CN117580947A

CN117580947A - Reprogramming cells to type 1 classical dendritic cells or antigen presenting cells

Info

Publication number: CN117580947A
Application number: CN202280047592.5A
Authority: CN
Inventors: F·弗乌扎洛萨; O·齐默尔曼诺瓦; A·G·巴罗斯费雷拉; E·艾西克; C·弗雷拉皮雷斯; C·菲利普·雷蓓罗·勒莫斯·佩雷拉
Original assignee: Asgard Treatment Co ltd
Current assignee: Asgard Treatment Co ltd
Priority date: 2021-05-19
Filing date: 2022-05-19
Publication date: 2024-02-20

Abstract

The present invention relates to compositions comprising transcription factors under the control of a promoter region, wherein the compositions can be used to reprogram cells into type 1 classical dendritic cells or antigen presenting cells. The invention further relates to a method for reprogramming a cell to a type 1 classical dendritic cell or an antigen presenting cell.

Description

Reprogramming cells to type 1 classical dendritic cells or antigen presenting cells

Technical Field

The present invention relates to compositions and methods for reprogramming cells to type 1 classical dendritic cells or antigen presenting cells.

Background

Cell reprogramming relies on the re-routing (rewiring) of the epigenetic and transcriptional network of one cell state to that of a different cell type. Transcription Factor (TF) overexpression experiments have highlighted the plasticity of adult somatic or differentiated cells, providing a new technique for generating any desired cell type. Reprogramming somatic or differentiated cells to Induced Pluripotent Stem Cells (iPSCs) very similar to embryonic stem cells is possible by forced expression of TF (Takahashi et al, 2007; takahashi & Yamanaka, 2006). Alternatively, somatic cells can also be transformed directly into another specific cell type (Pereira, lemischka, & Moore, 2012). Direct lineage conversion has proven successful in reprogramming mouse and human fibroblasts into several cell types, such as neurons, cardiomyocytes and hepatocytes, using TF specifying the identity of the target cells (Xu, du, & Deng, 2015). Direct cell conversion was also demonstrated in the hematopoietic system, where forced expression of TF induced macrophage fate in B cells and fibroblasts (Xie, ye, feng, & Graf, 2004), and direct reprogramming of mouse fibroblasts to clonal hematopoietic progenitor cells was achieved with Gata2, gfi B, cFos, and Etv6 (Pereira et al, 2013). These four TFs induce a dynamic, multi-stage hematopoiesis process, which proceeds through endothelial-like intermediates, reproducing developmental hematopoiesis in vitro (Pereira et al 2016).

Reprogrammed cells are a very promising therapeutic tool for regenerative medicine, and cells obtained by iPSC differentiation have been tested in clinical studies (Pires et al, 2019). Recently, it has been demonstrated that antigen presenting Dendritic Cells (DCs) can be reprogrammed from unrelated cell types by small combinations of TF (Rosa et al, 2018), opening the opportunity to apply cell reprogramming to modulate immune responses and develop new immunotherapies.

Traditionally, the division of DC can be divided into two functionally distinct DC subsets: it is a classical DC (DCs) of professional Antigen Presenting Cells (APCs), as well as a plasmacytoid DC (pDC). Dcs drive antigen-specific immune responses, while pdcs are specialized producers of type I interferons during viral infection. However, the timing and exact mechanisms of regulating differentiation of the different subsets during DC development remain to be established.

DCs are a class of bone marrow derived cells derived from lymphoid-myeloid hematopoiesis that scan for pathogens in organisms, forming an important interface between the activation of the innate immune system and adaptive immunity. DC act as professional APC, able to activate T cell responses by displaying peptide antigens complexed with Major Histocompatibility Complex (MHC) on the surface, along with all necessary soluble and membrane-associated costimulatory molecules. DCs induce a primary immune response by eliciting naive T lymphocytes, boost effector functions of previously sensitized T lymphocytes, and coordinate communication between innate and adaptive immunity. DCs are found in most tissues where they constantly sample the environment of the pathogen and several types of receptors are used to monitor the invading pathogen. In steady state, and at an increased rate after pathogen detection, sentinel DCs in non-lymphoid tissues migrate to lymphoid organs where they present their collected and processed antigens to T cells. The phenotype obtained by T cells depends on the environment of antigen presentation. If the antigen is derived from a pathogen or from an injured person, the DC will receive a danger signal, become activated and subsequently stimulate T cells to become effector cells necessary to provide protective immunity.

An important aspect of controlling immune responses is the presence of different types of DCs, each specifically responding to a particular pathogen and interacting with a particular subset of T cells. In this context, DCs can be further classified into myeloid/classical DC type 1 (adc 1 or adc 1 s) and myeloid/classical DC type 2 (adc 2). This expands the flexibility of the immune system to respond appropriately to a wide range of different pathogens and danger signals.

Human cDC1 (Wculek et al, 2019; heidkamp et al, 2016; dutertre et al, 2019) characterized by surface expression of CD141, CLEC9A, XCR1 and CD226 is functionally defined by: secretion of immunomodulatory cytokines including IL-12 and Interferon (IFN) and chemokines such as CXCL10, and cross-presentation of antigens to CD8 ⁺ T cells (Lauterbach et al, 2010; poulin et al, 2010). In the context of anti-tumor immunity, batf 3-/-animals lacking cDC1 fail to reject immunogenic tumors (Hildner et al, 2008). This effect appears to be dependent on DCs 1 that the tumor resides, highlighting the importance of this subset of DCs at the tumor site to mediate immune rejection of established tumors (Bottcher et al, 2018) and response to treatment (Salmon et al, 2016, sprager et al, 2017). Accordingly, the abundance of cDC1 in human tumors correlates with patient survival and responsiveness to checkpoint inhibitors (barre et al, 2018, broz et al, 2014, hubert et al 2020, mayoux et al 2020, spranger et al 2017). Human primary dcs 1 are very rare in vivo, so their research and transformation applications require methods to generate functional dcs 1 in vitro. Human CD34 ⁺ Bone Marrow (BM) progenitor cells have been used to derive CD141 in vitro in the presence of FLT3L with SCF, GM-CSF, and IL-4 ⁺ cDC1 (Poulin et al, 2010). More recently, FLT3L has been combined with co-culture of Notch-expressing stromal cell lines to promote cDC1 differentiation (Kirkling et al, 2018; balan et al, 2018). The production of cDC1, cDC2 and pDC-like cells from Induced Pluripotent Stem Cell (iPSC) cultures was also confirmed (Sontag et al, 2017). However, these schemes are complex, require feeder layers and result in low yields and mixtures of different DC subsets with conflicting functions.

Thus, new strategies are needed to generate homogeneous populations of differentiated human dcs 1 in vitro.

Disclosure of Invention

Provided herein are compositions and methods for reprogramming cells to dendritic cells or antigen presenting cells. The inventors have found that by expressing the transcription factors BATF3, IRF8, PU.1 under certain promoters, reprogramming of cells can be significantly improved. The inventors have also found additional transcription factors (i.e., IRF7 and BATF) that increase reprogramming efficiency when co-expressed with pu.1, IRF8 and BATF 3.

Thus, provided herein are compositions comprising one or more constructs or vectors that, upon expression, encode the following transcription factors:

a) BATF3 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO:10 (BATF 3), e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100% identity to SEQ ID NO:10 (BATF 3);

b) IRF8 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 11 (IRF 8), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 11 (IRF 8); and

c) Pu.1 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID No. 12 (pu.1), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 12 (pu.1);

Wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, wherein the promoter region comprises a Spleen Focus Forming Virus (SFFV) promoter.

Further provided herein are cells comprising one or more constructs or vectors according to the compositions described herein.

Also provided herein is a method of reprogramming or inducing cells into dendritic cells or antigen presenting cells, the method comprising the steps of:

a) Transducing cells with a composition comprising a construct or vector according to the compositions described herein.

b) Expressing the transcription factor;

thereby obtaining reprogrammed or induced cells.

Further provided herein are reprogrammed or induced cells obtained according to the methods disclosed herein.

Also provided herein are methods of treating cancer, the methods comprising administering to a subject in need thereof a composition according to the invention; a cell; a pharmaceutical composition; and/or reprogrammed or induced cells.

Further provided herein are compositions according to the present invention; a cell; a pharmaceutical composition; and/or the use of reprogrammed or induced cells for the manufacture of a medicament for the treatment of cancer.

Drawings

FIG. 1.PU.1, IRF8 and BATF3 induce global cDC1 gene expression program in human fibroblasts. (A) Human Embryonic Fibroblasts (HEF) were co-transduced with Dox-induced lentiviral particles encoding PU.1, IRF8 and BATF3 (PIB, tetO-PIB) and M2rtTA (UbC-M2 rtTA). On day 3 (d 3, CD 45) ⁺ ) Day 6 (d 6, CD 45) ⁺ ) And day 9 (CD 45) ⁺ HLA-DR ^- ，d9 DR ^- ；CD45 ⁺ HLA-DR ⁺ ，d9 DR ⁺ ) Purified PIB-transduced HEF (hiDC) was profiled by single cell RNA-seq. HEF and peripheral blood cDC1, cDC2 and pDC were included as controls. (B) Flow cytometry analysis of hiDC on days 3 and 9 after addition of Dox, and (C) DR ^- (Top) and DR ⁺ Kinetics of (bottom) cell appearance (n=2-8, mean ± SD). (D) scanning electron microscopy at day 9. Scale bar, 10 μm. The t-SNE panel (E) shows 45,870 single cells.(F) Integration of scPred (Alquiura-Hernandez et al 2019) with published DC subset data (Villani et al 2017) was used. The heat map shows the percentage of single cells that are affiliated with the subset cDC1-DC 6. (G) t-SNE map of single cells to which cDC1 belongs. (H) Violin plots show the distribution of gene expression of the dcs 1 specific genes. The log of gene counts is shown. (I) The heatmap shows genes differentially expressed across the profiling population in 5 clusters. (J) Violin map for genes selected from Cluster 3. (K) The first 5 response group (reactiome) pathways enriched in each cluster. The (L) heat map and (M) violin map show the expression of genes associated with antigen cross presentation.

Figure 2. Pseudo-temporal ordering of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. (A) HEF, at days 3, 6 and 9 (DR ^- And DR ⁺ ) Is classified as non-affiliated or affiliated to cDC1 with scppred (alquinura-Hernandez et al 2019), and single cell locus single cell 3 reconstruction of filtered cDC 1. (B) The trace was reprogrammed with cDC1 colored at the relative trace position (pseudo-time, left). The box and whisker plot shows pseudo-time distribution by cell type (right). (C) the tSNE plot shows the single cell velocity generated with scVelo (Bergen et al 2020). Arrows indicate the direction along the track and the thickness speed. (D) The heat map highlights 6 gene clusters (A-F) with dynamic expression along the scVelo latency. (E) the first 5 response group pathways enriched in each cluster. (F) The heat map shows the average expression values of gene modules per cell type differentially expressed along the trace. (G) Violin plots show the distribution of gene expression associated with unsuccessful and successful DC reprogramming. The log of gene counts is shown. (H) Transcription factors were reprogrammed with Chea3 unsuccessful (left) and successful (right) cDC 1. SPI1, IRF8 and BATF are highlighted in bold. (I) flow cytometry analysis of CD226 expression in hiDC. (J) With classification of DC subset data (Villani et al 2017). (K) By CD45 ⁺ HLA-DR ⁺ CD226 ⁺ And CD45 ⁺ HLA-DR ⁺ CD226 ^- The dead cells of hidcs phagocytized (n=6-7, mean ± SD). (L) reprogramming Cheng Xiaolv on day 9 generated by cotransduction of PIB with indicated transcription factor (n=4, average ±SD). M2 rtTA-and PIB-transduced cells were included as controls. * P<0.005；****p<0.00005。

Fig. 3 inflammatory cytokine signaling allows reprogramming of human cDC1 with high efficiency. (A) The obtained hiDC (CD 45) at day 9 in the presence of a combination of individual cytokines and (B) 2-3 cytokines ⁺ HLA-DR ⁺ ) Is a quantitative measure of (3). The untransduced HEF was included as a control (n=2-10, mean±sd).

FIG. 4 forced expression of transcription factors allows reprogramming of human cDC1 with high efficiency. (A) Reprogrammed cells obtained by reporting mouse embryonic fibroblasts with PIB-IRES-GFP transduction Clec9a-tdTomato (tdT) (tdT ⁺ MHC-II ⁺ ) The PIB-IRES-GFP was driven by either a Dox inducible (TetO) or constitutive promoter (UbC, SFFV, PGK, EF1S, EF and EF1 i). Expression of GFP (tetO-GFP) was used as a control (n=2-6, mean ± SD). (B) Quantification of hiDC produced with TetO-PIB or SFFV-PIB in the presence or absence of IFN- γ, IFN- β and TNF- α at day 9 (n=4-19, mean ± SD). (C) hiDC yield/input into fibroblasts (n=10-12, mean ± SD). (D) The hidcs generated on day 9 under four conditions were purified and analyzed by scRNA-seq. The heat map shows the percentage of single cells that are affiliated with the subset cDC1-DC 6.

Fig. 5. Anti-inflammatory cytokine signaling did not impair cDC1 reprogramming. CD45 at day 9 produced by transduction of HEF with SFFV-PIB in the presence of anti-inflammatory cytokines ⁺ HLA-DR ⁺ Cell (left) and in CD45 ⁺ HLA-DR ⁺ Cell (right) gated CD40 ⁺ Flow cytometry quantification of cells (n=3, mean±sd).

FIG. 6 optimized reprogramming protocol allows the generation of functional human cDC 1-like cells. (A) hiDC (CD 45) at day 9 generated with SFFV-PIB in the absence or presence of IFN-gamma, IFN-beta and TNF-alpha (hidc+cyt) ⁺ HLA-DR ⁺ ) And peripheral blood CD141 ⁺ CLEC9A ⁺ Median Fluorescence Intensity (MFI) of CD40 and CD80 in cDC 1. Cells were stimulated overnight with individual TLR agonists LPS, poly I: C (polyinosinic acid: polycytidylic acid), R848 or combinations (all) (n=2-14, plainMean ± SD). (B) Quantification of phagocytosis of dead cells by hidcs on day 9 after 2 hours incubation. Including HEF and CD141 ⁺ CLEC9A ⁺ cDC1 served as a control (n=3-12, mean±sd). (C) Cytokine secretion of purified hiDC on day 9 after overnight incubation with TLR agonist. Including HEF, monocyte derived DC (moDC) and CD141 ⁺ CLEC9A ⁺ XCR1 ⁺ cDC1 served as a control (n=2-11, mean±sd). (D) Cells were incubated with LPS, polyI: C and R848 overnight, pulsed with CMV proteins for 3 hours, washed and incubated with CMV ⁺ CD8 ⁺ T cell co-culture. Antigen cross presentation was quantified by measuring IFN- γ after 24 hours (n=2-4, mean ± SD). * P is p<0.05；**p<0.005；***p<0.0005；****p<0.00005。

Fig. 7. Effective cDC1 reprogramming of adult fibroblasts. Human Dermal Fibroblast (HDF) generated hiDC (CD 45) at day 9 in the absence (SFFV-PIB) or presence (SFFV-PIB+cyt) of IFN-gamma, IFN-beta and TNF-alpha from three independent donors ⁺ HLA-DR ⁺ ) The flow cytometry analysis (A) and the quantification (B). HDF was included as a control (n=3-13, mean ± SD). (C) expression of CD40 and CD 80. (D) HDF-derived hidcs were purified on day 9 and profiled by scRNA-seq. The heat map shows the percentage of single cells that are affiliated with the subset cDC 1-6. (E) The heat map shows the expression of genes that were up-regulated during reprogramming and expressed in cDC 1. The cDC1 and antigen presenting genes are highlighted in bold and shown as violin plots in (F). The log of gene counts is shown.

Fig. 8. Effective cDC1 reprogramming of mesenchymal stromal cells. (A) Strategy for deriving hidcs from human Mesenchymal Stromal Cells (MSCs) under xeno-free conditions. MSC were isolated from three healthy donors and FACS purified (Lin-CD 45-CD271 ⁺ ) Amplification was performed in pHPL medium, and transduction was performed and culture was performed in X-VIVO 15. dX = day X. (B and C) quantification of MSC-derived hiDC on day 9 with or without cytokine production (n=3-14, mean ± SD). (D) flow cytometry analysis of CD40 and CD 80. ns-insignificant; * P <0.00005。

FIG. 9 induced DC elicited anti-tumor immunity in vivo. (A) Dynamics of cross-presentation capability are obtained during DC reprogramming. Representative flow cytometry patterns showed that at reprogrammed d4, d7, and d9, with 50,000 tdT ⁺ Cell co-cultured TCR ⁺ CD8 ⁺ CD44 ⁺ CTV markers of T cells. MEF was included as a control. (B) As well as tdT sorted at different time points and in three different ratios ⁺ Quantification of proliferating T cells after cell co-culture (n=4, mean±sd). (C) Sorted tdT after stimulation with LPS or polyI: C ⁺ Cytokine secretion by cells (n=2, mean ± SD). Including MEF and CD103 ⁺ Bone marrow derived DC (BM-DC) served as a control. (D) Purified tdT was then used to prepare the tumor cells prior to subcutaneous implantation into C57BL/6 mice ⁺ The iDC was mixed with 0.5m b16ova cells. Tumor volumes (n=5-6, mean ± SEM) were assessed over a 14 day period. (E) Purified tdT was taken 8 days after establishment ⁺ intratumoral injection of iDC into B16OVA tumor. Tumor volumes were assessed until day 20 (n=4-9, mean±sem,2 independent experiments). Including PBS, MEF and CD103 injections ⁺ Animals of BM-DC served as controls. (F) CTV-labeled OT-I CD8 ⁺ T cells were injected intravenously on the same day as iDC and after 4 days tumors and tumor draining lymph nodes were analyzed. Tumor infiltration (left) and OVA-restricted CD8 ⁺ IFN-. Gamma.and granzyme B (GzmB) expression (right) following in vitro re-stimulation of T cells was quantified (n=2-4, mean.+ -. SD).

