi-quantitative real-time PCR assays. A cellular parameter can also refer to a functional parameter or functional activity. Most cellular parameters provide quantitative readouts, although in some cases semi-quantitative or qualitative results may be acceptable. The readout may include a single determined value, or may include a mean, median, variance, or the like. Typically, a range of parameter readouts is obtained for each parameter from multiple identical assays. Variability is expected, and the range of values ​​for each of the set of test parameters is obtained using standard statistical methods, with the common statistical method used to provide a single value.

[0170] In some embodiments of the compositions, methods, and kits described herein, additional factors and agents can be used to enhance the reprogramming of inducible cDC2s. For example, factors and agents that alter epigenetic pathways can be used to promote reprogramming to inducible cDC2s.

[0171] Essentially any primary somatic cell type can be used to generate inducible cDC2s or reprogram somatic cells into inducible cDC2s in accordance with the compositions, methods, and kits described herein. Such primary somatic cell types include other stem cell types, including pluripotent stem cells such as induced pluripotent stem cells (iPS cells); other multipotent 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, neuronal 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 they are derived. Cells can be primary cells isolated from any somatic tissue, including, but not limited to, spleen, bone marrow, blood, brain, liver, lung, intestine, stomach, intestinal tract, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc. The term "somatic cells" also encompasses, in some embodiments, primary cells grown in culture, provided that the somatic cells are not immortalized. When cells are maintained under in vitro conditions, conventional tissue culture conditions and methods can be used and are known to those skilled in the art. Methods for isolating and culturing various primary somatic cells are well within the capabilities of those skilled in the art.

[0172] In some embodiments of these and all such aspects described herein, the somatic cells are fibroblasts.

[0173] In some embodiments of these and all such aspects described herein, the somatic cells may be hematopoietic lineage cells.

[0174] In some embodiments of these and all such aspects described herein, the somatic cell may be a cancer cell or a tumor cell.

[0175] In some embodiments of the compositions, methods, and kits described herein, the somatic cells that are reprogrammed or made into inducible cDC2 cells are cells of hematopoietic origin. As used herein, the terms "hematopoietic-derived cells," "hematopoietic-derived differentiated cells," "hematopoietic lineage cells," and "cells of hematopoietic origin" refer to cells derived or differentiated from multipotent hematopoietic stem cells (HSCs). Thus, hematopoietic lineage cells for use with the compositions, methods, and kits described herein include multipotent, differentiation-restricted, and lineage-restricted hematopoietic progenitor cells, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, and lymphocytes (e.g., T lymphocytes bearing a T cell receptor (TCR), B lymphocytes or B cells that express immunoglobulins and produce antibodies, NK cells, NKT cells, and innate lymphocytes). As used herein, the term "hematopoietic progenitor cell" refers to a multipotent, differentiation-restricted, and lineage-restricted hematopoietic cell that can differentiate into two or more cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, and lymphocytes, B cells, and T cells. Hematopoietic progenitor cells include multipotent progenitors (MPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-monocyte progenitors (GMPs), and promegakaryocyte-erythroid progenitors. Lineage-restricted hematopoietic progenitor cells include megakaryocyte-erythroid progenitors (MEPs), pro-B cells, pre-B cells, pre-Pro-B cells, pro-T cells, double-negative T cells, pro-NK cells, pre-granulocyte / macrophage cells, granulocyte / macrophage progenitor (GMP) cells, and pro-mast cells (ProMCs).

[0176] Cells of hematopoietic origin for use in the compositions, methods, and kits described herein can be obtained from any source known to contain these cells, such as fetal tissue, umbilical cord blood, bone marrow, peripheral blood, mobilized peripheral blood, spleen, liver, thymus, lymph, etc. Cells obtained from these sources can be expanded ex vivo using any method acceptable to those of skill in the art before use with the compositions, methods, and kits for generating inducible cDC2s described herein. For example, cells can be sorted, fractionated, treated to remove specific cell types, or otherwise manipulated using any procedure acceptable to those of skill in the art to obtain cell populations for use in the methods described herein. Mononuclear lymphocytes can be collected by repeated lymphocyte exchange using a continuous flow cell separator, as described in U.S. Pat. No. 4,690,915, for example, or isolated using flow cytometry using a cytometer, magnetic separation using antibody- or protein-coated beads, affinity chromatography, or affinity purification steps of CLP methods, such as solid support affinity separation in which cells are retained on a substrate due to the expression or lack of expression of a specific protein or specific types of proteins, 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 collecting cells from peripheral blood, the subject can be treated with cytokines, such as granulocyte colony-stimulating factor, to promote cell migration from the bone marrow to the blood compartment and / or promote activation and / or proliferation of the desired population. For example, any method suitable for distinguishing surface proteins can be used to isolate hematopoietic cells of heterogeneous population origin. 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.

[0177] Furthermore, for various aspects and embodiments of the compositions, 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.

[0178] Generally, the methods for generating inducible cDC2s described herein involve culturing or expanding somatic cells, such as cells of hematopoietic origin, in any 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®, and serum-free media for the culture and expansion of DCs. Many media are also available as low-glucose formulations with or without sodium. In some embodiments, media used with the methods described herein can be supplemented with 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. Additionally, 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.

[0179] Cells in culture can be maintained either in suspension or attached to a solid support, e.g., seeded on extracellular matrix components or feeder cells. Cells used in the methods described herein may, in some embodiments, require additional factors to promote their attachment to a solid support, such as 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, cells are suitable for growth in suspension culture. Suspension-competent host cells are generally monodisperse or grow in loose aggregates without substantial aggregation. Suspension-competent host cells include cells suitable for suspension culture without adaptation or manipulation (e.g., cells of hematopoietic origin, such as lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of anchorage-dependent cells (e.g., epithelial cells, fibroblasts).

[0180] In some embodiments of these and all such aspects described herein, the isolated inducible cDC2s further comprise a pharmaceutically acceptable carrier for administration to a subject in need thereof.

[0181] In some embodiments, methods for treating a subject in need of treatment to induce an antigen-specific immune response to eliminate cancer cells or infectious pathogens or to generate immune tolerance to self-antigens using a cDC2-inducing composition and a method for preparing inducible cDC2 described herein, or using isolated inducible cDC2s and cell clones thereof generated using any of the DC inducer combinations, DC inducer compositions, or methods for preparing inducible cDC2 described herein are also provided herein. In such treatment methods, somatic cells such as fibroblasts or hematopoietic lineage cells are first isolated from a subject, and the isolated cells can be transduced or transfected with a DC inducer composition comprising an expression vector or synthetic mRNA, respectively, as described herein. Next, isolated inducible cDC2s generated using any of the cDC2-inducing factor combinations, cDC2 inducer compositions, or methods for preparing inducible cDC2 described herein can be administered to the subject, such as by systemic injection of the inducible cDC2 into the subject.

[0182] In some embodiments, methods for treating a subject in need of treatment to induce an antigen-specific immune response that eliminates cancer cells or infectious pathogens using any of the cDC2-inducing compositions and combinations of cDC2 inducers described herein are also provided herein. In such treatment methods, cancer cells are transduced with a cDC2-inducing composition comprising an expression vector as described herein. Cancer cells can be first isolated from a subject, transduced with a cDC2-inducing composition comprising an expression vector, and then administered to the subject via systemic injection, for example. Alternatively, cancer cells can be transduced in situ or in vivo with a cDC2-inducing composition comprising a viral expression vector.

[0183] Inducible cDC2s generated and reprogrammed using the compositions, methods, and kits described herein can be used directly in some embodiments of the therapeutic methods described herein or can be administered to a subject in need of immunotherapy. Accordingly, various embodiments of the methods described herein involve administering an effective amount of induced cDC2s or a population of induced cDC2s generated using any of the compositions, methods, and kits described herein to an individual or subject in need of cell therapy. The administered cells or population of cells can be an autologous population or can be derived from one or more heterologous sources. Furthermore, such induced cDC2s can be administered in a manner that allows them to migrate to lymph nodes and activate effector T cells.

[0184] Inducible cDC2s generated and reprogrammed using the compositions, methods, and kits described herein can be used directly in some embodiments of the therapeutic methods described herein or can be administered to a subject suffering from an autoimmune or hypersensitivity disorder. Accordingly, various embodiments of the methods described herein involve administering an effective amount of induced cDC2s or a population of induced cDC2s generated using any of the compositions, methods, and kits described herein to an individual or subject in need of cell therapy. The administered cells or population of cells can be an autologous population or can be derived from one or more heterologous sources. Furthermore, such induced cDC2s can be loaded with autoantigens and administered in a manner that allows them to migrate through the thymus, promote the negative selection of autoreactive T cells, migrate to lymph nodes, restrict effector T cells, or promote Treg differentiation.