Fig. 10.Pu.1 has independent chromatin targeting ability and recruits IRF8 and BATF3 to the same binding site. (A) Strategies for profiling the chromatin binding sites of pu.1, IRF8 and BATF3 (PIB) at the early stage of reprogramming. HDF was transduced with PIB (left) or individual factors (right) and analyzed by ChIP-seq after 48 hours. (B) The heat map shows the whole genome distribution of pu.1, IRF8 and BATF3 when expressed in combination (left) or individually (right). The signal is shown within an 8kb window centered on each peak. The number of peaks under each condition is shown. The average signal intensity (bottom) of the peaks is depicted. (C) The analysis of the slave motif predictions for pu.1, IRF8 and BATF3 target sites when expressed in combination or individually. The motif for PU.1 is highlighted in bold.

Fig. 11.Pu.1, IRF8 and bat f3 bind at open chromatin to inhibit fibroblast genes and to apply the cDC1 transcription procedure. (A) Venturi shows the genome-wide peak overlap between PU.1, IRF8 and BATF3 (PIB). (B) When expressed in combination, analysis was predicted from the head motif with respect to pu.1, IRF8 and BATF3 co-binding sites. Motifs for PU.1-IRF and BATF are highlighted in bold. (C) motif comparison between PU.1-IRF and BATF. Jiecade similarity coefficient (Jaccard similarity coefficient) =0.02. (D) Immunoblots showed Immunoprecipitation (IP) on pu.1 (top), IRF8 (middle) and BATF3 (bottom) in HEK293T cells 24 hours after transfection with PIB (left). Co-immunoprecipitation (Co-IP) was performed with 1, 2, and 5 million (M) cells (right). Input (10%) and IgG isotype served as controls. (E) The heat map shows genes differentially expressed between HDF and hiDC on day 9 bound by pu.1, IRF8 and BATF3 or three factors (crossing). (F) Thermal map of normalized read coverage of chromatin markers in HDF with respect to co-binding sites. The signal is displayed within an 8kb window and centered at the transcription factor binding site. The average signal strength is shown (upper graph). (G) model of the mechanism for initiating cDC1 reprogramming.

FIG. 12.PU.1, IRF8 and BATF3 reprogrammed mouse cancer cells with cDC 1-like cells. (A) Flow cytometry analysis of mouse Lewis lung carcinoma (3 LL) and melanoma (B16) cells at day 9 after transduction with SFFV-PIB-GFP lentiviral particles (tumor antigen presenting cells, tumor-APCs). A SFFV-GFP transduced parent cell line was included as a control. (B) On day 9 (d 9) Lewis Lung Carcinoma (LLC) and melanoma B16 derived reprogrammed 2 cells (GFP) were purified by FACS ⁺ CD45 ⁺ MHC-II ⁺ ). Cancer cell 3 transduced with GFP vector was included as a control (d 0). The heat map shows the expression of 4 genes associated with the IFN-. Gamma.and STING (right) pathways in reprogrammed LLC and induced 5 dendritic cells (iDC). Splenic dendritic cell type 1 (cDC 1) was included as reference 6 (GSE 103618). (C) When indicated, on day 3 of reprogramming after overnight stimulation with poly (I: C) (P (I: C)) following co-culture with FACS-purified B16-OVA cells transduced with PIB or eGFP lentivirusesIs CD8 ⁺ T cell proliferation (CTV dilution) and activation (CD 44) ⁺ ) Flow cytometry analysis (left) and quantification (right) of endogenous antigen presentation measured (n=3-4). (D) After 72 hours of co-culture, T cell mediated B16-OVA target cells (mOrange ⁺ ) Killing was analyzed by flow cytometry (left) and quantified (right), and the B16-OVA target cells were PIB transduced or IFN-treated (n=6-9). (E) When indicated, as CD44 after co-culture with B16 cells transduced with SFFV-PIB-GFP lentiviral particles and incubated overnight with OVA protein in the presence of P (I: C) and/or interferon gamma (IFN-g) ⁺ Proliferative OT-I CD8 ⁺ Flow cytometry quantification of antigen cross-presentation capacity measured as a percentage of 9T cells (n=4-8). (F) B16-derived tumor-APCs on day 5 of reprogramming were pulsed with OVA protein and P (I: C) and intratumorally injected into pre-established B16-OVA tumors on days 7, 10 and 13. Tumor growth (G) and survival (H) in mice injected with tumor-APC (PIB), PBS, or B16 cells transduced with control lentivirus (MCS) (n=6). Expressed as mean ± SD. * P<0.01，****p<0.0001。

Fig. 13, pu.1, IRF8 and bat f3 reprogrammed human cancer cells to cDC 1-like cells. (A) EGFP as transduction by flow cytometry when transduced with SFFV-PIB-GFP or control SFFV-GFP lentivirus ⁺ Cell percentage analysis of the cells (red) that gate the cell lines co-expressing CD45 and HLA-DR, glioblastoma (T98G), rectal cancer (ECC 4) and mesothelioma (ACC-Meso-1, accm 1) were reprogrammed efficiently. (B) cDC1 reprogramming efficiency across 28 solid tumor cell lines. Shows the expression (CD 45) ⁺ HLA-DR ⁺ ) Is a reprogramming population and an intermediate population (CD 45) ⁺ HLA-DR ^- Or CD45-HLA-DR ⁺ ) (n=2-8). Expressed as mean ± SD. (C) Flow cytometry quantification of cDC1 surface markers (CLEC 9A, CD141, CD11 c) in human glioblastoma (T98G) cells 9 days after transduction with SFFV-PIB-GFP lentiviral particles. A SFFV-GFP transduced parent cell line was included as a control. (D) At 9 days after SFFV-PIB-GFP lentiviral particle transduction overnight stimulated with poly-I-C and LPS, at CD45 ⁺ HLA-DR ⁺ Surface expression kinetics of co-stimulatory molecules CD40, CD80 and CD86 in cancer cells. ComprisingThe SFFV-GFP transduced parent cell line and the unstimulated SFFV-PIB-GFP transduced parent cell line served as controls. An example plot on day 9 is shown in (E). (F) Human primary tonsil cancer tissue (JCA 10) and patient xenograft derived bladder cancer cells (U3P 2E 2) were transduced with hPIB-IRES-EGFP or EGFP control vector (day 0) and analyzed by flow cytometry at day 9 to determine reprogrammed CD45 ⁺ HLA-DR ⁺ Percentage of cells (black) and partially reprogrammed cells expressing CD45 or HLA-DR. (G) The reprogramming efficiency in primary human tumor cells from melanoma (n=2), lung cancer (n=2), head and neck cancer (tonsil, n=2; tongue, n=3), pancreatic cancer (n=2), breast cancer (n=2) and bladder cancer (n=2), and cancer-associated fibroblasts (CAF, n=2) is shown. Shows partially reprogrammed cells (CD 45 ⁺ HLA-DR ^- ，CD45-HLA-DR ⁺ )。

Fig. 14.Pu.1, IRF8 and bat f3 induced rapid global transcription and apparent genetic reprogramming. (A) Experimental design to evaluate the kinetics of transcriptome and epigenetic reprogramming. Human glioblastoma cell line (T98G) was transduced with SFFV-hPIB-IRES-EGFP. Reprogramming (CD 45 at day 3 (d 3), day 5 (d 5), day 7 (d 7) and day 9 (d 9) ⁺ HLA-DR ⁺ ++) and partial reprogramming (CD 45-HLA-DR) ⁺ The cells of +) were FACS sorted and profiled with mRNA sequencing and ATAC sequencing. Control cells transduced with empty EGFP vector were indicated as day 0 (d 0). cDC1 donor cells were used as reference. (B) Principal Component Analysis (PCA) based on the time course of reprogramming cancer cells with differentially expressed genes (left panel). Reprogramming of Human Embryonic Fibroblasts (HEF) is also included as a reference to the kinetics of this process. The arrow highlights the reprogramming track. PCA based on differential accessible chromatin regions (right panel). For peripheral blood cDC1 as a reference. (C) Establishment of tumor-APC transcriptome features in reprogrammed and partially reprogrammed T98G cells (left). Chromatin accessibility at the tumor-APC gene set is shown on the right.

FIG. 15 histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. (A) PU.1, IRF8 and BATF3 (SFFV-PI) for Lewis Lung Cancer (LLC) and B16 cancer cellsB-eGFP), cultured in the presence or absence of valproic acid (VPA), and analyzed by flow cytometry on day 9 for CD45 and MHC-II expression. (B) In eGFP ⁺ Quantification of reprogramming efficiency in the presence of VPA gated in transduced cells (% CD 45) ⁺ MHC-II ⁺ Cells) (n=6-16). Cancer cells transduced with the eGFP vector (green, striped) were included as controls. (C) In eGFP ⁺ Gated MHC-I in transduced cells ⁺ Quantification of cell percentages (n=6-11). (D) CD44 after co-culture with reprogrammed LLC-OVA and B16-OVA cells ⁺ Proliferative OT-I CD8 ⁺ Quantification of T cells (n=4-11). (E) Quantification of T cell mediated reprogrammed B16-OVA target cells by flow cytometry (mOrange ⁺ ) And after 0 and 72 hours of co-culture with non-target B16-OVA cells at a 1:1 ratio of activated OT-I T cells to cancer cells (n=5-7). (F) CD44 after co-culture with reprogrammed LLC or B16 cells pre-incubated with OVA peptide (SIINFEKL) ⁺ Proliferative OT-I CD8 ⁺ Quantification of T cells (n=4-12). Expressed as mean ± SD. * P<0.01，****p<0.0001. (G) On day 9, the efficiency of cDC1 reprogramming in the presence and absence of valproic acid (VPA) was analyzed and quantified by flow cytometry in 6 human cancer cell lines (upper panel) and quantified (lower panel). Cancer cell lines were transduced with SFFV-hPIB-IRES-EGFP lentiviral particles or EGFP as a control and cultured in the presence or absence of VPA from day 1 to day 4 of reprogramming.

Fig. 16.Spib and SPIC compensate for the role of pu.1 in cDC1 reprogramming. (A) Clec9a reporter activated flow cytometry quantification in Mouse Embryonic Fibroblasts (MEFs) 5 days after transduction with pu.1 homologs alone or in combination with IRF8 and BATF 3. (B) Flow cytometry quantification of CD45 and MHC-II expression levels (in tdTomato ⁺ Gating in cells). Bar graph indicates mean ± SEM (n=4). * P<0.01，****p<0.0001。

Fig. 17 pu.1, IRF8 and BATF3 delivered by adenovirus and adeno-associated virus allow for cDC1 reprogramming in mouse and human cells. Quantification of (A) Clec9a reporter activation and (B) CD45 and MHC-II expression in Mouse Embryonic Fibroblasts (MEFs) 9 days after transduction with lentiviruses (Lenti), adenoviruses (Ad 5 and Ad 5/F35) and adeno-associated viruses (AAV-DJ and AAV 2-qYF) encoding PU.1, IRF8 and BATF3 (PIB) and GFP (PIB-GFP). Viruses encoding GFP alone were included as controls and tdTomato expression was not induced in transduced cells. Bar graph indicates mean ± SEM (n=4). (C) The flow cytometry quantification of the efficiency of cDC1 reprogramming in the B2905 mouse melanoma cell line measured as CD45 and MHC-II expression 9 days after transduction with PIB-GFP encoding virus. Lentiviruses encoding GFP alone were included as controls. Bar graph indicates mean ± SEM (n=4). (D) The flow cytometry quantification of the efficiency of cDC1 reprogramming in 2 human cancer cell lines (IGR-39 and T98G) and 1 primary melanoma sample (2778) was measured as expression of CD45 and HLA-DR 9 days after transduction with PIB-GFP encoding virus. Lentiviruses encoding GFP alone were included as controls. Bar graph indicates mean ± SEM (n=4).

Detailed Description

Definition of the definition

"biologically active variant" herein refers to a biologically active variant of a Transcription Factor (TF) that retains at least some activity of the parent TF. For example, the biologically active variants of basic leucine zipper ATF-like transcription factor 3 (bat f 3), interferon regulatory factor 8 (IRF 8) and pu.1 may serve as the separate TFs, and induce or inhibit expression of the same gene in cells as the bat f3, IRF8 and pu.1, respectively, although the induction efficiency may be different, e.g. a decrease or increase in the efficiency of inducing or inhibiting the gene compared to the parent TF.

In terms of polynucleotides or polypeptides, "identity and homology" is defined herein as the percentage of nucleic acids or amino acids in a candidate sequence that are identical or homologous to the residues of the corresponding natural nucleic acid or amino acid, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the greatest percent identity/similarity/homology, and considering any conservative substitutions as part of the sequence identity according to the NCIUB rules (hftp:// www.chem.qmul.ac.uk/iubmb/misc/naseq. Html; NC-IUB, eur J Biochem (1985)). Neither 5 'or 3' extension or insertion (for nucleic acids) nor N 'or C' extension or insertion (for polypeptides) results in a decrease in identity, similarity or homology. Methods and computer programs for alignment are well known in the art. In general, a given homology between two sequences means that the identity between these sequences is at least equal to homology; for example, if two sequences are 70% homologous to each other, they have not less than 70% identity to each other, but may share 80% identity.

"mesenchymal stem cells" or "mesenchymal stromal cells" (both referred to as "MSCs") are used interchangeably herein and are referred to herein as pluripotent stromal cells, which can differentiate into various cell types, including but not limited to: osteoblasts (bone cells), chondrocytes (chondrocytes), and adipocytes (fat cells).

"murine" herein refers to any and all members of the murine family (Muridae), including rats and mice.

"reprogramming" refers herein to the process of transforming differentiated cells from one cell type to another. In particular, reprogramming in this context refers to the conversion or transdifferentiation of any type of cell into a classical dendritic cell type 1 or an antigen presenting cell.

"Treating" or "Treatment" herein refers to any administration or application of a therapeutic agent to a disease, disorder, and condition disclosed in a subject, and includes inhibiting the progression of the disease, slowing the disease or its progression, preventing its progression, partially or fully alleviating the disease, or partially or fully alleviating one or more symptoms of the disease.

As used herein, the term "adenovirus" is used to refer to any and all viruses that can be classified as adenoviruses, including any adenovirus that infects humans or non-human animals, including all groups, subgroups, and serotypes, unless otherwise required. Thus, as used herein, "adenovirus" refers to the virus itself or derivatives thereof, and encompasses all serotypes and subtypes, naturally occurring (wild-type), forms to be used as modifications of an adenovirus vector, such as gene delivery vehicles, modified in a manner known in the art, such as capsid mutations, and recombinant forms that are replication competent, conditionally replication competent, or replication defective, unless otherwise indicated.

As used herein, the term "adeno-associated virus" may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term is used to refer to any and all viruses that can be classified as adeno-associated viruses, including any adeno-associated virus that infects humans or non-human animals, and encompasses all subtypes, serotypes, and pseudotypes, as well as naturally occurring, modified, and recombinant forms, such as modifications, e.g., gene delivery vehicles, to be used as adeno-associated viral vectors, unless otherwise required.

As used herein, the abbreviation "Ad" in the context of viral vectors refers to adenovirus, and is typically followed by a number indicating the serotype of adenovirus. For example, "Ad5" refers to adenovirus serotype 5.

Any Ad suitable for this purpose may be used herein, such as, but not limited to, ad of any serotype from any of the A, B, C, D, E, F, G Ad subgroups, such as Ad2, ad5 or Ad35, avian Ad, bovine Ad, canine Ad, caprine Ad, equine Ad, primate Ad, non-primate Ad and ovine Ad. "primate Ad" refers to primate-infected Ad, "non-primate Ad" refers to non-primate-infected Ad, and "Niu Zu Ad" refers to bovine-infected Ad.

The genomic sequences of the various serotypes of Ad and the sequences of the natural Terminal Repeats (TR) and capsid subunits are known in the art.

As used herein, the abbreviation "AAV" in the context of viral vectors refers to adeno-associated virus, and is typically followed by a number indicating the serotype of the adeno-associated virus. For example, "AAV2" refers to adeno-associated virus serotype 2.

Any AAV suitable for this purpose may be used herein, such as, but not limited to, AAV serotype 1 (AAV 1), AAV serotype 2 (AAV 2), AAV serotype 3A (AAV 3A), AAV serotype 3B (AAV 3B), AAV serotype 4 (AAV 4), AAV serotype 5 (AAV 5), AAV serotype 6 (AAV 6), AAV serotype 7 (AAV 7), AAV serotype 8 (AAV 8), AAV serotype 9 (AAV 9), AAV serotype 10 (AAV 10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "primate AAV" refers to a primate-infected AAV, "non-primate AAV" refers to a non-primate-infected AAV, and "bovine AAV refers to a bovine-infected AAV.

The genomic sequences of the various serotypes of AAV, and the sequences of the natural Terminal Repeats (TR), rep proteins, and capsid subunits are known in the art.

As used herein, a "hybrid" Ad or AAV vector refers to an Ad or AAV-based vector that is engineered in such a way that the Ad or AAV vector contains proteins derived from two or more different Ad or AAV serotypes.

As used herein, "AAV2-qYF" or "AAV 2-quadryf" refers to a quadruple tyrosine to phenylalanine mutant of AAV 2.

As used herein, "AAV-DJ" refers to a hybrid capsid that is derived from DNA family shuffling of 8 wild-type serotypes of AAV (including AAV2, 4, 5, 8, 9, avian, bovine, and capriaceae AAV). AAV-DJ is a synthetic serotype, type 2/type 8/type 9 chimera, differing from its closest natural relatives (AAV-2) by 60 capsid amino acids.

Composition and method for producing the same

The present invention relates to compositions and their use in methods for reprogramming or inducing cells into dendritic cells or antigen presenting cells. The inventors have surprisingly found that reprogramming can be significantly improved by expressing TF bat f3, IRF8 and pu.1 under specific promoters.

a) BATF3 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 10 (BATF 3);

b) IRF8 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 11 (IRF 8); and

c) Pu.1 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID No. 12 (pu.1);

wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, wherein the promoter region comprises a Spleen Focus Forming Virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, a deleted negative control region, a substituted dl587rev primer binding site) promoter, a CAG (CMV early enhancer/chicken beta actin) promoter, a Cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1alpha (EF-1 a) promoter, an EF-1a short (EF 1S) promoter, an EF-1a with an intron (EF 1 i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter exhibiting substantially the same effect.