[0185] Various means for administering cells to a subject are known to those skilled in the art. Such methods can include systemic injection, e.g., IV injection, or transplantation of cells into a target site in a subject. Cells can be inserted into a delivery device that facilitates introduction by injection or transplantation into a subject. Such a delivery device can include a tube, e.g., a catheter, for injecting cells and fluids into the recipient subject's body. In one preferred embodiment, the tube further includes a needle, e.g., through which the cells can be introduced into the subject at a desired location. Cells can be prepared for delivery in a variety of different forms. For example, when included in such a delivery device, the cells can be suspended in a solution or gel or embedded in a support matrix. Cells can be mixed with a pharmaceutically acceptable carrier or diluent that allows the cells to remain viable.

[0186] Thus, the cells produced by the methods described herein may be used to treat breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian carcinoma, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic carcinoma, gastric cancer, colon carcinoma, prostatic cancer, The present invention can be used to prepare cells for treating or ameliorating several cancers and tumors, including, but not limited to, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, 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, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

[0187] In addition to the above, the methods of the present disclosure can be used to prevent or eliminate infection by pathogens known to predispose to certain cancers. Pathogens of particular interest for use in the cancer vaccines provided herein include hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (hepatocellular carcinoma), Epstein-Barr virus (EBV) (Burkitt's lymphoma, nasopharyngeal carcinoma, PTLD in immunosuppressed individuals), HTLV-L (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, more particularly humans, are described in detail in the literature, for example, CGA Thomas, Medical Microbiology, Bailliere Tindall, (1983).

[0188] In addition to the above, the methods of the present disclosure can be used for viral infections. Exemplary viral pathogens include, but are not limited to, infectious viruses that infect mammals, and more particularly 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); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (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); Bungaviridae (e.g., Hantaan virus, Bungawi virus, Rus, Phlebovirus, and Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus P. oxyiridae (variola virus, vaccinia virus, variola virus); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., pathogens of spongiform encephalopathy, pathogens of hepatitis delta (thought to be defective satellites of hepatitis B virus), non-A, non-B hepatitis pathogens (Class 1 = internally transmitted; Class 2 = parenterally transmitted (i.e., hepatitis C); Norwalk and related viruses, and astroviruses).

[0189] In addition to the above, the disclosed methods can be used to target Gram-negative and Gram-positive bacteria in vertebrates. Such Gram-positive bacteria include, but are not limited to, Pasteurella, Staphylococcus, and Streptococcus. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, and Salmonella. Specific examples of infectious bacteria include Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacteria (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), and Streptococcus viridans. group), Enterococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthrax, Corynebacterium diphtheriae, Corynebacterium species, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsiae, and Actinomyces israelii.

[0190] In addition to the above, the methods of the present disclosure can be used to target pathogens, including, but not limited to, infectious fungi and parasites that infect mammals, more particularly humans. Examples of infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0191] In addition to the above, the methods of the present disclosure can be used to target parasites such as intracellular parasites and obligate intracellular parasites, including, but not limited to, Plasmodium falciparum, Plasmodium ovalveolar, Plasmodium vivax, Plasmodium vivax, Plasmodium vivax, Babesia murine, Babesia polymorpha, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis, Leishmania major, Leishmania donovani, Leishmania brasiliensis, Leishmania tropicalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Wuchereria bancrofti, Brugia malayi, Brugia timoli, Ascaris lumbricoides, Onchocerca volvulus, and Schistosoma mansoni.

[0192] The engineered inducible cDC2s can be used to induce tolerance responses, including the suppression of future or existing immune responses to one or more target antigens. Therefore, inducible cDC2s are useful for treating or preventing unwanted 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 disclosure include rejection associated with bone marrow transplants and transplants of organs such as the 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. Autoimmune diseases that can be treated or prevented by the present disclosure include, for example, psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff-person 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), primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, and the like. Diseases that may be present include: insulin-dependent diabetes mellitus (IDDM), Goodpasture's syndrome, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, sympathetic ophthalmia, Hashimoto's disease / hypothyroidism, 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.

[0193] Pharmaceutically acceptable carriers and diluents include physiological saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably a sterile fluid. Preferably, prior to introduction of the cells, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0194] Preferably, the mode of cell administration is relatively non-invasive, for example, by intravenous injection, pulmonary delivery by inhalation, topical administration, or intranasal administration. However, the route of cell administration will depend on the tissue being treated and may include implantation. Methods of cell delivery are known to those skilled in the art, and their use with the methods and compositions described herein can be predicted by those skilled in the art of medicine.

[0195] Also provided herein, in some embodiments, are kits for generating inducible cDC2s, the kits comprising any of the DC-inducing compositions comprising one or more expression vector components described herein.

[0196] Also provided herein, in some embodiments, are kits that include one or more of the cDC2 inducers described herein as components for the methods of generating inducible cDC2s described herein.

[0197] Thus, in some aspects, provided herein are kits for preparing inducible dendritic cells, comprising the following components: (a) one or more expression vectors encoding at least one, two, three, four, five, six, or more cDC2 inducers selected from PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), IRF2 (SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47), RELB (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41), and (B) packaging and instructions therefor.

[0198] The kits described herein, in some embodiments, can further provide synthetic mRNA or one or more expression vectors encoding DC inducers, either mixed or in separate aliquots.

[0199] In some embodiments, the kit can further comprise an agent that enhances the efficiency of reprogramming. In some embodiments, the kit can further comprise one or more antibodies or primer reagents for detecting cell type-specific markers to identify cells induced to the cDC2 state.

[0200] In some embodiments, the kit can further comprise a buffer. In some such embodiments, the buffer is an RNase-free TE buffer at pH 7.0. In some embodiments, the kit further comprises a container having a cell culture medium.

[0201] All kits described herein can further include buffers, cell culture media, transduction or transfection media, and / or media supplements. In preferred embodiments, the buffers, cell culture media, transfection media, and / or media supplements are DNAse and RNAse free. In some embodiments, the synthetic modified RNA provided in the kit can be in a non-solution form of a specific amount or mass, e.g., 20 μg, such as a lyophilized powder form, such that the end user adds an appropriate amount of buffer or media to bring the component to a desired concentration, e.g., 100 ng / μl.

[0202] All kits described herein can include a device that facilitates single administration or repeated or frequent infusion of cells generated using the kit components described herein, such as a non-implantable delivery device, e.g., a needle, syringe, or pen device, or an implantable delivery device, e.g., a pump, a semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal, or intrasaccular), or a reservoir. In some such embodiments, the delivery device can include a mechanism for dispensing a unit dose of a pharmaceutical composition comprising inducible cDC2s. In some embodiments, the device releases the composition continuously, e.g., by diffusion. In some embodiments, the device can include a sensor that monitors a parameter within the subject. For example, the device can include, for example, a pump and optionally associated electronics.

[0203] In one embodiment, inducible cDC2s are generated by hand, for example, by altering the gene expression of at least one of the factors disclosed herein in somatic cells, pluripotent cells, progenitor cells, or stem cells, or by exposing any one of these cell types to at least one protein or RNA that produces at least one protein disclosed herein. The cells can also be generated by exposing them to a small molecule that turns on at least one of the factors disclosed herein. In some aspects, at least two, three, four, five, or six factors are used to generate inducible cDC2s.

[0204] In one embodiment, mouse embryonic fibroblasts (MEFs) were isolated and purified as follows: Clec9aCre / Cre animals (Schraml et al., 2013) were crossed with Rosa26-stopflox-tdTomato reporter mice (The Jackson Laboratory) to generate double homozygous Clec9aCre / Cre RosatdTomato / tdTomato (Clec9a-tdTomato) mice. All animals were housed under controlled temperature (23±2°C), subjected to a fixed 12-h light / dark cycle, and allowed free access to food and water.

[0205] In one experiment, primary cultures of MEFs were isolated from E13.5 embryos of Clec9a-tdTomato or C57BL / 6 mice. The head, fetal liver, and all internal organs were removed, and the remaining tissues were mechanically dissociated. Dissected tissues were enzymatically digested using 0.12% trypsin / 0.1 mM ethylenediaminetetraacetic acid (EDTA) solution (3 mL per embryo) and incubated at 37°C for 15 minutes. An additional 3 mL of the same solution was added per embryo, followed by an additional 15 minutes of incubation. Single-cell suspensions were obtained and plated in growth medium in 0.1% gelatin-coated 10 cm tissue culture dishes. Cells were grown for 2–3 days until confluent, dissociated with Tryple Express, and frozen in fetal bovine serum (FBS) 10% dimethyl sulfoxide (DMSO). Prior to plating for lentiviral transduction, MEFs were sorted for residual CD45 expression, which may represent cells with hematopoietic potential. + and tdTomato + The MEFs used for screening and the following experiments were >99% pure tdTomato-CD45 - and were expanded for up to 4 passages.

[0206] 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) CO. All tissue culture reagents were from Thermo Fisher Scientific unless otherwise noted.