TF may be as defined in the section "transcription factor" herein.

The promoter region may be as defined in the section "promoter" herein.

TF may be expressed from one or more vectors or constructs that are polycistronic constructs, bicistronic (or bicistronic) constructs, and/or monocistronic constructs. When an mRNA molecule contains genetic information that translates only a single protein chain, it is said to be monocistronic. Polycistronic mrnas, on the other hand, carry several Open Reading Frames (ORFs), each of which is translated into a polypeptide. Bicistronic mRNA encodes only two proteins. Polycistronic and bicistronic mRNAs are expressed from a single promoter or promoter region.

In one embodiment, the composition further comprises one or more constructs or vectors that, upon expression, encode one or more transcription factors selected from the group consisting of:

a) IRF7 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 21 (IRF 7);

b) BATF or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 19 (BATF);

c) SPIB or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 23 (SPIB);

d) SPIC or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 25 (SPIC);

e) CEBP alpha or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 13 (CEBP alpha);

In one embodiment, the composition comprises:

a) A construct or vector encoding the transcription factors BATF3, IRF8 and PU.1 after expression;

b) A construct or vector encoding the transcription factors BATF3, IRF8 and SPIB after expression;

c) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factor PU.1 after expression;

d) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factor SPIB after expression;

e) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factor IRF8 and PU.1 after expression;

f) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factors IRF8 and SPIB after expression;

g) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factor BATF3 and pu.1 after expression;

h) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factors BATF3 and SPIB after expression;

i) A first construct or vector encoding the transcription factor BATF3 after expression, a second construct or vector encoding the transcription factor IRF8 after expression, and a third construct or vector encoding the transcription factor PU.1 after expression;

and/or

j) A first construct or vector encoding the transcription factor BATF3 after expression, a second construct or vector encoding the transcription factor IRF8 after expression, and a third construct or vector encoding the transcription factor SPIB after expression.

In one embodiment, the one or more constructs or vectors further encode the transcription factor CCAAT/enhancer binding protein α (cbppα) or a biologically active variant thereof after expression. cEBP alpha may be as defined in the segment "transcription factor" herein.

In another embodiment, the one or more constructs or vectors further encode the transcription factor interferon regulatory factor 7 (IRF 7) or a biologically active variant thereof after expression. IRF7 may be as defined in the section "transcription factor" herein.

In another embodiment, the one or more constructs or vectors further encode the transcription factor basic leucine zipper ATF-like (bat) or biologically active variant thereof after expression. BATF may be as defined herein in the section "transcription factors".

In another embodiment, the one or more constructs or vectors further encode the transcription factor Spi-C (SPIC) or a biologically active variant thereof after expression. SPIC may be as defined in the section "transcription factor" herein.

In one embodiment, the composition comprises:

a) A construct or vector encoding the transcription factors BATF3, IRF8, PU.1 and IRF7 after expression;

b) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factors PU.1 and IRF7 after expression;

c) A first construct or vector encoding the transcription factors BATF3 and PU.1 after expression, and a second construct or vector encoding the transcription factors IRF8 and IRF7 after expression;

d) A first construct or vector encoding the transcription factors pu.1 and IRF8 after expression, and a second construct or vector encoding the transcription factors bat f3 and IRF7 after expression;

e) A first construct or vector encoding the transcription factors BATF3, IRF8 and PU.1 after expression, and a second construct or vector encoding the transcription factor IRF7 after expression;

f) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factors IRF8, PU.1 and IRF7 after expression;

g) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factors BATF3, pu.1 and IRF7 after expression;

h) A first construct or vector encoding the transcription factor pu.1 after expression, and a second construct or vector encoding the transcription factors BATF3, IRF8 and IRF7 after expression;

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor pu.1 after expression, and a fourth construct or vector encoding the transcription factor IRF7 after expression;

In one embodiment, the composition comprises:

a) A construct or vector encoding the transcription factors BATF3, IRF8, PU.1 and BATF after expression;

b) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factors PU.1 and BATF after expression;

c) A first construct or vector encoding the transcription factors BATF3 and PU.1 after expression, and a second construct or vector encoding the transcription factors IRF8 and BATF after expression;

d) A first construct or vector encoding the transcription factors pu.1 and IRF8 after expression, and a second construct or vector encoding the transcription factors bat 3 and bat after expression;

e) A first construct or vector encoding the transcription factors BATF3, IRF8 and PU.1 after expression, and a second construct or vector encoding the transcription factor BATF after expression;

f) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factors IRF8, PU.1 and BATF after expression;

g) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factors BATF3, pu.1 and BATF after expression;

h) A first construct or vector encoding the transcription factor pu.1 after expression, and a second construct or vector encoding the transcription factors BATF3, IRF8 and BATF after expression;

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor pu.1 after expression, and a fourth construct or vector encoding the transcription factor bat after expression;

in one embodiment, the composition comprises:

a) A construct or vector encoding the transcription factors BATF3, IRF8, SPIB and IRF7 upon expression;

b) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factors SPIB and IRF7 after expression;

c) A first construct or vector encoding the transcription factors BATF3 and SPIB after expression, and a second construct or vector encoding the transcription factors IRF8 and IRF7 after expression;

d) A first construct or vector encoding the transcription factors SPIB and IRF8 after expression, and a second construct or vector encoding the transcription factors BATF3 and IRF7 after expression;

e) A first construct or vector encoding the transcription factors BATF3, IRF8 and SPIB after expression, and a second construct or vector encoding the transcription factor IRF7 after expression;

f) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factors IRF8, SPIB and IRF7 after expression;

g) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factors BATF3, SPIB and IRF7 after expression;

h) A first construct or vector encoding the transcription factor SPIB after expression, and a second construct or vector encoding the transcription factors BATF3, IRF8 and IRF7 after expression;

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor SPIB after expression, and a fourth construct or vector encoding the transcription factor IRF7 after expression;

in one embodiment, the composition comprises:

a) A construct or vector encoding the transcription factors BATF3, IRF8, SPIB and BATF after expression;

b) A first construct or vector encoding the transcription factors BATF3 and IRF8 after expression, and a second construct or vector encoding the transcription factors SPIB and BATF after expression;

c) A first construct or vector encoding the transcription factors BATF3 and SPIB after expression, and a second construct or vector encoding the transcription factors IRF8 and BATF after expression;

d) A first construct or vector encoding the transcription factors SPIB and IRF8 after expression, and a second construct or vector encoding the transcription factors BATF3 and BATF after expression;

e) A first construct or vector encoding the transcription factors BATF3, IRF8 and SPIB after expression, and a second construct or vector encoding the transcription factor BATF after expression;

f) A first construct or vector encoding the transcription factor BATF3 after expression and a second construct or vector encoding the transcription factors IRF8, SPIB and BATF after expression;

g) A first construct or vector encoding transcription factor IRF8 after expression, and a second construct or vector encoding transcription factors bat 3, SPIB and bat after expression;

h) A first construct or vector encoding the transcription factor SPIB after expression, and a second construct or vector encoding the transcription factors BATF3, IRF8 and BATF after expression;

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor SPIB after expression, and a fourth construct or vector encoding the transcription factor bat after expression;

The one or more constructs and vectors disclosed herein may be any type of construct and vector, such as a plasmid.

In one embodiment, the one or more constructs or vectors are one or more viral vectors. In other embodiments, the viral vector is selected from the group consisting of: lentiviral vectors, retroviral vectors, adenoviral vectors, herpesviral vectors, poxviral vectors, adeno-associated viral vectors (adeno-associated virus vectors), paramyxoviridae vectors, rhabdoviral (rabdoviral) vectors, alphaviral vectors, flaviviral vectors and adeno-associated viral vectors (adeno-associated viral vectors). In another embodiment, the viral vector is a lentiviral vector.

Adenovirus (Ad) and adeno-associated virus (AAV) vectors may be vectors derived from any Ad or AAV serotype known in the art, and may allow for gene expression in specific cells (e.g., neural cells, myocytes, and hepatocytes), tissues, and organs, for example, by employing specificity for target cells to be infected for each serotype. Ad or AAV may be wild-type or have one or more wild-type genes deleted in whole or in part. Ad or AAV can be further engineered by any method known in the art, such as pseudotyping, resulting in hybrid (or chimeric) viral particles, such as hybrid viral capsids.

AAV or Ad viral particles may also, for example, have been mutated at one or more amino acid residues, for example at one or more tyrosine residues.

In one embodiment, the adenovirus vector is selected from the group consisting of: wild-type Ad vectors, hybrid Ad vectors, and mutant Ad vectors.

In another embodiment, the adeno-associated viral vector is selected from the group consisting of: wild-type AAV vectors, hybrid AAV vectors, and mutant AAV vectors.

In a further embodiment, the wild-type Ad vector is Ad5 and the hybrid Ad vector is Ad5/F35.

In yet another embodiment, the hybrid AAV vector is AAV-DJ and the mutant AAV vector is AAV2-QuadYF.

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

In one embodiment, the lentiviral vector comprises a chimeric 5' Long Terminal Repeat (LTR) fused to a heterologous enhancer/promoter, such as the Rous Sarcoma Virus (RSV) or CMV promoter.

In one embodiment, the lentiviral vector comprises a deletion within the U3 region of the 3' LTR, whereby the vector is replication defective and self-inactivating upon integration.

In one embodiment, the one or more constructs or vectors are one or more plasmids.

In one embodiment, the backbone of one or more constructs or vectors is selected from the following: FUW, pRRL-cPPT, pRLL, pCCL, pCLL, pHAGE2, pWPXL, pLKO, pHIV, pLL, pCDH and pLenti.

pRRL, pRLL, pCCL and pCLL are lentiviral transfer vectors containing a chimeric Rous Sarcoma Virus (RSV) -HIV or CMV-HIV 5'ltr, and wherein the simian virus 40 polyadenylation and (without enhancer) replication origin sequences have been included in the vector backbone downstream of the HIV 3' ltr, replacing most of the human sequences remaining from the HIV integration site. In pRRL, enhancers and promoters from the U3 region of RSV (nucleotides-233 to-1 relative to the transcription initiation site; genBank accession J02342) are linked to the R region of HIV-1 LTR. In pRLL, the RSV enhancer (nucleotide-233 to-50) sequence is linked to the promoter region of HIV-1 (from position-78 relative to the transcription initiation site). In pCCL, the enhancer and promoter of CMV (nucleotides-673 to-1 relative to the transcription initiation site; genBank accession number K03104) are linked to the R region of HIV-1. In pCLL, the CMV enhancer (nucleotides-673 to-220) is linked to the promoter region of HIV-1 (position-78).

One or more constructs or vectors disclosed herein may comprise any type of element in addition to the polynucleotide encoding the TF and the promoter region driving expression of the TF. For example, one or more constructs or vectors may comprise regulatory, selectable, and/or structural elements and/or sequences.

In one embodiment, one or more constructs or vectors comprise a self-cleaving peptide operably linked to at least two of the at least three coding regions, thereby forming a single open reading frame. The self-cleaving peptide may be any type of self-cleaving peptide. In one embodiment, the self-cleaving peptide is a 2A peptide. In one embodiment, the 2A peptide is selected from the group consisting of equine rhinitis virus (E2A), foot and mouth disease virus (F2A), porcine teschovirus-1 (P2A), and echinococcosis minor virus (Thosea asigna virus) (T2A) peptides.

In one embodiment, one or more constructs or vectors comprise a post-transcriptional regulatory element (PRE) sequence. In a preferred embodiment, the PRE sequence is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

In one embodiment, one or more constructs or vectors comprise 5 'and 3' terminal repeats. In a preferred embodiment, at least one of the 5' and 3' terminal repeats is a lentiviral long terminal repeat or a self-inactivating (SIN) design of U3 with partial deletion of the 3' long terminal repeat.

In one embodiment, one or more constructs or vectors comprise a central polypurine region (cPPT).

In one embodiment, one or more constructs or vectors comprise a nucleocapsid protein packaging target site. In a preferred embodiment, the protein packaging target site comprises HIV-1psi sequence.

In one embodiment, one or more constructs or vectors comprise a REV protein responsive element (RRE).

The compositions disclosed herein may further comprise additional components, such as components that improve the efficiency of reprogramming cells according to the methods disclosed herein. The additional component may be a macromolecule, such as a protein, for example a cytokine.

Cytokines are small proteins (peptides) that are important in cell signaling. Cytokines cannot cross the lipid bilayer of cells to enter the cytoplasm, but rather act by modulating surface receptors of intracellular signaling pathways. They have been shown to be involved in autocrine, paracrine and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines and tumor necrosis factors.

In one embodiment, the composition further comprises one or more pro-inflammatory cytokines. In one embodiment, the composition further comprises one or more hematopoietic cytokines. In one embodiment, the composition further comprises one or more cytokines selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM). In a preferred embodiment, the one or more cytokines are selected from the group consisting of: ifnβ, ifnγ, and tnfα.

Additional components may also include, for example, small molecules. Small molecules are low molecular weight molecules that differ from macromolecules such as proteins, including lipids, monosaccharides, second messengers, other natural products and metabolites, as well as drugs and other xenobiotics. The small molecules have high level of cell permeability, are inexpensive to produce, and are easy to synthesize and standardize.

In one embodiment, the composition further comprises one or more small molecules.

The small molecule may be, for example, a small molecule that acts as an epigenetic regulator. The small molecule may also be, for example, a small molecule targeting epigenetic regulation of gene expression, such as a histone deacetylase inhibitor (HDACi), a DNA methyltransferase inhibitor, a Histone Methyltransferase (HMT) inhibitor, or a histone demethylase inhibitor.

Thus, in one embodiment, the composition further comprises one or more histone deacetylase inhibitors.

In one embodiment, the composition further comprises valproic acid, suberoylanilide hydroxamic acid (SAHA), trichostatin a (TSA), sodium butyrate.

Thus, in one embodiment, the composition further comprises one or more DNA methyltransferase inhibitors, such as 5 '-azacytidine (5' -azaC) or RG108.

In one embodiment, the composition further comprises one or more inhibitors of Histone Methyltransferase (HMT), such as BIX-0194, an inhibitor that inhibits G9 a-mediated H3K9me2 methylation.

In one embodiment, the composition further comprises one or more histone demethylase inhibitors, such as a parnate (LSD 1 inhibitor).

Such additional components may also include, for example, nucleic acids encoding additional TFs or genes associated with successful reprogramming.

Thus, in one embodiment, the composition further comprises one or more additional TFs and/or genes encoding one or more additional TFs, wherein the one or more TFs are associated with successful reprogramming. In one embodiment, the one or more TFs associated with successful reprogramming are selected from the TFs associated with successful reprogramming listed in table 1.

In one embodiment, the composition further comprises one or more additional TFs and/or genes encoding additional TFs associated with successful reprogramming, wherein the one or more TFs associated with successful reprogramming are selected from the list in table 1.

In one embodiment, the composition comprises cells expressing one or more additional surface markers, wherein the one or more additional surface markers are selected from the group consisting of the surface markers listed in table 1.

Table 1. A list of genes encoding transcriptional regulators and surface markers, associated with successful cDC1 reprogramming.

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As set forth above, the composition may further comprise a gene encoding a protein associated with successful reprogramming. In one embodiment, the gene encodes a protein other than TF.

Gene expression can be tested using methods known in the art, such as transcriptomics or other methods described herein.

In one embodiment, the composition is a pharmaceutical composition.

Cells

Provided herein are cells comprising one or more constructs or vectors that, upon expression, encode the following transcription factors:

b) IRF8 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 11 (IRF 8);

d) IRF7 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 21 (IRF 7);

e) BATF or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 19 (BATF);

f) SPIB or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 23 (SPIB);

g) SPIC or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 25 (SPIC); and/or

h) CEBP alpha or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 13 (CEBP alpha); or any combination thereof;

In one embodiment, the cell comprises:

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; and a third construct or vector encoding the transcription factor pu.1 after expression; and/or

j) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; and a third construct or vector encoding the transcription factor SPIB after expression;

TF may be as defined in the section "transcription factor" herein.

The promoter region may be as defined in the section "promoter" herein.

One or more constructs or vectors may be as defined in the section "composition" herein.

The cells may be any type of cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In another embodiment, the cell is a murine cell.

In one embodiment, the cell is selected from the group consisting of: stem cells, differentiated cells, and cancer cells.

In one embodiment, the stem cells are selected from the following: pluripotent stem cells, endodermal derived cells, mesodermal derived cells, ectodermal derived cells and multipotent stem cells, such as mesenchymal stem cells and hematopoietic stem cells.

In one embodiment, the differentiated cell is a cancer cell, such as a solid tumor cell, hematopoietic tumor cell, melanoma cell, bladder cancer cell, breast cancer cell, lung cancer cell, pleural cancer cell, colon cancer cell, rectal cancer cell, colorectal cancer cell, prostate cancer cell, liver cancer cell, pancreatic cancer cell, cholangiocarcinoma cell, gastric cancer cell, testicular cancer cell, brain cancer cell, ovarian cancer cell, lymphoma cancer cell, sarcoma cancer cell, skin cancer cell, brain cancer cell, bone cancer cell, oral cancer cell, head and neck cancer cell, or soft tissue cancer cell, such as glioblastoma cell, rectal cancer cell, or mesothelioma cell.

In one embodiment, the differentiated cell is any somatic cell.

In one embodiment, the somatic cell is selected from the group consisting of: fibroblasts and hematopoietic cells, such as monocytes.

The cells disclosed herein may be further engineered or modified in a manner that improves reprogramming efficiency according to the methods disclosed in the section "methods" herein. Such modifications may for example include overexpression or silencing of genes encoding TF associated with successful reprogramming, respectively. It may also include overexpression or silencing of other genes associated with reprogramming efficiency, such as genes that are differentially expressed after expression of TF as disclosed in the section "transcription factors" herein. Methods for over-expressing or silencing genes are well known in the art.