[0207] In one embodiment, viral transduction and reprogramming experiments were performed as follows: Clec9a-tdTomato MEFs were seeded onto 0.1% gelatin-coated 6-well plates at a density of 40,000 cells per well. Cells were incubated overnight with a 1:1 ratio of FUW-TetO-TF and FUW-M2rtTA lentiviral particles in growth medium supplemented with 8 μg / mL polybrene. When testing combinations of TFs, equal MOIs of individual viral particles were applied. Cells were transduced twice on consecutive days, and after overnight incubation, the medium was replaced with fresh growth medium. After the second transduction, the growth medium was supplemented with doxycycline (1 μg / mL) - day 0. The medium was replaced every 2-3 days for the duration of the culture. The emergence of tdTomato + Cells were analyzed 5 to 9 days after transduction.

[0208] 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 5% FBS, and kept at 4°C before analysis on a BD Accuri C6 (BD Biosciences). For analysis of MHC-II, CD45, and CD11b cell surface marker expression, dissociated cells were incubated with APC-conjugated rat anti-mouse IA / IE, anti-mouse CD45, and anti-mouse CD11b antibodies (Biolegend), respectively, in the presence of rat serum (1 / 100, GeneTex) diluted in PBS 5% FBS for 30 minutes at 4°C to block nonspecific binding. Cells were washed with PBS 5% FBS, resuspended in PBS 5% FBS, and analyzed on a BD Accuri C6. Flow cytometry data were analyzed using FlowJo software (FLOWJO, LLC, version 7.6).

[0209] 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 5% FBS for 30 minutes at 4° C. Subsequently, MEFs were washed with PBS 5% FBS and resuspended in PBS 5% FBS, and tdTomato-CD45-MEFs were purified using a BD FACSAria III (BD Biosciences).

[0210] In one embodiment, cytokine secretion analysis was performed as follows: tdT produced by PI4P overexpression +Cells were FACS-sorted on day 9 of reprogramming. The following day, LPS (100 ng / mL), PiC (1 μg / mL), R848 (1 μg / mL), or CpG ODN1585 (0.5 μM) (Invivogen) were added to the medium for overnight stimulation. Culture supernatants were then collected for further analysis using the LEGENDplex™ Mouse Th Cytokine Panel (13-plex) kit according to the manufacturer's instructions. Acquisition was performed on a BD Accuri C6, and data were then analyzed using LEGENDplex™ v8.0 software (BioLegend).

[0211] In one embodiment, bone marrow was isolated from C57BL6 mice and used to generate bone marrow-derived dendritic cells. Briefly, whole bone marrow (BM) cells were harvested from long bones (tibia and femur) by grinding with a pestle and mortar. Cells were collected in phosphate-buffered saline (PBS) supplemented with 2% FBS and filtered through a 70 μm cell strainer (BD Biosciences). Red blood cells were lysed using BD Pharm Lyse (BD Biosciences) for 8 minutes at room temperature. Lysis was stopped by adding 5 or more volumes of PBS with 2% FBS. Total BM cells were seeded in Petri dishes (15 × 10 cells per 10 cm plate) in RPMI complete medium supplemented with Flt3l (200 ng / ml) and GM-CSF (5 ng / ml). 6 After 5 days of culture, 5 mL of complete RPMI medium was added, and on day 9, 3 × 10 cells were cultured. 6 The cells were replated in 10 ml of fresh medium containing Flt31 and GM-CSF. BM-DCs were used after 15 days of culture.

[0212] In one embodiment, antigen presentation assays were performed as follows: CD4+ T cells were obtained by spleen removal from OT-II mice and subsequent MACS purification using the Miltenyi Naive CD4+ T cell isolation kit. Purified CD4+ T cells were labeled with 5 mM CTV (Thermo Fisher), washed at room temperature for 20 minutes, and counted. FACS-sorted tdT +PIP-producing cells, MEFs, or BM-DCs were pre-cultured overnight with OVA 323-339 peptide (10 μg / ml). After thorough washing, 20,000 tdT cells were added. + PIP-producing cells, MEFs, or BM-DCs were co-cultured with 100,000 CTV-labeled CD4+ T cells in 96-well U-bottom culture plates in the presence or absence of TLR stimuli LPS (100 ng / mL), PiC (1 μg / mL), R848 (1 μg / mL), or CpG ODN1585 (0.5 μM) (Invivogen). After 5 days of culture, T cells were harvested, stained, and analyzed using a BD LSRFortessa™. T cell proliferation was assessed by the expression of LifeSingle TCRβ. + CD4 + Determined by gating on T cells.

[0213] 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 to the above Description, but rather is set forth in the claims.

[0214] Where the singular form of an element or feature is used in a claim recitation, the plural is also included, and vice versa, unless specifically excluded. For example, the term "a transcription factor" or "the transcription factor" includes the plural "transcription factor" or "those transcription factors," and vice versa. In the claims, articles such as "a," "an," and "the" can mean one or more, unless the context indicates otherwise or is otherwise clear from the context. A claim or description including one or more members of a group "or" members of a group is considered satisfied if one, more than one, or all group members are present in, employed in, or otherwise associated with a given product or process, unless the context indicates otherwise or is otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, employed in, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one, or all, of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0215] Furthermore, it should be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or from the relevant portion of the description are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same base claim.

[0216] Furthermore, when a claim recites a composition, it should be understood that unless otherwise indicated or unless a contradiction or inconsistency would arise apparent to one of ordinary skill in the art, it also includes methods of using the composition for any of the purposes disclosed herein, and includes methods of making the composition according to either the methods of manufacture disclosed herein or other methods known in the art.

[0217] When ranges are specified, endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any particular value within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It should also be understood that unless otherwise indicated or apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any subrange within the given range, with the endpoints of the subranges being expressed to the same degree of precision as the tenth of the unit of the lower limit of the range.

[0218] The present disclosure is in no way limited to the described embodiments, and those skilled in the art will envision many possibilities for modifying it.

[0219] The above embodiments can be combined.

[0220] The following claims further describe and describe particular embodiments of the present disclosure.