In one embodiment, the cells are engineered to overexpress one or more genes encoding TFs associated with successful reprogramming, such as one or more genes encoding TFs associated with successful reprogramming listed in table 1. In one embodiment, the cell is engineered to overexpress one or more genes encoding TF associated with successful reprogramming, wherein the one or more genes encoding TF associated with successful reprogramming are selected from the list in table 1.

Method

Provided herein are methods of reprogramming or inducing cells into dendritic cells or antigen presenting cells, the method comprising the steps of:

a) Transducing a cell with one or more constructs or vectors which, upon expression, encode the following transcription factors:

i) BATF3 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 10 (BATF 3);

ii) IRF8 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID NO. 11 (IRF 8);

iii) Pu.1 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID No. 12 (pu.1);

iv) IRF7 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID NO. 21 (IRF 7);

v) BATF or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 19 (BATF);

vi) SPIB or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO. 23 (SPIB);

vii) SPIC or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 25 (SPIC); and/or

viii) cebpα or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO:13 (cebpα):

wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, wherein the promoter region comprises a Spleen Focus Forming Virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, a deleted negative control region, a substituted dl587rev primer binding site) promoter, a CAG (CMV early enhancer/chicken beta actin) promoter, a Cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1alpha (EF-1 a) promoter, an EF-1a short (EF 1S) promoter, an EF-1a with an intron (EF 1 i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter exhibiting substantially the same effect;

b) Expressing the transcription factor;

thereby obtaining reprogrammed or induced cells.

In one embodiment, the cells to be reprogrammed or induced are not dendritic cells or antigen presenting cells.

In one embodiment, the reprogramming or induction is in vivo, e.g., in an animal or human.

In another embodiment, the reprogramming or induction is in vitro.

In another embodiment, reprogramming or induction is ex vivo.

In one embodiment, the method further comprises the step of culturing the transduced cells in a cell culture medium. The step of culturing the transduced cells in a cell culture medium may be performed before or after step b) of the method, i.e. before or after expression of the transcription factor. In one embodiment, the step of culturing the transduced cells in a cell culture medium is performed prior to expression of the transcription factor, i.e., after step a) and before step b) in the methods presented herein. In one embodiment, the transduced cells are cultured for a period of at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

For example, it may be beneficial for the efficiency of reprogramming that the cell culture medium contains one or more additional components.

In one embodiment, the method further comprises culturing the transduced cells in a medium comprising one or more cytokines. In one embodiment, the one or more cytokines are pro-inflammatory cytokines. In one embodiment, the one or more cytokines are hematopoietic cytokines. In one embodiment, the one or more cytokines are selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM). In a preferred embodiment, the one or more cytokines are selected from the group consisting of: ifnβ, ifnγ, and tnfα.

The method may further comprise culturing the transduced cells in a cell culture medium comprising the small molecule. The small molecule may be, for example, a small molecule that acts as an epigenetic regulator. The small molecule may also be, for example, a small molecule targeting epigenetic regulation of gene expression, such as an epigenetic modifier, for example, a histone deacetylase inhibitor (HDACi), a DNA methyltransferase inhibitor, a Histone Methyltransferase (HMT) inhibitor, or a histone demethylase inhibitor, or any small molecule falling within these categories, such as small molecules falling within the categories disclosed herein.

In some embodiments, the method further comprises culturing the transduced cells in a cell culture medium comprising one or more histone deacetylase inhibitors.

In one embodiment, the one or more histone deacetylase inhibitors is valproic acid.

In one embodiment, the cells are transduced with the following:

TF may be as defined in the section "transcription factor" herein.

The promoter region may be as defined in the section "promoter" herein.

Cells may be as defined in the section "cells" herein.

The method may comprise the further step of improving the efficiency of reprogramming.

In one embodiment, the method further comprises overexpressing in the transduced cells one or more genes encoding TF associated with successful reprogramming, e.g., one or more genes encoding TF associated with successful reprogramming listed in table 1.

In one embodiment, the method further comprises overexpressing one or more genes encoding TF associated with successful reprogramming in the transduced cells, wherein the one or more genes encoding TF associated with successful reprogramming are selected from the list in table 1.

In another embodiment, the method further comprises overexpressing one or more genes encoding proteins associated with successful reprogramming in the transduced cells. In one embodiment, the gene encodes a protein other than TF.

Overexpression and silencing of the gene can be accomplished using methods known in the art, for example, by expressing the gene from a vector or by deleting a portion or the entire gene from the cell, respectively.

In one embodiment, the resulting reprogrammed cell is a type 1 classical dendritic cell (DC 1). cDC1 is a specific subset of DCs that express, for example, the human leukocyte antigen DR isoform (HLA-DR) and hematopoietic marker cluster of differentiation 45 (CD 45). cDC1 further has a typical RNA expression profile and expresses the surface markers cluster of differentiation 141 (CD 141), C lectin domain family 9 member a (CLEC 9A), X-C motif chemokine receptor 1 (XCR 1) and cluster of differentiation 226 (CD 226).

Thus, in one embodiment, the resulting reprogrammed cell is enriched in one or more surface markers selected from the list in table 1.

In one embodiment, the resulting reprogrammed cell is CD45 positive. In one embodiment, the resulting reprogrammed cell is HLA-DR positive. In one embodiment, the resulting reprogrammed cell is CD141 positive. In one embodiment, the resulting reprogrammed cell is CLEC9A positive. In one embodiment, the resulting reprogrammed or induced cell is CD226 positive. In one embodiment, the resulting reprogrammed or induced cell is XCR1 positive. In one embodiment, the resulting reprogrammed or induced cell is positive for CD45, HLA-DR, CD141, CLEC9A, XCR1 and/or CD 226.

Methods for determining whether one or more cells are cDC1 cells are well known in the art. For example, whether the cell expresses CD45, HLA-DR, CD226, CD141, XCR1 and/or CLEC9A can be determined by: cells were incubated with fluorophore conjugated antibodies specific for CD45, HLA-DR, CD226, CD141, XCR1 and/or CLEC9A, and then screened using flow cytometry. For example, it may be determined whether the cell expresses one or more surface markers selected from the list in table 1 and identified as successfully reprogramming the cell to cDC1 by: cells are incubated with fluorophore conjugated antibodies specific for the surface markers and subsequently screened using flow cytometry.

In addition, single cell RNA seq can be used to determine the RNA profile of a cell and to classify a cell as dcs 1 if the RNA profile is the same or similar to that of a native dcs 1 cell. In addition, the cells can be characterized by their functional properties, such as the ability to respond to TLR stimulation and up-regulate surface expression of CD40, CD80 and other co-stimulatory molecules, the ability to secrete pro-inflammatory cytokines and chemokines, and the ability to activate antigen-specific T cells.

Thus, provided herein are reprogrammed or induced cells obtained by the methods presented herein. In one embodiment, the cell is a dendritic cell or an antigen presenting cell.

Transcription factor

Transcription Factors (TF) are proteins that control the rate of transcription of genetic information from DNA to mRNA by binding to specific DNA sequences. The function of TF is to regulate (i.e., turn on and off) the expression of genes. The TF group functions in a coordinated manner, directing cell division, cell growth and cell death throughout life; cell migration and organization during embryonic development; and intermittently responds to signals from outside the cell, such as hormones. Up to 1600 TFs are present in the human genome. Transcription factors are members of the proteome and regulatory group (regulatory).

TF acts alone or in combination with other proteins in the complex by promoting (as activators) or blocking (as repressors) the recruitment of RNA polymerase to a particular gene. The decisive feature of transcription factors is that they contain at least one DNA Binding Domain (DBD) attached to a specific DNA sequence adjacent to the gene they regulate.

Presented herein are TF that can be used to reprogram cells into dendritic cells or antigen presenting cells. Such TFs include BATF3, IRF8, pu.1, IRF7, BATF, SPIB, SPIC, and CEBPA.

BATF3 is a nuclear basic leucine zipper, which belongs to the AP-1/ATF superfamily of TF. It controls CD8 in the immune system ⁺ Differentiation of thymic classical dendritic cells. It functions via the formation of heterodimers with JUN family proteins that recognize and bind specific DNA sequences to regulate expression of target genes.

IRF8 is a TF belonging to the family of Interferon Regulatory Factors (IRFs). It plays a role in the regulation of lineage commitment and myeloid cell maturation. IRF8 and other TFs in the IRF family bind to IFN-stimulated response elements and regulate expression of genes stimulated by type I IFNs.

PU.1 is TF belonging to the erythropoiesis transformation-specific (ETS) domain family. It is a transcriptional activator that binds to PU-box, a purine-rich DNA sequence that can act as a lymphoid specific enhancer. Pu.1 may be specifically involved in differentiation or activation of myeloid cells such as macrophages and dendritic cells as well as B cells.

IRF7 is TF belonging to the family of Interferon Regulatory Factors (IRFs). IRF7 has been shown to play a role in the transcriptional activation of viral-induced cellular genes, including type I interferon genes. IRF7 is constitutively expressed in lymphoid tissues and is inducible in many other tissues throughout the body.

BATF is a nuclear basic leucine zipper, which belongs to the AP-1/ATF superfamily of TF. BATF can interact with partner transcription factors, including IRF8 and IRF4, via leucine zipper domains to mediate synergistic gene activation. Compensation in the BATF factor has been previously demonstrated in the context of cDC1 development.

SPIB is TF belonging to the Erythroblast Transformation Specific (ETS) domain family. Like pu.1, SPIB is a sequence-specific transcriptional activator that binds to PU-box, a purine-rich DNA sequence that can act as a lymphoid-specific enhancer. Promote the development of plasmacytoid dendritic cells (pDC) and dcs precursors.

SPIC is TF belonging to the Erythroblast Transformation Specific (ETS) domain family. Like PU.1 and SPIB, SPIC is a sequence-specific transcriptional activator that binds to PU-box, a purine-rich DNA sequence. SPIC controls the development of red marrow macrophages required for red blood cell recirculation and iron homeostasis.

CEBP alpha (CCAAT enhancer binding protein alpha) is TF which contains a basic leucine zipper (bZIP) domain and recognizes a CCAAT motif in the promoter of a target gene. CEBP alpha coordinates the proliferation retardation and differentiation of myeloid progenitor cells, adipocytes, hepatocytes, and lung and placental cells.

Biologically active variants of BATF3, IRF8, PU.1, IRF7, BATF, SPIC and SPIB are also disclosed. Biologically active variants are variants of the TF that retain at least some of the activity of the parent TF. For example, a biologically active variant of SPIB, SPIC, BATF, BATF, IRF8 or pu.1 is capable of inducing and/or inhibiting the expression of the same gene as the parent BATF3, IRF8 or pu.1, respectively. SPIB, SPIC, BATF, BATF3, IRF8 and pu.1 three biologically active variants are capable of reprogramming or inducing cells into dendritic cells or antigen presenting cells according to the methods disclosed herein. However, biologically active variants of the respective TF may be more or less potent than the respective parent TF. For example, the efficiency of inducing and/or inhibiting gene expression and/or the efficiency of reprogramming or inducing cells into dendritic cells may be increased or decreased compared to the respective parent TF.

In one embodiment, the biologically active variant of BATF3 has at least 60% identity to SEQ ID NO:10, e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 96%, e.g., at least 97%.

In one embodiment, the biologically active variant of IRF8 has at least 60% identity to SEQ ID No. 11, e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%.

In one embodiment, the biologically active variant of pu.1 has at least 60% identity with SEQ ID No. 12, e.g. at least 61%, e.g. at least 62%, e.g. at least 63%, e.g. at least 64%, e.g. at least 65%, e.g. at least 66%, e.g. at least 67%, e.g. at least 68%, e.g. at least 69%, e.g. at least 70%, e.g. at least 71%, e.g. at least 72%, e.g. at least 73%, e.g. at least 74%, e.g. at least 75%, e.g. at least 76%, e.g. at least 77%, e.g. at least 78%, e.g. at least 79%, e.g. at least 80%, e.g. at least 81%, e.g. at least 82%, e.g. at least 83%, e.g. at least 84%, e.g. at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%.

In one embodiment, the biologically active variant of IRF7 has at least 60% identity to SEQ ID No. 21 (IRF 7), e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%.

In one embodiment, a biologically active variant of a BATF has at least 60% identity to SEQ ID NO:19 (BATF), e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%.

In one embodiment, the biologically active variant of the SPIB has at least 60% identity to SEQ ID NO:23 (SPIB), e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.100%.

In one embodiment, a biologically active variant of the SPIC has at least 60% identity to SEQ ID No. 25 (SPIC), e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%.

In one embodiment, a biologically active variant of CEBPA has at least 60% identity to SEQ ID NO:13 (CEBP. Alpha.) such as at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

In one embodiment, BATF3 is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO:14, e.g., at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

In one embodiment, IRF8 is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO. 15, e.g., having at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

In one embodiment, PU.1 is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO. 16, e.g., having at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

In one embodiment, IRF7 is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO:20, e.g., having at least 61%, such as at least 62%, such as at least 63%, such as at least 64%, such as at least 65%, such as at least 66%, such as at least 67%, such as at least 68%, such as at least 69%, such as at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.

In one embodiment, the BATF is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO:18, e.g., at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at 100%..100.

In one embodiment, the SPIB is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO. 22, e.g., having at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.100%.

In one embodiment, the SPIC is encoded by a polynucleotide sequence having at least 60% sequence identity to SEQ ID NO:24, e.g., having at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.100%.

In one embodiment, CEBP alpha is encoded by a polynucleotide sequence having at least 60% sequence identity with SEQ ID NO:17, e.g., having at least 61%, e.g., at least 62%, e.g., at least 63%, e.g., at least 64%, e.g., at least 65%, e.g., at least 66%, e.g., at least 67%, e.g., at least 68%, e.g., at least 69%, e.g., at least 70%, e.g., at least 71%, e.g., at least 72%, e.g., at least 73%, e.g., at least 74%, e.g., at least 75%, e.g., at least 76%, e.g., at least 77%, e.g., at least 78%, e.g., at least 79%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at 100%.

Promoters

A promoter or promoter region is a DNA sequence to which a protein binds to initiate transcription of a single RNA from downstream DNA. Such RNA may be mRNA encoding a protein, or it may itself be functional, such as transfer RNA (tRNA) or ribosomal RNA (rRNA). The promoter is located near the transcription initiation site of the gene, upstream of the gene on the DNA.

In addition to the core promoter, the eukaryotic promoter region may further comprise other elements, such as a transcription initiation site (TSS); binding sites for RNA polymerase; TF binding site; as well as other regulatory and/or structural elements. Eukaryotic gene promoter regions are typically located upstream of the gene and may have regulatory elements several kilobases away from the TSS. Such regulatory elements may for example be enhancers.

The TF disclosed herein is controlled by a promoter region comprising a core promoter. The inventors have surprisingly shown that by expressing TF disclosed herein under certain promoters or promoter regions, cell reprogramming according to the methods disclosed herein can be significantly improved. Such promoter regions include those comprising SFFV promoter, MND promoter, CAG promoter, CMV promoter, EF-1 alpha promoter, EF1S promoter, EF1i promoter, PGK promoter, and other promoters exhibiting substantially the same effect.

A promoter or promoter region that exhibits substantially the same effect is defined herein as a promoter or promoter region that exhibits the same level of expression of the gene it controls as the promoter or promoter region disclosed herein. Thus, whether a promoter region exhibits substantially the same effects as the promoter regions disclosed herein can be measured by: the expression level of the gene controlled by the promoter region is measured and compared to the expression level of the same gene controlled by the promoter region disclosed herein, wherein the expression level of the tested promoter region and the expression level of the promoter region disclosed herein are tested under the same conditions. Methods for measuring expression levels are well known in the art and can be accomplished using routine experimentation. For example, the level of expression of a gene controlled by a certain promoter region can be measured by measuring the amount of messenger RNA (mRNA) produced by expression of the gene. The amount of mRNA can be measured, for example, using reverse transcription-polymerase chain reaction (RT-PCR) or transcriptomics. The expression level of a gene controlled by a certain promoter region can also be measured by measuring the amount of protein, i.e. the amount of gene product, generated by the expression of said gene using proteomics or western blotting. A promoter that exhibits substantially the same effect as a disclosed promoter region is defined herein as a promoter region that produces an expression level that is 50% or 50% lower than the expression level of the promoter region disclosed herein, e.g., 45% higher or lower than the expression level of the promoter disclosed herein, e.g., 40%, e.g., 35%, e.g., 30%, e.g., 25%, e.g., 20%, e.g., 15%, e.g., 10%, e.g., 5%.

The TF disclosed herein may be controlled by any of the disclosed promoter regions. In one embodiment, the same promoter region controls the expression of at least one TF, e.g., at least two TFs, e.g., three TFs. In one embodiment, the first promoter region controls expression of the first TF; the second promoter region controls expression of the second TF; and the third promoter region controls expression of the third TF. In one embodiment, the first promoter region controls the expression of the first TF and the second TF, and the second promoter region controls the expression of the third TF. TF may be as disclosed in the section "transcription factor" herein.

In one embodiment, the SFFV promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID No. 1, e.g., having at least 75%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100% identity to SEQ ID No. 1.

In one embodiment, the MND promoter comprises or consists of a polynucleotide sequence having at least 70% identity with SEQ ID NO. 2, e.g. having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID NO. 2.

In one embodiment, the CAG promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID No. 3, e.g. having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 3.

In one embodiment, the CMV promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID NO. 4, e.g., at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO. 4.

In one embodiment, the UbC promoter comprises or consists of a polynucleotide sequence having at least 70% identity with SEQ ID No. 5, e.g., having at least 75%, e.g., at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100% identity with SEQ ID No. 5.

In one embodiment, the EF-1. Alpha. Promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID NO. 6, e.g., having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO. 6.

In one embodiment, the EF1S promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID NO. 7, e.g., having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO. 7.

In one embodiment, the EF1i promoter comprises or consists of a polynucleotide sequence having at least 70% identity to SEQ ID NO. 8, e.g., having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO. 8.

In one embodiment, the PGK promoter comprises or consists of a polynucleotide sequence having at least 70% identity with SEQ ID No. 9, e.g. having at least 75%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID No. 9.

Therapeutic method

The compositions and methods disclosed herein may be used to treat and/or prevent diseases or disorders, such as tumors and cancers or infectious diseases.

In one embodiment, the compositions and methods disclosed herein are used to treat tumors and/or cancers or infectious diseases.

In one embodiment, the tumor and/or cancer is selected from the following: benign tumors, malignant tumors, early stage cancers, basal cell carcinomas, cervical atypical hyperplasia, sarcomas, germ cell tumors, retinoblastomas, glioblastomas, lymphomas, hodgkin's lymphomas, non-hodgkin's lymphomas, hematological cancers, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, uterine cancer, vaginal cancer, breast cancer, head and neck cancer, gastric cancer, oral cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, pleural cancer, bladder and urothelial cancer, hollow organ cancer, esophageal cancer, gastric cancer, cholangiocarcinoma, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureteral cancer, renal cancer, liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, skin cancer, melanoma or myeloma. In a preferred embodiment, the cancer is selected from melanoma, head and neck cancer, bladder cancer and urothelial cancer, pancreatic cancer and glioblastoma.

Accordingly, cells produced by the methods described herein can be used to prepare cells for treating or alleviating several cancers and tumors including, but not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, esophageal cancer, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, wilms 'tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal gland cancer, renal cell carcinoma, endometrial cancer, adrenocortical cancer, malignant pancreatic insulinoma, malignant carcinomatosis, choriocarcinoma, mycosis, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute lymphoblastic leukemia, chronic leukemia, lymphoblastic leukemia, hodgkin's sarcoma, lymphoblastic sarcoma, lymphosarcoma, lymphoblastic sarcoma, and the like.

Provided herein are compositions as presented herein for use in medicine; a cell; a pharmaceutical composition; and/or reprogrammed or induced cells.

Also provided herein are compositions as presented herein for use in treating cancer or infectious disease; a cell; a pharmaceutical composition; and/or reprogrammed or induced cells.

Further provided herein are methods of treating cancer or infectious disease comprising administering to an individual in need thereof a composition as presented herein; a cell; a pharmaceutical composition; and/or reprogrammed or induced cells.

Also provided herein are compositions as presented herein; a cell; a pharmaceutical composition; and/or the use of reprogrammed or induced cells for the manufacture of a medicament for the treatment of cancer or infectious disease.

Examples

Example 1 general methods and materials

Cell culture

Human embryonic kidney HEK293T cells, human Embryonic Fibroblasts (HEF) (3 rd-8 th generation) and Human Dermal Fibroblasts (HDF) (3 rd-8 th generation) were maintained in a growth medium, darby Modified Eagle's Medium (DMEM), supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS), 2mM L-glutamine and antibiotics (10U/ml penicillin 10. Mu.g/ml streptomycin) -DMEM was complete. Mouse Embryonic Fibroblasts (MEFs) (3-5 passages) were isolated from E13.5 embryos of Clec9a-tdTomato report mice as previously described (Rosa et al 2020) and cultured in DMEM complete in 0.1% gelatin coated dishes. Monocyte-derived dendritic cells (moDCs) were cultured in RPMI 1640-RPMI complete supplemented with 10% heat-inactivated FBS, 2mM L-glutamine and antibiotics. cDC1 isolated from peripheral blood was maintained in RPMI complete supplemented with 50 μm 2-mercaptoethanol, 1mM sodium pyruvate and antibiotics. Mesenchymal stromal cells (MSC, 3 rd to 5 th generation) were cultured in Minimal Essential Medium (MEM) supplemented with 10% pooled human platelet lysate, 2U/ml heparin (STEMCELL Technologies), 2mM L-glutamine and antibiotics. All tissue culture reagents were from Thermo Fisher Scientific unless otherwise indicated.

A mouse

C57BL/6J and OT-I mice were purchased from Janvier and Taconic, respectively. Clec9a ^Cre/Cre Rosa ^{tdTomato/tdTomato} (Clec 9 a-tdTomato) animals were Caetano Reis e Sousa, francis Crick Institute, london, uk (Rosa et al, 2018) friendly gifts and were re-derived by Janvier prior to introduction into Lund University animal houses. All animals were kept at controlled temperature (23±2 ℃) and subjected to a fixed 12 hour light/dark cycle with free access to food and water. Animal care and experimental procedures were performed following approval from the local commission in accordance with swedish guidelines and regulations.

Lentivirus production

HEK293T cells were co-transfected with a mixture of transfer plasmid, packaging and envelope construct encoding VSV-G with Polyethylenimine (PEI) as previously described (Rosa et al 2020). The virus supernatants were harvested after 36, 48 and 72 hours, filtered (0.45 μm, low protein binding), concentrated 40-fold with a Lenti-X concentrator and stored at-80 ℃.

Virus transduction and reprogramming

HEF, HDF and Clec9 a-tdtomo MEF at a density of 40,000 cells/well and MSCs at a density of 50,000 cells/well were seeded on 0.1% gelatin coated 6 well plates. On the next day, cells were incubated overnight with TetO-PIB and M2rtTA, SFFV-PIB-GFP or SFFV-GFP lentiviral particles at a ratio of 1:1 in medium supplemented with 8 μg/ml polybrene. Cells were transduced twice overnight for several consecutive days with medium changes in between. After the second transduction, the medium was replaced with normal growth medium (day 0). When TetO-PIB was used, the medium was supplemented with Dox (1. Mu.g/ml). For the duration of the culture, the medium was changed every 2-3 days. At the time of presentation, the medium was supplemented with LPS (100 ng/ml, enzo), poly I: C (25. Mu.g/ml, invivoGen) and R848 (3. Mu.g/ml, invivoGen) overnight. Cytokines were added on day 2 and retained for the duration of the culture; for reprogramming in the absence of heterologous conditions, MSCs were cultured in X-Vivo15 (Lonza) after transduction and medium was changed every 2-3 days for the duration of culture.

Molecular cloning

For the doxycycline (Dox) inducible overexpression system, the coding regions of human pu.1, IRF8 and bat f3 (PIB) were cloned in this order into the pFUW-TetO plasmid separated by the 2A self-cleaving peptide. The first two coding sequences lack a stop codon. The coding regions of PU.1, IRF8, BATF3, ID2, TXNIP, ZFP36PLEK, SUB1, JUNB, CREM, KLF, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP110, IRF5, STAT2, BATF, ZNF267, IRF1, RELB and BATF2 were individually cloned into pFUW-TetO plasmids. Lentiviral vectors (pFUW-UbC-M2 rtTA) containing the reverse tetracycline transactivator M2rtTA under the control of a constitutively active human ubiquitin C promoter were used for co-transduction (Rosa et al 2018). For constitutive overexpression, the human PIB polycistronic cassette was subcloned into a lentiviral vector with a constitutive promoter: pFUW-UbC, pRRL. PPT-SFFV, pRRL. PPT-PGK, pRRL. PPT-EF1S, pHAGE-EF 1 and pWPXL-EF1i (Addgene plasmid # 12257) (Sommer et al 2009; dahl et al 2015; schambach et al 2006).

Human MSC, moDC andgeneration of cDC1 cultures

Human Bone Marrow (BM) cells were collected from consented healthy donors by aspiration from the iliac crest at Hematology Department, lund University (sweden). The use of human samples was approved by the Lund University institutional review board (Institutional Review Board) according to the declaration of helsinki (Declaration of Helsinki). Monocytes from BM aspirate samples were isolated by density gradient centrifugation (LSM 1077 lymphocytes, PAA) with prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies) for lineage depletion (CD 3, CD14, CD19, CD38, CD66b, glycophorin a) by Magnetically Activated Cell Sorting (MACS) as previously described (Li et al 2014). MSC purification was followed by FACS sorting (additional information below). To generate moccs, fresh leukocyte concentrate was diluted 1x in Phosphate Buffered Saline (PBS) at a ratio of 1:1 and Peripheral Blood Mononuclear Cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (STEMCELL Technologies). Enrichment of CD14 from PBMC by positive selection using MACS with CD14 microbeads (Miltenyi Biotec) according to the manufacturer's protocol ⁺ Monocytes. CD14 ⁺ Monocytes were cultured in X-VIVO 15 medium (Lonza) supplemented with 5% FBS for 7 days. Cells at 1X10 ⁶ The individual cells/ml density was seeded and 8ml of cell suspension was added to the T75 flask. The medium was supplemented with IL-4 (350 ng/ml) and GM-CSF (850 ng/ml) on day 0, and medium was changed every 2-3 days. On day 6, IL-6 (15 ng/ml), PGE2 (10. Mu.g/ml), TNF-. Alpha.10 ng/ml, and IL 1-. Beta.5 ng/ml were added to the medium for 24 hours to produce mature moDCs. Mature moccs were dissociated using TrypLE Express (Gibco) and used for functional characterization. To isolate cDC1 for functional assays, DCs were enriched from PBMCs by MACS using a Pan-DC enrichment kit (Miltenyi Biotec), followed by further purification with anti-CLEC 9A antibodies conjugated to biotin and avidin microbeads (Miltenyi Biotec).

Flow cytometry analysis

To analyze surface marker expression, human and mouse cells were dissociated and incubated with antibodies diluted in PBS 5% FBS in the presence of mouse or rat serum (1/100, genetex) for 30 min at 4 ℃ for human and mouse cells, respectively, to block non-specific binding. Unless otherwise indicated, cells were washed and resuspended in PBS 5% FBS and analyzed in BD FACSCanto II or BD LSRFortessa flow cytometer (BD Biosciences). DAPI was used for dead cell exclusion. Flow cytometry data was analyzed using FlowJo software (FlowJo, LLC, version 10.6.1). All flow cytometry analyses were performed in a living single cell gate.

Fluorescence Activated Cell Sorting (FACS)

DC were enriched from PBMC using negative selection for MACS using Pan-DC Enrichment Kit (Miltenyi Biotec) according to the manufacturer's protocol. HLA-DR ⁺ CD11C ⁺ CD141 ⁺ cDC1、HLA-DR ⁺ CD11C ⁺ CD141 ^- CD1C ⁺ cDC2 and HLA-DR ⁺ CD11C ^- CD123 ⁺ pDC was purified in FACSAria III (BD Biosciences) and used for single cell RNA-seq profiling. To purify CD45 ⁺ 、CD45 ⁺ HLA-DR ^- 、CD45 ⁺ HLA-DR ⁺ And CD45 ⁺ HLA-DR ⁺ CD226 ⁺ hiDC, cells were dissociated using TrypLE Express, resuspended in PBS 5% FBS, incubated with anti-CD 45, anti-HLA-DR and anti-CD 226 antibodies at 4 ℃ for 30 min in the presence of mouse serum, and purified in FACSAria III. To isolate human primary MSCs, lineage depleted BM monocytes were subjected to blocking buffer [ Ca2/Mg2 free PBS, 3.3Mg/ml human normal immunoglobulin (Octaphaman), 1% FBS]Followed by antibody staining. CD45 ^- CD271 ⁺ MSCs were purified in FACSAria III (BD Biosciences) and used in reprogramming experiments. Dead cells were excluded by 7-amino actinomycin staining (7-AAD) or 4', 6-diamidino-2-phenylindole (DAPI).

Single cell RNA sequencing

HEF from peripheral blood (from 3 individual donors), hidC (CD 45) on days 3, 6 and 9 ⁺ HLA-DR ^- And CD45 ⁺ HLA-DR ⁺ ) FAC by cDC1, cDC2 and pDC S-sorting was used for scRNA-seq. Purified cells were loaded onto 10×chromasum (10×genomics) according to the manufacturer's protocol. The scRNA-seq index library was prepared using Chromium Single Cell '3 v2 and v3 Reagent Kit (10. Times. Genomics) according to the manufacturer's protocol. hiDC on day 9 and CD45 were also reprogrammed in the presence and absence of cytokines from HEF and HDF ⁺ HLA-DR ⁺ CD226 ⁺ hiDC was profiled. Library quantification and quality assessment were determined using High Sensitivity DNA assay kit (Agilent) using Agilent Bioanalyzer. Index libraries were pooled at equimolar concentration (equimolar) and sequenced on Illumina NextSeq 500. Coverage of approximately 130,000 reads per single cell was obtained. Details about the scRNA-seq data analysis can be found in the supplementary material.

Analysis of RNA sequencing data

A total of 51,903 single cell transcriptomes were profiled with approximately 130,000 reads per cell (R1 reads: technical, length: 26 to 28bp; R2 reads: biological, length: 90 to 98 bp). Double-ended sequencing reads of single-Cell RNA-seq were processed using 10Xgenomics software Cell Ranger v2.2.0 (https:// support.10 xgenomics.com/single-Cell-gene-expression/software). First, cellranger mkfastq is used to convert the binary base call file into a FASTQ file and simultaneously decode the multiplexed sample. Next, cellrange counts were applied to FASTQ files and alignment using STAR v2.5.3a with human (hg 38) genome assembly was performed. The output files from each run are then combined using a cellrange aggregation to produce a single matrix. The sparse expression matrix generated by the cellrange analysis procedure was used as input for a Scater library (http:// bioconductor. Org/packages/release/bioc/html/scan) and included cells and genes that passed the quality control threshold according to the following criteria: 1) The total number of Unique Molecular Identifiers (UMI) detected per sample is greater than 3 lower Median Absolute Deviations (MADs); 2) The number of genes detected in each single cell is greater than 3 lower MADs; 3) The percentage of counts in mitochondrial genes was <7.5%. The resulting expression matrix was filtered through a Scater analysis procedure and used as input to a Seurat library v4 (https:// satijalab. Org/setup). To address the technological variations, batch integration is performed. First, each batch was normalized using a "lognormal" scale factor of 10,000, respectively, and 9,000 variable features were identified. Next, batch integration is performed by looking up the corresponding anchor points between batches using 30 dimensions. Then, 50 principal components were calculated and tested for significance by JackStraw. The first 30 principal components are selected for subsequent tSNE visualizations. For differential expression analysis between cell types, the serat v4 findalmarkers function was used with LR assays, with assignment of latent variables (sequencing runs and donors) to reduce batch effects and define the following parameters: logfc.threshold=0.5, min.pct=0.5, bh-adjusted p <0.05. In addition, scaling is performed with the designation of potential variables in the vars.to.regress parameter, and the resulting genes are visualized using the setup v4 dohetmap function. For the other samples, all data were normalized together using a "lognormal" scale factor of 10,000. The first 30 principal components were selected for subsequent tSNE and UMAP visualizations. For the differential expression analysis comparing the membership hiDC with the control, the serat v4 findmarks function was used with the Wilcox test and the following parameters were defined: logfc.threshold=0.25, min.pct=0.25, bh adjusted p <0.05. The resulting intersection was visualized using a Vennerrable R library (https:// gitsub. Com/js 229/Vennerrable).

Example 2 pu.1, IRF8 and BATF3 induce global cDC1 Gene expression procedure in human fibroblasts

To characterize DC reprogramming of human fibroblasts at the transcriptional level, untransduced HEF (d 0) was performed by using single cell RNA-seq of the 10X Chromium system, on day 3 (CD 45 ⁺ D 3), day 6 (CD 45 ⁺ D 6) and day 9 (CD 45) ⁺ HLA-DR ⁺ ，d9 DR ⁺ ；CD45 ⁺ HLA-DR ^- ，d9 DR ^- ) Is shown, and peripheral blood cDC1, cDC2 and pDC (fig. 1. A).

Method

Carrier body

To induce a DC fate in HEF, polycistronic constructs encoding PIBs (PU.1, IRF8, and BATF 3) separated by 2A sequences were cloned into a doxycycline (Dox) inducible lentiviral vector (tetO-PIB) and introduced into cells (Rosa et al, 2018) (FIG. 1A).

Single cell RNA sequencing

Transduced and untransduced HEF cells were FACS sorted for scRNA-seq. Purified cells were loaded onto 10×chromasum (10×genomics) according to the manufacturer's protocol. The scRNA-seq library was prepared using the Chromium Single Cell 3'v2 Reagent Kit (10. Times. Genomics) according to the manufacturer's protocol. Index sequencing libraries were constructed using reagents from Chromium Single Cell' 3 v2 Reagent Kit. Library quantification and quality assessment were determined using High Sensitivity DNA assay kit using Agilent Bioanalyzer. The index libraries were pooled equimolar and sequenced on Illumina NextSeq 500 using a double ended 26x98bp sequencing mode. Coverage of approximately 100,000 reads per single cell was obtained.

Scanning Electron Microscopy (SEM)

HEF transduced with PIB factor was sorted on day 8 (CD 45 ⁺ HLA-DR ⁺ ) Plates were plated on 0.1% gelatin coated coverslips and analyzed on day 9 along with M2rtTA transduced HEF. The samples were washed in 0.1M Sorensen's phosphate buffer and fixed with 0.1M Sorensen's phosphate buffer pH 7.4, 1.5% formaldehyde and 2% glutaraldehyde at room temperature for 30 minutes. After fixation, the samples were washed in 0.1M Sorensen's buffer. The samples were then dehydrated in a gradient series of ethanol (50%, 70%, 80%, 90% and twice 100%), critical dried and fixed on a 12.5mm aluminum stub shaft (stub). The samples were then sputtered with 10nm Au/Pd (80/20) in a Quorum Q150T ES turbo-pumped sputter coater and inspected in a Jeol JSM-7800F FEG-SEM.

DC subset classification

The scPred library (Alquicira-Hernandez et al, 2019) and publicly available DC single cell expression data (Villani et al, 2017) were used for subset membership. To train the classifier using the scppred method (implemented as an R library), default parameters for getFeatureSpace, trainModel are used as defined in the tool vignette. To predict the assignment of DCs isolated from PBMCs to publicly available DC subsets, a scPredict function with default parameters was used. For classification of hiDC, the scpredictt function with threshold = 0.99 was applied separately for each donor, and then the cell numbers belonging to each subset were combined.