[0221] Example To further characterize the induced cells described in this composition and their similarity to bona fide DC subsets, mRNA sequencing can be performed at the population level. Population RNA-seq is typically performed as follows: total RNA is extracted with TRIzol reagent, and cDNA is generated and further amplified using a specific RNA kit (e.g., Takara SMARTSeq Ultra Low Input RNA Kit). The resulting cDNA is then analyzed using appropriate reagents (e.g., Agilent High Sensitivity DNA Kit). Following library preparation, the resulting cDNA is tagged, forward and reverse indexed by PCR, and sequenced on an appropriate instrument (e.g., Illumina NextSeq 500). The resulting data can then be analyzed for differential expression analysis, DC2-specific gene enrichment, and integrated with existing public datasets. Alternatively, single-cell RNA sequencing (scRNA-seq) may complement this analysis by providing expression profiles of individual cells, ideal for better defining the phenotype and cellular state of the derived cells described herein. References Brown, CC, Gudjonson, H., Pritykin, Y., Deep, D., Lavalle' e, VP, Mendoza, A., Fromme, R., Mazutis, L., Ariyan, C., Leslie, C., Pe'er, D., & Rudensky, AY (2019). Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity. Cell, 179(4), 846-863.e24. https: / / doi.org / 10.1016 / j.cell.2019.09.035 Datta, J., Terhune, J. H., Lowenfeld, L., Cintolo, J. A., Xu, S., Roses, R. E., & Czerniecki, B. J. (2014). Optimizing dendritic cellbased approaches for cancer immunotherapy. The Yale Journal of Biology and Medicine, 87(4), 491-518. Retrieved from http: / / www.ncbi.nlm.nih.gov / pubmed / 25506283 Gabriele, L., & Ozato, K. (2007). The role of the interferon regulatory factor (IRF) family in dendritic cell development and function. Cytokine & Growth Factor Reviews, 18(5-6), 503-510. https: / / doi.org / 10.1016 / j.cytogfr.2007.06.008 Kopf, M., Gros, G. Le, Bachmann, M., Lamers, M. C., Bluethmann, H., & Koehler, G. (1993). Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nature, 362(6417), 245-248. https: / / doi.org / 10.1038 / 362245a0 Laoui, D., Keirsse, J., Morias, Y., Van Overmeire, E., Geeraerts, X., Elkrim, Y., … Van Ginderachter, J. A. (2016). The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity. Nature Communications, 7, 13720. https: / / doi.org / 10.1038 / ncomms13720 Merad, M., Sathe, P., Helft, J., Miller, J., & Mortha, A. (2013). The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annual Review of Immunology, 31, 563-604. https: / / doi.org / 10.1146 / annurev-immunol-020711-074950 Mosmann, T. R., & Coffman, R. L. (1989). TH1 and TH2 Cells: Different Patterns of Lymphokine Secretion Lead to Different Functional Properties. Annual Review of Immunology, 7(1), 145-173. https: / / doi.org / 10.1146 / annurev.iy.07.040189.001045 Pereira, C. -F., Chang, B., Gomes, A., Bernitz, J., Papatsenko, D., Niu, X., … Moore, K. A. (2016). Hematopoietic Reprogramming In Vitro Informs In Vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells. Developmental Cell, 36(5), 525-539. https: / / doi.org / 10.1016 / j.devcel.2016.02.011 Pereira, C. -F., Chang, B., Qiu, J., Niu, X., Papatsenko, D., Hendry, C. E., … Moore, K. (2013). Induction of a Hemogenic Program in Mouse Fibroblasts. Cell Stem Cell, 13(2), 205-218. https: / / doi.org / 10.1016 / j.stem.2013.05.024 Pereira, C. -F., Lemischka, I. R., & Moore, K. (2012). Reprogramming cell fates: insights from combinatorial approaches. Annals of the New York Academy of Sciences, 1266(1), 7-17. https: / / doi.org / 10.1111 / j.1749-6632.2012.06508.x Plantinga, M., Guilliams, M., Vanheerswynghels, M., Deswarte, K., Branco-Madeira, F., Toussaint, W., … Lambrecht, B. N. (2013). Conventional and Monocyte-Derived CD11b+ Dendritic Cells Initiate and Maintain T Helper 2 Cel-Mediated Immunity to House Dust Mite Allergen. Immunity, 38(2), 322-335. https: / / doi.org / 10.1016 / j.immuni.2012.10.016 Rosa, F. F., Pires, C. F., Kurochkin, I., Ferreira, A. G., Gomes, A. M., Palma, L. G., … Pereira, C. -F. (2018). Direct reprogramming of fibroblasts into antigen-presenting dendritic cells. Science Immunology, 3(30), eaau4292. https: / / doi.org / 10.1126 / sciimmunol.aau4292 Sakaguchi, S. (2004). N aturally A rising CD4 + R egulatory T C ells for I mmunologic S elf -T olerance and N egative C ontrol of I mmune R esponses. Annual Review of Immunology, 22(1), 531-562. https: / / doi.org / 10.1146 / annurev.immunol.21.120601.141122 Schraml, B. U., van Blijswijk, J., Zelenay, S., Whitney, P. G., Filby, A., Acton, S. E., … Reis e Sousa, C. (2013). Genetic tracing via DNGR-1 expression history defines dendritic cells as a hematopoietic lineage. Cell, 154(4), 843-858. https: / / doi.org / 10.1016 / j.cell.2013.07.014 Subklewe, M., Geiger, C., Lichtenegger, F. S., Javorovic, M., Kvalheim, G., Schendel, D. J., & Bigalke, I. (2014). New generation dendritic cell vaccine for immunotherapy of acute myeloid leukemia. Cancer Immunology, Immunotherapy:CII, 63(10), 1093-1103. https: / / doi.org / 10.1007 / s00262-014-1600-5 Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell, 131(5), 861-872. https: / / doi.org / 10.1016 / j.cell.2007.11.019 Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 126(4), 663-676. https: / / doi.org / 10.1016 / j.cell.2006.07.024 Weaver, C. T., Harrington, L. E., Mangan, P. R., Gavrieli, M., & Murphy, K. M. (2006). Th17: An Effector CD4 T Cell Lineage with Regulatory T Cell Ties. Immunity, 24(6), 677-688. https: / / doi.org / 10.1016 / j.immuni.2006.06.002 Xie, H., Ye, M., Feng, R., & Graf, T. (2004). Stepwise reprogramming of B cells into macrophages. Cell, 117(5), 663-676. https: / / doi.org / 10.1016 / s0092-8674(04)00419-2 Xu, J., Du, Y., & Deng, H. (2015). Direct lineage reprogramming: strategies, mechanisms, and applications. Cell Stem Cell, 16(2), 119-134. https: / / doi.org / 10.1016 / j.stem.2015.01.013 Zhu, J., & Paul, W. E. (2008). CD4 T cells: fates, functions, and faults. Blood, 112(5), 1557-1569. https: / / doi.org / 10.1182 / blood-2008-05-078154

Claims

1. One or more constructs or vectors encoding a combination of at least three transcription factors, wherein the encoded combination of transcription factors is PU.1, IRF4 and PRDM1; PU.1, IRF4 and POU2F2; PU.1, IRF4 and TGIF1; and PU.1, IRF4 and RBPJ and one or more constructs or vectors selected from the group consisting of: (PU.1) SEQ ID NO:3 or SEQ ID NO:6, (IRF4) SEQ ID NO:9 or SEQ ID NO:12, (PRDM1) SEQ ID NO:15, SEQ ID NO:18, (POU2F2) SEQ ID NO:27, SEQ ID NO:30, (TGIF1) SEQ ID NO:33, SEQ ID NO:36, and (RBPJ) SEQ ID NO:45, SEQ ID NO:48, wherein the transcription factor is at least 90% identical to the sequence of:

2. 2. The one or more constructs or vectors of claim 1, wherein the encoded combination of transcription factors is PU.1, IRF4, and PRDM1, arranged in the 5' to 3' order of PU.1, IRF4, PRDM1.