Results

To induce a DC fate in Human Embryonic Fibroblasts (HEF), polycistronic constructs (Rosa et al 2018) cloned into a doxycycline (Dox) inducible lentiviral vector (TetO-PIB) encoding PU.1, IRF8, and BATF3 (PIB) separated by a 2A sequence were used (FIG. 1A). CD45 was observed on day 3 after transduction of HEF with PIB ⁺ Cell populations, and small CD45 with DC-like morphology from day 6 to day 9 ⁺ HLA-DR ⁺ The appearance of a cell population, designated human induced DC (hidC), was shown (FIGS. 1B-D). To elucidate transcriptional changes, scRNA-seq was performed using a 10 Xchromium system. Profiling 45,870 cells from 3 donors, including peripheral blood cDC1, cDC2, pDC, untransduced HEF (d 0), on day 3 (CD 45 ⁺ D 3), day 6 (CD 45 ⁺ D 6) and day 9 (CD 45) ⁺ HLA-DR ^- ，d9 DR ^- ；CD45 ⁺ HLA-DR ⁺ ，d9 DR ⁺ ) Is not shown in the drawings). The t-distribution random neighborhood embedding (t-SNE) of the dataset reveals four clusters highlighted: HEF, cDC1, cDC2 and pDC (fig. 1E). While hiDC d3 and d6 are not specifically mapped to any cluster, hiDC d9 is mapped by cDC1, where DR ⁺ Ratio DR ^- Closer to cDC1. These data suggest that 9 days of time frames are required for human cDC1 reprogramming, and CD45 ⁺ HLA-DR ^- Cells represent partially reprogrammed cell states. Next, the single cell data obtained was integrated with publicly available DC data sets using scPred (Alquiria-Hernandez et al 2019) (Villani et al 2017). As expected, 53.8% of purified DCs 1 were observed to be assigned to the DCs 1 subset, 66.9% and 29.8% of DCs 2 were assigned to the DCs 2 and 3 subsets, respectively, and 66.5% of pdcs were assigned to the DCs 6 subset (fig. S3). Although it is HEF is non-affiliated but 1.3% d3, 5.3% d6, 14.4% d9 DR ^- And a d9 DR of 36.7% ⁺ The specific assignment of hidcs to the cDC1 subset suggests that pu.1, IRF8, and BATF3 gradually applied cDC1 features, with a degree of heterogeneity that did not cross the subset boundaries (fig. 1F-G). When compared to its non-affiliated counterpart, the cDC 1-affiliated cell expressed higher levels of cDC 1-specific genes CADM1 and WDFY4 (Villani et al 2017) (fig. 1H). Next, the genes that vary the most between the datasets were extracted and grouped in 5 clusters (fig. 1I). Cluster 1 contains genes that are highly expressed in HEF and that are silenced during reprogramming. Cluster 2 highlights early transcriptional changes during reprogramming, and cluster 3 includes the highly expressed cDC 1-specific genes C1orf54, ANPEP, TACSTD2, and SLAMF8 in d9 hiDC and cDC1 (heikkamp et al 2016; see et al 2017; villani et al 2017) (fig. 1J, table 2). Cluster 4 and cluster 5 contain genes highly expressed in dcs 2 and pDC, respectively. Interestingly, d9 hiDC expressed high levels of antigen processing and cross-presentation genes, including PSMB9, TAP1, and HLA-C (fig. 1K-M), suggesting that reprogrammed cells have acquired cross-presentation capability.

Table 2. Genes included in cluster 3 from FIG. 1I.

Conclusion(s)

Taken together, these data demonstrate that PIB factor exerts cDC1 characteristics in human fibroblasts.

Example 3 Single cell analysis highlights pathways related to successful and unsuccessful cDC1 reprogramming

It is assumed that single cell RNA-seq can be used to profile human DC reprogramming trajectories and reveal pathways or factors associated with successful reprogramming and thus allow optimization of cDC1 reprogramming in human cells.

Method

Pseudo-time reconstruction

Monocle3 library (Cao et al 2019) was used to order cells at the reprogramming pseudo time. Monocle3 was run on tSNE with the following parameters: use_part=false, which assumes that all cells in the dataset originate from a common transcriptional ancestor. The root of the trajectory is automatically selected. To mitigate batch effects in gene identification along trajectory variation, batch correction was performed using regress batches function from the batch R library (http:// bioconductor. Org/packages/release/bioc/html/batch. Html). Next, genes that vary on the trajectory were identified using the graph_test function and grouped into 21 different modules using the find_gene_modules function, and clustered using the Ward.D2 method in the pheeatmap R library (https:// scan.r-project.org/web/packages/pheeatmap/index.html). Genes are defined as successful and unsuccessful reprogramming groups according to the clustering result. Next, these gene lists were additionally filtered by: the average value for each gene in each cell population was calculated and the following criteria were used: for successful reprogramming-hiDC d9 DR ⁺ (of the membership)>hiDC d9 DR ⁺ (non-affiliated)&hiDC d9 DR ^- (of the membership)>hiDC d9 DR ⁺ (non-affiliated)&hiDC d9 DR ⁺ (of the membership)>HEF&hiDC d9 DR ^- (of the membership)>HEF and vice versa for all comparisons of unsuccessful reprogramming. For the reconstruction of reprogramming kinetics, sc velo v0.2.4 (Bergen et al 2020) was used. For these analyses, velocyto v0.17.17 (http:/-for example) was usedvelocyto.org) The sparse expression matrix generated by the cellrange analysis procedure was converted to a spliced and non-spliced matrix. scello runs with default settings. In addition, scello was also used to restore latency, the first 1000 genes that changed along latency were selected and visualized on a heatmap. For additional samples (fig. 3), the Monocle3 library was used to rank cells over pseudo-time during HEF to hiDC reprogramming using the same method.

Transcription Factor (TF) co-regulatory network analysis

To construct the TF network, genes associated with successful or unsuccessful cDC1 reprogramming were submitted for analysis using ChEA3 (https:// maayanlab. Closed/ChEA 3 /), and integration by average ranking across the library. TF networks appear as network graphs based on their co-expression similarity, with dots representing human TF.

Results

The inventors used Monocle3 to reconstruct the cDC1 reprogramming trace (Cao et al 2019). HEF and cDC1 were placed at the beginning and end of the pseudo-time respectively (fig. 2A-B). While d9 hiDC is placed at the end of the trace with respect to cDC1, d3 and d6 hiDC are positioned in the middle highlighting the gradual transition of the single cell transcriptome during cDC1 reprogramming. Importantly, the d9 hiDC of membership was located later in pseudo-time when compared to its non-affiliated counterpart, suggesting that trace reconstruction is capturing a successful cDC1 reprogramming path. It was also observed that there was a "dead-end" trace mapping non-affiliated hidcs closer to HEF, suggesting that these cells failed to enter a successful reprogramming path. To infer the expression dynamics and directionality of individual cells during reprogramming, a scello analysis (Bergen et al 2020) was applied to the reprogramming trajectories. The hiDC speed was mainly compared to the cDC1 reprogramming trajectory predicted by Monocle3 (fig. 2C). Nonetheless, it was observed that the hiDC mapping was closer to the blind end trajectory display speed directed to HEF, consistent with previous reports describing unsuccessful reprogramming pathways through gene expression markers correlated with primitive cell status (Biddy et al 2018; zhou et al 2019; treutlein et al 2016). Dynamic reconstitution of gene expression using latency showed down-regulation of cell cycle genes during cDC1 reprogramming (fig. 2D-E), suggesting a hiDC exit cycle. The inventors also observed an enrichment of cytokines and type I and type II interferon (IFN- γ) signaling pathways late in latency. Down-regulation of the cell cycle and up-regulation of IFN gene characteristics were consistent with our previous findings in the mouse system (Rosa et al 2018), suggesting a species-conserved role for IFN signaling in cDC1 reprogramming. To map the phase-specific gene changes along the reprogramming, the inventors clustered genes differentially expressed along pseudo-time in 21 modules (fig. 2F). Non-affiliated hidcs failed to down-regulate several gene modules enriched in HEF, including modules 1, 2, 4, and 7 (unsuccessful reprogramming). The affiliated hidcs and cdcs 1 were enriched for genes expressed in modules 3, 9, 13, 15 and 17 (successful reprogramming). Extraction of genes encoding surface molecules and transcriptional regulators from these modules highlights fibroblast genes (CD 248 and PRRX 1) that were enriched in unsuccessful reprogramming, as well as DC genes that were up-regulated in successfully reprogrammed cells, including cDC1 markers CD226 (heikkamp et al 2016) and IRF7 (Honda et al 2005) (fig. 2G, table 1). Transcription factor enrichment analysis for unsuccessful reprogramming genes identified previously described obstacles to direct reprogramming, including TWIST1, TWIST2, PRRX1, PRRX2 and OSR1 (Tomaru et al 2014) (FIG. 2H). Interestingly, the same analysis of successful reprogramming genes underscores the importance of pu.1, IRF8 and bat in establishing successful cDC1 reprogramming gene signatures.

Conclusion(s)

These data demonstrate that forced expression of transcription factors pu.1, IRF8 and bat 3 can improve reprogramming efficiency and highlight the role of cytokines and IFN signaling in cDC1 reprogramming.

Example 4. Single cell analysis highlights surface markers associated with successful reprogramming, allowing identification and prospective isolation of successfully reprogrammed hidcs.

The inventors hypothesize that surface markers enriched in successful cDC1 reprogramming (table 1) will allow identification and prospective separation of successfully reprogrammed hidcs. As proof of concept, one of these surface markers-CD 226 was selected, and CD226 was purified ⁺ Reprogrammed hiDC and associating its cDC1 identity with CD226 ^- The reprogrammed hidcs were compared.

Method

First, the inventors evaluated CD226 at partially reprogrammed CD45 ⁺ HLA-DR ^- And reprogrammed CD45 ⁺ HLA-DR ⁺ Surface expression in hiDC. Then, CD45 was purified ⁺ HLA-DR ⁺ CD226 ⁺ hiDC and uses the scppred system to match their cDC1 identity to CD45 ⁺ HLA-DR ⁺ CD226 ^- The cDC1 identity of the hidcs was compared. See previous examples for experimental details.

Results

First, CD45 was observed ⁺ HLA-DR ⁺ hiDC expression ratio CD45 ⁺ HLA-DR ^- hiDC is higher in CD226 (fig. 2I). Then, to verify the utility of CD226 to identify cDC 1-like cells, CD45 was identified ⁺ HLA-DR ⁺ CD226 ⁺ The hidcs were purified and profiled by scRNA-seq. Interestingly, CD226 ⁺ Cells showed increased cDC1 membership (from 19.5% to 40.9%) (fig. 2J). In addition, it was observed that when it was identical to CD226 ^- CD226 when compared to hiDC ⁺ hiDC performed better in terms of phagocytosis of dead cells, suggesting that CD226 marks functional hidcs (fig. 2K).

Conclusion(s)

These data suggest that the inclusion of CD226 in the surface marker associated with successful reprogramming allows the isolation of more functional hidcs with a fine cDC1 identity.

Example 5 Single cell analysis identified transcription factors associated with successful cDC1 reprogramming that were able to cooperate with PU.1, IRF8 and BATF3 and increase cDC1 reprogramming Cheng Xiaolv

The inventors hypothesize that transcription factors enriched in successful cDC1 reprogramming (table 1) can cooperate with pu.1, IRF8 and BATF3 to increase cDC1 reprogramming efficiency. As proof of concept, 22 transcription factors (ID 2, TXNIP, ZFP36, PLEK, SUB1, JUNB, CREM, KLF, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP110, IRF5, STAT2, BATF, ZNF267, IRF1, rebab, and BATF 2) were selected, which correlated with successful cDC1 reprogramming or were predicted to regulate successful cDC1 reprogramming gene characteristics (see Transcription Factor (TF) co-regulatory network analysis described in example 3), and evaluated for their ability to increase cDC1 reprogramming efficiency when co-expressed with pu.1, IRF8, and BATF 3.

Method

The coding regions of ID2, TXNIP, ZFP36, PLEK, SUB1, JUNB, CREM, KLF, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP, IRF5, STAT2, BATF, ZNF267, IRF1, RELB and BATF2 were cloned into pFUW-TetO plasmids, respectively. Lentiviral particles encoding each individual transcription factor, either PU.1, IRF8 and BATF3 (pFUW-tetO-PIB), or the reverse tetracycline transactivator M2rtTA under the control of a constitutively active human ubiquitin C promoter (pFUW-UbC-M2 rtTA) were used for co-transduction (Rosa et al 2018). The reprogramming efficiency in HEF was assessed by flow cytometry 9 days after transcription factor overexpression.

Results

To evaluate whether additional modulators enhance reprogramming, the inventors supplemented pu.1, IRF8, and BATF3 with individual transcription factors associated with successful cDC1 reprogramming, and observed that IRF7 and BATF increased cDC1 reprogramming Cheng Xiaolv (fig. 2L). IRF7 is a transcriptional regulator downstream of inflammatory signaling (Honda et al 2005). BATF is highly homologous to BATF3 and is shown to compensate for BATF3 during cDC1 development (Tussiwand et al 2012).

Conclusion(s)

These data suggest that transcription factors associated with successful reprogramming, including IRF7 and BATF, may increase the efficiency of cDC1 reprogramming.

Example 6 use of external cytokines to increase cDC1 reprogramming Cheng Xiaolv

Through enrichment of cytokine signaling in successfully reprogrammed hidcs and its role in human DC specialization, the inventors hypothesized that cytokines could act synergistically with pu.1, IRF8 and BATF3 in DC reprogramming.

Method

At 2 days post PIB induction in HEF, 17 human hematopoietic cytokines including inflammatory mediators (table 3) were added individually to the medium and reprogramming efficiency was measured at day 9. A single cytokine and a combination of two or three cytokines were used to analyze changes in reprogramming efficiency.

Table 3. List of 17 human hematopoietic cytokines used in the current experiment.

Results

IFN-gamma has the most significant effect, promoting CD45 ⁺ HLA-DR ⁺ 20-fold increase in cell production (7.9% ± 2.2% versus 0.4% ± 0.2% without cytokine) (fig. 3A). Other inflammatory cytokines also increase reprogramming efficiency, including IL-1 beta (3-fold), IL-6 (2.5-fold), oncostatin M (4-fold), TNF-alpha (3-fold), and IFN-beta (4-fold). FLT3L, IL-4 and GM-CSF for DC in vitro differentiation from progenitor cells (Balan et al, 2018) or monocytes (Chapuis et al, 1997) did not affect reprogramming efficiency. Combining IFN-gamma with IFN-beta or TNF-alpha induces additional 2.5-fold and 2-fold increases in reprogramming efficiency. Combining the three cytokines IFN-gamma, IFN-beta and TNF-alpha resulted in a 70-fold increase in 28.1% + -3.4% hidC when compared to the cytokine-free condition (FIG. 3B).

Conclusion(s)

These data strongly suggest that the efficiency of cDC1 reprogramming increases with the provision of inflammatory cytokines.

Example 7 use of stronger constitutive promoters to augment cDC1 reprogramming Cheng Xiaolv

Whereas the trajectory reconstruction of the cDC1 reprogramming correlated pu.1, IRF8 and BATF3 expression with successful establishment of a cDC1 fate, the inventors examined whether forced expression of reprogramming factors using stronger constitutive promoters increased reprogramming efficiency.

Method

The PIB polycistronic cassette was then cloned into a constitutive vector as IRES-GFP using multiple promoters on the lentiviral backbone, and DC reprogramming Cheng Xiaolv in MEFs harboring the Clec9a-tdTomato reporter gene was evaluated (Rosa et al, 2018). The following vector backbones and promoters were used: pFUW-UbC, pRRL. PPT-SFFV, pRRL. PPT-PGK, pRRL. PPT-EF1S, pHAGE-EF 1 and pWPXL-EF1i.

Results

Overexpression of PIB driven by SFFV promoter induced excellent efficiency in mouse cells (46.6% + -16.7% tdTomato) ⁺ MHC-II ⁺ Cells) (fig. 4A). In HEF, the appearance of 21.3±6.1% hidc was observed with the SFFV system (fig. 4B). Furthermore, constitutive overexpression of PIB induced surface expression of CD45 in most fibroblasts, suggesting that a larger cell line initiated the DC reprogramming process when compared to the Dox-inducible system (fig. 4B).

Conclusion(s)

These data indicate that lentiviral vectors with SFFV promoters can be used to improve reprogramming in human cells.

Example 8 use of a combination of cytokines and stronger constitutive promoters to increase cDC1 reprogramming Cheng Xiaolv

In view of the increased ctc 1 reprogramming Cheng Xiaolv by IFN- γ, IFN- β and TNF- α signaling and SFFV mediated constitutive overexpression of pu.1, IRF8 and bat 3, the inventors examined whether inflammatory cytokine signaling synergistically works with constitutive overexpression of reprogramming factors to achieve higher reprogramming efficiency.

Method

The effect of reprogramming efficiency after a combination of cytokine treatment and SFFV-driven PIB induction was evaluated. The scppred system is used for integration with "natural" DCs. See previous examples for experimental details.

Results

The induction of SFFV drive and combination of treatment with IFN- γ, IFN- β and TNF- α produced 76.9.+ -. 11.9% of hidC, which is a 190-fold increase in efficiency of cDC1 reprogramming compared to the original protocol (FIG. 4B). Furthermore, an increase in the absolute number of hidcs generated was observed with the improved protocol (fig. 4C). To characterize the cDC1 identity of hidcs generated with improved reprogramming protocols, hidcs obtained with and without cytokine induction (tetO-PIB) and constitutive (SFFV-PIB) systems were profiled and scppred was used for integration with peripheral blood DCs (fig. 4E). Interestingly, 61.4% and 53.2% of hidcs generated with and without cytokines with SFFV, respectively, were affiliated to the cDC1 lineage, in contrast to only 33.4% and 22.0% generated with the inducible system (tetO-PIB). These data suggest that the forced expression of cytokine signaling, pu.1, IRF8 and BATF3, was synergistic for the successful establishment of cDC 1-like cell fate. IL-10 did not affect reprogramming efficiency, whereas TGF- β reduced it by 1/2 (FIG. 5). IL-10 and TGF- β signaling do not affect CD40 expression in hidCs.

Conclusion(s)

These data suggest that: 1) The improved regimen increased both the efficiency of cDC1 reprogramming and the identity of cDC1, and 2) the forced expression of cytokines and pu.1, IRF8, and bat 3 was synergistic for successful reprogramming.

Example 9 functional reprogramming of human cDC 1-like cells

cDC1 coordinates adaptive immunity through multiple mechanisms, including secretion of cytokines and antigen presentation to T cells. The inventors examined whether hiDC could act as a naturally occurring cDC1 by elicitation of cDC 1-like gene expression profile in human fibroblasts after pu.1, IRF8 and BATF3 overexpression.

Method

To investigate whether hidcs share the same functionality as naturally occurring DCs, hidcs were challenged with all TLRs of toll-like receptor 4 (TLR 4) [ Lipopolysaccharide (LPS) ], TLR3[ polyinosinic-polycytidylic acid (poly I: C) ] or TLR7/8[ resiquimod (R848) ], or a combination. The co-stimulatory molecules CD40 and CD80 (required for T cell activation) were analyzed for surface expression by flow cytometry and used as markers for T cell activation.

To assess dead cell phagocytosis, HEK293T cells were exposed to Ultraviolet (UV) radiation (50J/m 2) to induce cell death and labeled with CellVue Claret Far Red Fluorescent Cell Linker Kit (Sigma). hiDC, HEF and cDC1 on day 9 were incubated with far infrared labeled dead cells for 2 hours, washed with PBS 5% FBS, and analyzed in BD LSRFortessa X-20. In-vivo CD45 using far infrared channels ⁺ HLA-DR ⁺ hiDC、CD45 ⁺ HLA-DR ⁺ CD226 ^- hiDC、CD45 ⁺ HLA-DR ⁺ CD226 ⁺ hiDC、CD141 ⁺ CLEC9A ⁺ Dead cell incorporation was quantified in peripheral blood cDC1 or control populations. For delayed fluorescence microscopy imaging of dead cell phagocytosis, far infrared labeled dead cells were added to FACS-sorted HEF-derived CD45 immediately prior to initiation of image acquisition on Zeiss Celldiscoverer 7 ⁺ HLA-DR ⁺ In hiDC culture. Microscopy images were acquired every 10 minutes for a total of 16 hours.

To evaluate inflammatory cytokine secretion, CD45 produced in the presence or absence of cytokines was sorted in FACS using a cell count bead array kit (LEGENDplex Human Anti-Virus Response Panel, bioLegend) according to manufacturer's instructions ⁺ HLA-DR ⁺ Day 9 hiDC, HEF cultured in the presence or absence of cytokines, moDC and FACS-sorted CD141 ⁺ CLEC9A ⁺ XCR1 ⁺ The level of human cytokines was quantified in 25 μl of culture supernatant of cDC 1. When stated, stimulus of LPS, poly I: C or R848 or a combination of the three was added overnight prior to analysis. The collection was performed in FACSCanto and the data was analyzed using LEGENDplex software (BioLegend).

To assess cross-presentation capacity, HEF, moDC, magnetically Activated Cell Sorting (MACS) enriched Clec9a ⁺ cDC1 and hiDC on day 8 of reprogramming were stimulated with LPS (3 ng/ml), poly I: C (25 μg/ml) and R848 (3 ng/ml). After overnight stimulation, cells were washed in PBS containing 2% FBS and pulsed with 2 μl/ml CMV protein (Miltenyi Biotec). After 3 hours, cells were washed and isolated from CMV seropositive donors for MACS-enriched CD8 ⁺ T cell co-culture. CMV positivity was verified by flow cytometry using CMV dexmer (Immudex). 1x10 ⁵ CD8 ⁺ T cells and 5x10 ⁴ The DCs were co-cultured in 200. Mu. l X-VIVO15 in 96-well plates. After 24 hours, T cell activation was measured by quantifying IFN- γ levels in the supernatant using ELISA (BD). Absorbance at 490nm was read in GloMax Discover Microplate Reader (Promega).

Results

The inventors observed that both hiDC and cDC1 up-regulated costimulatory molecules following TLR3 or combined stimulation (fig. 6A). In addition, hiDC triggered responses to TLR4 to a higher extent than cDC 1. Accordingly, from CD34 ⁺ The differentiation of hematopoietic progenitor cells into dcs 1 in response to LPS indicated the general characteristics of cDC 1-like cells generated in vitro (Balan et al 2018). To assess phagocytic capacity, a brief incubation with labeled dead cells was performed. The inventors observed that hiDC (47.5±12.0%) produced in the presence of IFN- γ, IFN- β and TNF- α (19.4±7.7%) and cDC1 (10.9±3.5%) incorporated into dead cell material (fig. 6B-D), cross-presenting key features of DCs. DC maturation and phagocytosis are often inversely related (Broz et al 2014). Accordingly, hidcs produced in the presence of cytokines expressed higher levels of costimulatory molecules and showed a decrease in the ability to incorporate into dead cells (fig. 6A-C). To confirm that hiDC also provided a third signal required for T cell activation, cytokine secretion was assessed (fig. 6F). It was first observed that hiDC and cDC1 respond to TLR3 challenge by secreting human cDC 1-specific cytokine IFN- λ1 (Hubert et al 2020). This is in contrast to modcs that do not respond to TLR3 agonists (Lauterbach et al 2010). In addition, hidcs also respond to TLR4 and 3 by secreting IL12p70, CXCL10, and TNF- α. Furthermore, IFN-gamma, IFN-beta and TNF-alpha were observed to increase the magnitude of cytokine secretion. The inventors then examined whether hidcs cross-present antigen to CD8 ⁺ T cells. HEF, moDC, cDC1 and hiDC pulsed with CMV proteins and secondary CMV ⁺ Isolated CD8 in donor ⁺ T cells were co-cultured. IFN-gamma secretion was quantified as a readout of T cell activation (FIG. 6G). As expected, cDC1 was observed to be effective in cross-presentation of CMV antigen to CD8 in contrast to mocc or HEF ⁺ T cells. Remarkably, the inventors observed that hidcs with or without cytokine production established cross-presentation of antigen to CD8 ⁺ T cell capacity. In summary, these data support reprogrammed hidcs are functionally cross-presented DCs.

Conclusion(s)

In summary, these data support the membership of hiDC to subset cDC1 and their acquisitionThe resulting ability to respond to inflammatory stimuli, phagocytose dead cells, secrete cytokines, and cross-present antigens, enables activation of antigen-specific CD8 ⁺ T cells.

Example 10 efficient reprogramming of human adult somatic cells

The generation of DCs 1 from human-available cell types may represent an additional source of DCs for cancer immunotherapy. Accordingly, the inventors tried to reprogram primary Human Dermal Fibroblasts (HDF) and Mesenchymal Stromal Cells (MSCs) into cDC 1-like cells with an improved DC reprogramming protocol.

Method

HDFs from 3 healthy donors were obtained and the cDC1 reprogramming efficiency was evaluated. Single cell transcriptomes were generated for HDF derived hidcs and scppred analysis was used for membership of the DC subset. Purified MSCs from 3 healthy donors were transduced with SFFV-PIB lentiviral particles and cultured in chemically defined serum-free X-VIVO 15 medium (fig. 8A). Cells were evaluated for the efficiency of cDC1 reprogramming. See previous examples for experimental details.

Results

The efficiency of hiDC production varied from donor to donor by 20-35% using SFFV-PIB (fig. 7A-B). When combined with IFN-gamma, IFN-beta and TNF-alpha, reprogramming efficiency increased approximately 2-fold (FIG. 7A-B), and also resulted in an increase in CD40 and CD80 (FIG. 7C). The scppred analysis assigned 60.6% and 59.3% of HDF-derived hidcs with and without cytokine production, respectively, to the cDC1 subset (fig. 7D). The identity of cDC1 was further confirmed by expression of the cDC1 specific genes C1orf54 and HLA-DPA1, as well as antigen processing and presentation genes CD74, HLA-C, B2M, PSMB9, NAAA and TAP1 (FIGS. 7E-F). 60-75% of MSCs from 3 donors were converted to hiDCs co-expressing CD40 and CD80 (CD 45) ⁺ HLA-DR ⁺ ) (FIGS. 8B-D). IFN-gamma, IFN-beta and TNF-alpha did not further improve hiDC1 production from MSC cultures.

Conclusion(s)

These data suggest that pu.1, IRF8 and BATF3 induce a cDC1 fate in human adult cells and MSCs, highlighting the consistency of the reprogramming method across multiple cell types and donors. The lack of effect of cytokine addition in MSCs suggests that inflammatory cytokine signaling may promote cDC1 reprogramming in a cell type specific manner.

EXAMPLE 11 Induction of anti-tumor immunity in vivo

Considering the interest in assessing whether or not iDC generated by direct cell reprogramming is functional in vivo, the inventors utilized a mouse system and examined mouse iDC using a syngeneic cancer mouse model (derived from Clec9a-tdTomato report MEF, tdTomato ⁺ Cells) induce anti-tumor immunity.

Method

To assess antigen cross-presentation, initial mouse CD8 was used ⁺ T cell isolation kit (Miltenyi) enriched for CD8 from spleen of OT-I mice ⁺ T cells. Enriched CD8 ⁺ T cells were labeled with 5 μ M Cell Trace Violet CTV (Thermo Fisher) for 20 min at room temperature, washed and counted. FACS sorted tdTomato+ (generated with SFFV-PIB) and cDC 1-like BM-DC at indicated time points were incubated with OVA protein (10. Mu.g/ml) in the presence of poly I: C (1. Mu.g/ml) for 10 hours at 37 ℃. After extensive washing, 20,000 DCs were incubated with 100,000 CTV-labeled OT-I CD8 ⁺ T cells were incubated together in 96-well round-bottomed tissue culture plates with poly I: C (1. Mu.g/ml). After 3 days of co-culture, T cells were collected, stained and analyzed in BD LSR Fortessa. By aligning live single TCRs ⁺ CD8 ⁺ T cells were gated to determine T cell proliferation (dilution of CTV staining).

tdTomato purified on day 9 using LEGENDplex Mouse Anti-Virus Response Panel (BioLegend) ⁺ The levels of mouse IFN-. Alpha.and Cxcl10 were evaluated in 50. Mu.l culture supernatant of cells. LPS (100 ng/ml) or poly I: C (1. Mu.g/ml) was added overnight. Acquisition was performed in FACSCanto and data was analyzed using LEGENDplex (BioLegend) software.

For in vivo experiments, B16-OVA (0.5x10 ⁶ ) Tumor cells were subcutaneously injected into the left flank of 6-10 week old C57Bl/6 females. Will be on day 9 prior to tumor implantationFACS-sorted tdTomato generated with SFFV-PIB ⁺ The cells were mixed with B16-OVA cells. Alternatively, tdTomato was taken at day 8 after tumor establishment ⁺ Cells, MEF or CD103 transduced with SFFV-GFP control ⁺ BM-DC was intratumorally injected into established tumors. On the previous day, cells were stimulated overnight with LPS (100 ng/ml) and poly I: C (1. Mu.g/ml). Cells were injected with OVA at day of the injection _257-264 The peptide (5. Mu.g/ml) was pulsed at 37℃for 30 minutes. After washing twice in PBS, 80,000 cells were resuspended in 60 μl PBS and intratumoral injection was performed on each tumor-bearing mouse on day 8 after B16-OVA tumor implantation. Tumor size [ volume=0.5 x length x width x height ] was measured with calipers every one to two days during the indicated period]. To assess T cell infiltration and activation, 1.5x10 was injected intravenously after iDC injection in tumor-bearing animals ⁶ OT-I CD8 labeled with CTV ⁺ T cells. After 4 days, animals were sacrificed and tumors and tumor draining lymph nodes were collected and mechanically and chemically digested with collagenase D (1 mg/ml) and dnase I (10 mg/ml). Dead cells were eliminated using Percoll. For intracellular cytokine analysis, cells were re-stimulated in the presence of phorbol 12-tetradecanoate 13-acetate (100 ng/ml) and ionomycin (1. Mu.g/ml) in complete RPMI medium at 37℃and 5% CO2 for 4 hours. For the last 2.5 hours, golgi plug solution (1. Mu.l/ml) was added to the medium. Cells were stained with the fixable vital dye FITC at 4 ℃ for 30 min. Using Intracellular Fixation&The Permeabilisation buffer pool was used to perform intracellular staining for IFN-. Gamma.and granzyme B. Data were acquired using Gallios and BD LSRFortessa.

Results

The iDC was already able to perform cross-presentation of antigen on day 4 and day 6 of reprogramming (fig. 9A-B). Furthermore, purified tdTomato ⁺ The iDC secretion was previously described as Cxcl10 and ifnα (fig. 9C) necessary for dcs 1-mediated tumor rejection (Diamond et al, 2011). It was further observed that co-injection with iDC reduced tumor growth during tumor establishment (fig. 9D). Notably, a single intratumoral injection of 80,000 iDC's in an established tumor is sufficient to slow down the swelling Tumor growth (fig. 9E). Un-reprogrammed MEF and CD103 ⁺ Intratumoral injection of BM-DC is not as effective in controlling tumor growth. In addition, in both models, injection of iDC increased antigen-specific CD8 in tumors ⁺ Infiltration of T cells and an increase in the cytotoxicity profile of T cells in tumor draining lymph nodes was promoted (fig. 9F).

Conclusion(s)

These data support the hypothesis that iDC induces an anti-tumor immune response. Taken together, these data suggest that iDC is able to activate antigen-specific CD8 by activating antigen-specific CD8 ⁺ T cells and promote their infiltration within tumors to control tumor growth.

Example 12 efficient DC reprogramming requires the combined action of PU.1, IRF8 and BATF3

To elucidate the molecular mechanisms underlying DC reprogramming mediated by PU.1, IRF8 and BATF3, the inventors transduced HDF with Dox-induced lentiviral particles encoding three reprogramming factors simultaneously or each separately, and performed ChIP-seq for PU.1, IF8 and BATF3 48 hours after TF induction (FIG. 10A)

Method

ChIP sequencing

TF is delivered with polycistronic lentiviral vector (pFUW-tetO-PIB) or individual vector (pFUW-tetO-PU.1, pFUW-tetO-IRF8 or pFUW-tetO-BATF 3) concomitantly with pFUW-M2 rtTA. ChIP was performed 48 hours after Dox addition.

Chromatin in cultured cells was fixed by adding 1/10 volume of freshly prepared formaldehyde solution [11% formaldehyde (Sigma), 0.1M NaCl, 1mM EDTA and 50mM HEPES ] to each cell suspension in complete DMEM. The tube was left at room temperature with agitation for 15 minutes. Immobilization was stopped by adding 1/20 volume of 2mM glycine solution (Sigma). After 5 min incubation, the cells were centrifuged at 800g for 10 min at 4 ℃. The cell pellet was resuspended in 10ml ice-cold PBS-Igepal, 100. Mu.l PMSF was added to each tube, and centrifuged at 800g for 10 min at 4 ℃. The cell pellet was snap frozen on dry ice and stored at-80 ℃. Active Motif (Carlsbad, CA) prepares chromatin, performs ChIP, generates a library, and sequences the library. Briefly, chromatin was isolated by addition of lysis buffer followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500bp (EpiShear probe sonicator for Active Motif). Genomic DNA (input) was prepared by: aliquots of chromatin were treated with rnase, proteinase K and heat for decrosslinking, followed by purification using Solid Phase Reversible Immobilization (SPRI) beads (Beckman Coulter), and quantification by Clariostar (BMG Labtech). Extrapolation to the original chromosome volume allows determination of total chromatin yield. 30 μg of chromatin was pre-cleared with protein A/G agarose beads (Invitrogen). Immunoprecipitation was performed with 4 μg of antibodies to human pu.1, IRF8 and bat 3 (rabbit anti-human pu.1, rabbit anti-human IRF8 or sheep anti-human bat 3). The complexes were washed, eluted from the beads with SDS buffer, and subjected to rnase and proteinase K treatment. The cross-linking was reversed by incubation at 65 ℃ overnight and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. To confirm ChIP enrichment, quantitative PCR (QPCR) reactions were performed in triplicate on specific genomic regions using SYBR Green Supermix (Bio-Rad). By performing QPCR for each primer pair using the input DNA, the resulting signal is normalized to primer efficiency. An Illumina sequencing library was prepared from ChIP and input DNA by standard sequential enzymatic steps of end polishing, dA addition and adaptor ligation. The steps are performed on an automated system (Apollo 342,Wafergen Biosystems/Takara). After the final PCR amplification step, the resulting DNA library was quantified and sequenced on the Illumina Next Seq500 (75 nt reading, single end).

ChIP sequencing analysis and data visualization

ChIP-seq analysis is performed on the original FASTQ file. The FASTQ file was mapped to the human hg38 genome using the Bowtie 2 program that allowed 2 base pair mismatches. The mapped output file was processed by MACS v2.1.0 analysis software to determine peaks. Peak annotation was performed using the ChIPseeker R library. For the genomic tracks, bigwig files are created from the bam file with deepto (https:// deeptols readthes. Io/en/devilop /), andexploration was performed using UCSC Genome Browser. For analysis of folded Enrichment of chromatin status (Chromatin State Fold-engineering), chromHMM Overlap Enrichment%http://compbio.mit.edu/ ChromHMM/) Enrichment scores were calculated for genomic features such as pu.1, IRF8 and BATF3 ChIP-seq peaks and histone marks. ChromHMM fragments containing 18 different chromatin states were downloaded from the Roadmap website (http:// www.roadmapepigenomics.org/tools) and used for analysis. The enrichment score is calculated as the ratio between the observed overlap and the predicted overlap for each feature and chromatin state based on its size and the size of the human genome. For the finding from the head motif, the findMotifsGenome.pl program from HOMER was used for PU.1, IRF8 and BATF3, respectively. HOMER was run using default parameters and an input sequence containing +/-100bp from the center of the first 2,500 peaks. Using the findOverlapsOfPeaks function in the ChuppeakAnno R library (http:// www.biomedcentral.com/1471-2105/11/237), a co-binding region was found by PU.1, IRF8 and BATF3. The co-binding region was used for the slave motif discovery using HOMER. To evaluate the similarity of the two sets based on intersection, the inventors calculated the Jaccard statistic using MACRO-APE (https:// opera. Auto-name. Ru/MACRO-APE/compare). To generate the heat and cross-section, deep tools in reference point mode were used, where each feature (e.g., TF or histone labeled peak) was aligned at the pu.1, IRF8 or BATF3 peak top and tiled upstream and downstream flanking regions within ±4 kb.