3. 3. One or more constructs or vectors according to any one of claims 1 to 2, wherein the vector is a viral vector.

4. The one or more constructs or vectors according to any one of claims 1 to 2, wherein said one or more constructs or vectors are synthetic mRNA, naked alphavirus RNA replicon or naked flavivirus RNA replicon.

5. A composition comprising one or more vectors according to any one of claims 1 to 4.

6. 6. The composition of claim 5, wherein the transcription factors are individually encoded by polynucleotides at least 90% identical to the following sequences: PU.1 (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5), IRF4 (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11), PRDM1 (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17), POU2F2 (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29), TGIF1 (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35), and RBPJ (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47).

7. The one or more vectors are a combination of: PU.1, IRF4 and PRDM1; PU.1, IRF4 and POU2F2; PU.1, IRF4 and TGIF1; and PU.1, IRF4 and RBPJ The composition according to any one of claims 5 to 6, wherein the composition encodes a combination of transcription factors selected from:

8. The composition according to any one of claims 5 to 6, wherein the combination of transcription factors is PU.1, IRF4 and PRDM1.

9. The composition according to any one of claims 5 to 8, wherein the one or more vectors are viral vectors.

10. The composition of any one of claims 5 to 9, wherein the one or more vectors are synthetic mRNA, naked alphavirus RNA replicon, or naked flavivirus RNA replicon.

11. A composition according to any one of claims 5 to 10 for use in veterinary or human medicine.

12. A composition according to any one of claims 5 to 11 for use in the treatment of cancer, infectious diseases or autoimmune diseases.

13. An in vitro or ex vivo method for reprogramming or inducing stem cells or differentiated cells into conventional dendritic cells type 2, 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 encoding at least three transcription factors, wherein the first and second transcription factors are PU.1 and IRF4, and the third transcription factor is selected from the group consisting of PRDM1, POU2F2, TGIF1, and RBPJ; Culturing the transduced cells in a cell culture medium that supports the proliferation of dendritic cells or antigen-presenting cells; A method comprising:

14. 14. The method of claim 13, wherein the transduced cells are cultured for 2 to 9 days.

15. The method according to any one of claims 13 to 14, wherein the stem cells or the differentiated cells or a mixture thereof are mammalian cells.

16. A method according to any one of claims 13 to 15, wherein the stem cells or differentiated cells or a mixture thereof are human cells.

17. The method according to any one of claims 13 to 16, wherein the stem cells or the differentiated cells or a mixture thereof are selected from the group consisting of pluripotent stem cells, multipotent stem cells, and differentiated cells, and 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, mesenchymal stem cells, hematopoietic stem cells, intestinal stem cells, and cell lines thereof.

18. The method described in claim 17, wherein the mesoderm-derived cells are fibroblasts or the hematopoietic stem cells are umbilical cord blood stem cells.

19. 19. The method of any one of claims 13 to 18, wherein the transduction step further comprises using at least one vector comprising 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, or a mixture thereof.

20. An induced dendritic cell comprising one or more constructs or vectors according to any one of claims 1 to 4 or a composition according to claims 5 to 10.

21. 21. A composition comprising a therapeutically effective amount of the induced dendritic cells of claim 20, or a mixture thereof, and further comprising a pharmaceutically acceptable excipient.

22. 22. The composition of claim 21 for use in veterinary or human medicine.

23. 22. The composition of claim 21 for use in the treatment of cancer, an infectious disease or an autoimmune disease.

24. A vaccine or injectable preparation for cancer, comprising the composition of claim 21, or the induced dendritic cells of claim 20, or a mixture thereof.

25. Ingredients:

21. The induced dendritic cell of claim 20.

22. The composition of claim 21 ; One or more constructs or vectors according to any one of claims 1 to 4; The composition according to any one of claims 5 to 10. or a mixture thereof A kit comprising at least one of:

Images