Co-immunoprecipitation (Co-IP)

Total cell extracts were prepared from HEK293T cells transfected with SFFV-PIB at three cell densities (1, 2, 5 million cells) in IP lysis buffer (Thermo Fisher) supplemented with protease inhibitor [1X Halt Protease Inhibitor Cocktail (Thermo Fisher), 1mM PMSF,5mM NaF ]. The ChIP-grade protein A/G beads were incubated with 5. Mu.g of each antibody (rabbit anti-human PU.1, rabbit anti-human IRF8 or sheep anti-human BATF 3) for 2 hours. Cell lysates were pre-clarified with ChIP grade protein a/G beads without antibody treatment for 1 hour and then incubated with antibody treated beads for 1 hour. The supernatant was removed and the beads were washed 3 times with Tris Buffered Saline (TBST) with 0.1% Tween 20 detergent. An input control was performed using 10% of the non-immunoprecipitated sample (200 ten thousand cell density). As a control, lysates (200 ten thousand cell density) were immunoprecipitated with 5. Mu.g of rabbit IgG isotype (Invitrogen). Samples were eluted by boiling in Laemmli sample buffer and treated for western blotting. For immunoblotting, the membranes were blocked with TBST buffer containing 3% milk, incubated overnight with primary antibody, washed five times with PBS (PBST) with 0.1% Tween 20 detergent, blocked with TBST buffer containing 3% milk for 45 min, incubated with HRP-conjugated secondary antibody for 1 hour, washed four times with PBST, and then detected by ECL (Thermo Scientific) in Chemidoc (Bio-Rad).

Results

PU.1-dominant chromatin targeting ability in cDC1 reprogramming

To elucidate the molecular mechanisms underlying DC reprogramming mediated by pu.1, IRF8 and bat f3, the inventors expressed three reprogramming factors in HDF in combination or individually and performed chromatin immunoprecipitation sequencing (ChIP-seq) at the early stage of reprogramming (48 hours, fig. 10A). First, pu.1 showed the highest chromatin binding (75,593 peaks) when the factors were co-expressed, followed by IRF8 (18,962 peaks) and BATF3 (11,505 peaks) (fig. 10B). Interestingly, >40% of the peaks of pu.1 binding were observed to be similar between individual and combined expression, suggesting that pu.1 has independent targeting ability, which is enhanced when IRF8 and BATF3 are available. In sharp contrast, when these transcription factors were expressed individually, IRF8 and BATF3 peaks were few (< 3% of peaks when compared to combined expression), suggesting that IRF8 and BATF3 need to synergistically bind to pu.1 to engage chromatin and induce a cDC1 fate. The analysis of the slave-head motif prediction for the pu.1 peak showed strong enrichment for the pu.1 motif when expressed individually or in combination (fig. 10C). While individually expressed IRF8 and BATF3 showed enrichment of IRF and AP-1 motifs, respectively, the pu.1 motif was highly enriched for these transcription factors when expressed in combination. These data are consistent with the recent discovery that pu.1 was described as a non-classical pioneer transcription factor that was able to redistribute partner transcription factors in human myeloid and lymphoid cells (Minderjahn et al 2020), and highlights the synergistic kinetics of transcription factors at the early stages of cDC1 reprogramming.

Synergistic binding of PU.1, IRF8 and BATF3 at promoters and enhancers at open chromatin

Next, the inventors studied the overlap between pu.1, IRF8 and bat f3 chromatin targets. When expressed together, 5,383 genomic positions were shared among the three reprogramming factors, representing 28% and 47% of the total IRF8 and BATF3 peaks, respectively (fig. 11A). The slave motif prediction for PIB overlapping peaks showed enrichment for pu.1-IRF and BATF motifs (fig. 11B), which also demonstrated some overlap and similarity (jaccard similarity index=0.02) (fig. 11C). These data suggest that pu.1, IRF8 and bat f3 physically interact. To test this hypothesis, co-immunoprecipitation (co-IP) was performed, and interactions between 3 factors were confirmed (fig. 11D). Next, genes differentially expressed between HDF and hiDC d9 bound by at least one reprogramming factor were plotted and were observed to contain both down-regulated fibroblast genes and up-regulated cDC 1-related genes, including SLAMF8 and TACSTD2 (fig. 11E). To profile whether pu.1, IRF8 and bat f3 binding occurs at the open or closed chromatin region, the inventors utilized publicly available ChIP-seq datasets concerning histone markers in HDF and used chrommmm chromatin fragments (Ernst and Kellis 2012) for visualization. PIB co-binding peaks were observed to be enriched predominantly at the promoter and enhancer regions (fig. 11F). A small fraction (12%) peak associated with bivalent chromatin marked by H3K4me1, H3K4me3 and H3K27me3 or H3K4me1 and H3K27me3 was also observed.

Conclusion(s)

Taken together, our data support a model in which pu.1 binds mainly to active promoters and enhancers located at open chromatin sites, and recruits IRF8 and BATF3 to silence primitive fibroblast genes and gradually applies the cDC1 transcription program (fig. 11G).

Example 13 efficient DC reprogramming of mouse and human cancer cells

Given that ectopic expression of pu.1, IRF8 and bat f3 induces a cDC 1-like fate in mouse and human fibroblasts, the inventors hypothesize that forced expression of the same TF combination in cancer cells converts it into antigen-presenting cDC1 and solves one of the major problems in tumor immunity-loss of antigen presentation mechanism.

Method

Cancer cell reprogramming

The cancer cell line was seeded at a density of 60 000 cells/mL in a 6-well plate and incubated overnight with SFFV-PIB-GFP lentiviral supernatant supplemented with polybrene (8 μg/mL). For the duration of the culture, the medium was changed every 2 days. Cells were plated at 1:6 dilution onto 10cm plates each time the cells reached 80-90% confluence. Flow cytometry was used to evaluate the efficiency of DC reprogramming in mouse and human cancer cells.

T cell priming and antigen cross presentation assay

Initial mouse CD8 use ⁺ T cell isolation kit (Miltenyi) enriched for CD8 from spleen of OT-I mice ⁺ T cells. Labelling of enriched CD8 with CTV according to manufacturer's protocol ⁺ T cells. MACS-sorted reprogrammed cells, non-reprogrammed cancer cells, eGFP-transduced cancer cells and CD103 ⁺ BM-DC was incubated with OVA peptide (SIINFEKL, T cell priming assay) or protein (cross presentation assay) at 37 ℃. Cells expressing OVA were not incubated with exogenous OVA. When indicated, cells were incubated overnight in the presence of poly (I: C) or IFN-gamma. By 5x10 ³ Antigen presenting cells and 1X10 ⁵ OT-I CD8 labeled with CTV ⁺ T cells were incubated together in 96-well round-bottomed untreated tissue culture plates. After 3 days of co-culture, T cells were collected, stained for viability (fixable viability dye eFluor-520, ebioscience), CD8 a, TCR- β and CD44, and analyzed by flow cytometry. By aligning live single TCR-beta ⁺ And CD8 ⁺ T cells were gated to determine T cell proliferation (CTV dilution) and activation (CD 44 expression). With respect to dataThe plotted threshold is fixed at 1,000 events within the living cell gate.

T cell killing assay

According to the manufacturer's protocol, mouse CD8 was used ⁺ T cell isolation kit (Miltenyi) enriched for CD8 from spleen of OT-I mice ⁺ T cells. Untreated 6-well plate for 2X10 ^-3 mg mL ^-1 Is coated at 37℃for 2 hours and washed 3x, then 1x10 ⁶ The individual T cells/mL were inoculated into complete growth medium (RPMI) supplemented with murine IL-2 (Peprotech, 100U mL-1) and murine IL-12p70 (Peprotech, 2.5X10-3 mg mL-1). After 24 hours of activation, T cells were plated at 1x10 ⁶ Individual cells/mL were re-seeded into fresh complete RPMI supplemented with murine IL-2 on fresh untreated plates for 48 hours to allow T cell expansion. MACS-sorted reprogrammed mOrange was performed 24 hours prior to co-culture with T cells ⁺ B16-OVA cells or IFN-gamma treated cells were seeded with an equal number of non-fluorescent B16-OVA (mOrange-). Expanded T cells are added at a ratio of 0:1, 1:1, 5:1, 10:1T cells/target cells. B16 cells that did not express OVA were used to evaluate assay specificity. For flow cytometry analysis, cells were resuspended and stained for viability (DAPI) and anti-CD 3, and measured at indicated time points after co-culture with T cells.

Tumor induction and injection

By mixing 2-5x10 ⁵ Individual tumor cells were subcutaneously injected into the right flank of 6-10 week old C57BL/6 females to establish B16-OVA tumors. Reprogrammed tumor-APCs were generated by transduction of B16 with SFFV-PIB. tumor-APCs were purified by MACS with anti-MHC-II antibodies at day 5 post transduction and at days 7, 10 and 13 post tumor establishment, and 2x10 ⁵ -3x10 ⁵ Individual cells were resuspended in 100 μl PBS and intratumoral injection was performed. PBS or cells transduced with control lentiviruses were injected into the tumor as controls. Tumor cells or tumor-APCs were stimulated with poly (I: C) and loaded with OVA 24 hours prior to injection. Mice were followed for survival and tumor size was measured with calipers every 2 days after tumor establishment [ volume = pi/6 x Lx WxH]Until the end point. When swellingMice were sacrificed when tumor volume exceeded 1500mm 3.

Results

First, SFFV-PIB-IRES-GFP lentiviral supernatants were used to overexpress PIB in 3LL and B16, murine lung adenocarcinoma, and melanoma cells, respectively. Both murine cancer cell lines were derived from the C57BL/6 background and were widely used in syngeneic mouse models for tumor immunization. Appearance of the double positive population with respect to MHC-II and CD45 was observed 9 days after transduction (fig. 12A). Recent CRISPR screening methods have highlighted the importance of IFN- γ signaling in unlocking anti-tumor immunity and Cytotoxic T Lymphocyte (CTL) sensitivity. Interestingly, both IFN and STING pathway gene signatures were up-regulated in both B16 and LLC cells transduced with PIB (fig. 12B), suggesting an immunogenic profile obtained during reprogramming. To further investigate whether tumor-APCs became immunogenic and presented endogenously expressed antigens, the inventors utilized a B16 cell line (B16-OVA) expressing Ovalbumin (OVA). Magnetically Activated Cell Sorting (MACS) enriched expression CD45 ⁺ MHC-II ⁺ tumor-APC and initial OT-I CD8 of OVA ⁺ T cells were co-cultured to evaluate priming. Although control eGFP transduced B16-OVA and LLC-OVA cells showed low OVA antigen presenting capacity after IFN-gamma or P (I: C) stimulation, tumor-APCs were responsible for priming of the original OT-I CD8 ⁺ T cells were significantly effective, independent of IFN-. Gamma.or P (I: C) treatment (FIG. 12C). Next, to evaluate whether tumor-APCs became prone to CTL killing, B16-OVA cells expressed the fluorescent protein mOrange. Generating tumor-APCs, or B16-OVA cells treated with IFN-gamma (target ⁺ ) Mix with untreated B16-OVA cells (non-target), with concomitant increased rates of activated OT-I CD8 ⁺ T cells were co-cultured for 3 days. First, tumor-APCs were observed to be more susceptible to passage through CD8 than untreated B16-OVA cells ⁺ T cell killing effect (fig. 12D). In addition, tumor-APCs were killed more effectively by T cells (42.42.+ -. 6.2%), especially at low ratios (1:1), compared to IFN-gamma stimulated B16-OVA cells (12.31.+ -. 7.1%). Interestingly, in tumor-APC co-cultures, non-linearities were also observed at higher T cell/target cell ratios and later time points (72 hours)Killing of the target population. This bystander killing effect may reflect the continued activation of T cells by reprogrammed cells, increasing clearance of non-target cancer cells. Next, the inventors assessed tumor-APC cross presentation after pulsing with OVA protein. Remarkably, it was observed that tumor-APC established cross-presentation of antigen to CD8 ⁺ The capacity of T cells, which was further enhanced by TLR3 stimulation (63.5±8.5 versus 27.5±20.9%) (fig. 12E). The inventors then examined whether OVA-loaded tumor-APCs induced tumor growth control in vivo following intratumoral injection in established B16-OVA tumors (fig. 12F). Notably, injection of tumor-APCs resulted in significantly reduced tumor growth and improved survival when compared to mice injected with PBS or control virus (fig. 12G-H).

Then, to assess whether the ctc 1 fate can be induced directly in human cancer cells, the expression of pu.1, IRF8 and BATF3 was forced in a group of subjects of 28 human cancer cell lines. After 9 days, EGFP was evaluated as a transducer for simultaneous expression of CD45 and HLA-DR ⁺ The efficiency of reprogramming in percent of cells reflects hematopoietic typing and antigen presenting capacity (FIGS. 13A-B).

Reprogramming CD45 was observed in all cell lines transduced with hPIB-IRES-EGFP ⁺ HLA-DR ⁺ The appearance of cells, but not observed in the EGFP-transduced control, suggested that dcs 1 were suitable for use with human cancer cells, cheng Anfan. Furthermore, these data show that the efficiency of cDC1 reprogramming ranges from 0.2±0.1% to 94.5±7.6% across cancer cell lines, independent of transduction levels and proliferation rates. Interestingly, despite the low reprogramming efficiency in cancer cell lines from lung and breast cancers, large cell populations were detected that obtained CD45 or HLA-DR expression, which may represent partially reprogrammed cells that have obtained dendritic cell characteristics (fig. 13A-B). Human cancer cell derived CD45 ⁺ HLA-DR ⁺ Cells expressed cDC1 surface markers including CLEC9A (59.1±3.6%), CD226 (67.5±1.8%) and CD11C (54.4±3.6%) (fig. 13C). Surface expression of CD40, CD80 and CD86 was evaluated in view of the expression of costimulatory molecules required for productive activation of primary T cells. Human cancer cell derived CD45 ⁺ HLA-DR ⁺ Cells expressed these costimulatory molecules starting on day 4 and gradually increasing until day 9. Importantly, tumor-APCs responded to TLR3/4 triggering (LPS and poly I: C resulted in increased surface expression of costimulatory molecules, particularly CD40 (88.2.+ -. 3.8% versus 31.3.+ -. 1.8%) (FIG. 13D, E).

An important consideration for the conversion of tumor-APCs into therapy is whether reprogramming can be initiated in human primary cancer cells (fig. 13f, g). To verify cDC1 reprogramming in primary cancer cells, 17 samples were collected from 7 different tumors and lung cancer-associated fibroblasts (CAF) from melanoma, lung cancer, tonsil cancer, tongue cancer, pancreatic cancer, breast cancer and PDX-derived bladder cancer patients. After transduction with hPIB, all primary cancer cells showed major phenotypic changes, initiating expression of CD45 and HLA-DR, reflecting reprogramming. Reprogramming efficiency ranges from 0.6% ± 0.4 to 47.0% ± 2.0. Samples from the same tumor type showed similar phenotype profiles, indicating relatively low variability across patients. Interestingly, primary cells were less resistant to reprogramming when compared to the cell line subject group, as shown by lung cancer cell lines (0.5% ± 0.1) and primary cells (47.0% ± 2.0). These data suggest that epigenetic disorders that limit reprogramming are less mandatory in primary cells, opening up a pathway for the broad applicability of cDC1 reprogramming to human tumors (fig. 13f, g).

Conclusion(s)

These data support the following assumptions: pu.1, IRF8 and BATF3 can reprogram mouse and human cancer cells into cDC 1-like cells, which can present tumor antigens and induce anti-tumor immune responses. The inventors also verified that reprogramming to dcs 1 is conserved across species and tissues and is viable in primary cancer cells from patients.

Example 14. Epigenetic modifiers enhance the efficiency of cDC1 reprogramming.

Given that ectopic expression of pu.1, IRF8 and bat f3 induces a cDC 1-like fate in unrelated cell types by forced transcription and epigenetic changes, the inventors hypothesize that epigenetic modifiers, i.e., histone deacetylase inhibition, can enhance the efficiency of cDC1 reprogramming.

Method

ATAC-seq library preparation

To study epigenetic changes induced by reprogramming, 5,000-10,000 cells were isolated from a defined population via FACS and processed to prepare a sequencing library. Quality was assessed using High Sensitivity DNA Chip (Agilent Technologies) and the library was sequenced using the NextSeq 500/550High Output Kit (150 cycles) on NextSeq500 (Illumina).

ATAC-seq data analysis

A total of 1,384,592,926 ATAC-seq readings were obtained with a median sample coverage of approximately 4670 tens of thousands of readings. To remove Illumina universal adaptors, NGmerge61 is used by setting the adaptor removal mode. Reads were mapped to GRCh38 reference genome using HISAT2 v2.0.462 with the following parameters: -ver-active-k 20. Peak calls were performed with Genrich (v0.6.1, available at https:// gitsub.com/jsh 58/Genrich, parameters: -m 30-j-y-r-echrM), respectively, for each sample. A list of combined peaks for all samples was obtained by using PEPATACr R library. Finally, the count of readings on the combined peak list was calculated with bedtools multicov 56. The resulting read counts were processed using R-pack DESeq252 and normalized using RLE method. PCA was performed using the plotdac function from DESeq2 packets. For peak annotation, the ChIPseeker R library 64 was used. To map common chromatin changes, a modification procedure on ATAC-seq data as described for tumor-APC gene expression profiles was used. Briefly, for each peak associated with an individual gene from a tumor-APC signature, the average difference between day 9 and day 0 was calculated and normalized to the difference between day 0 and cDC1 for the individual phenotype/reprogramming time point. After this, the inventors separately acquired the median of the normalized peaks for each phenotype/time point of the reprogramming and plotted. For genomic trajectories, the bigwig file was created from the bam file with deeptols. Genome trajectories were explored using the WashU Epigenome browser. For motif discovery, the findMotifsGenome. Pl program with default parameters from HOMER67 was used for the differential ATAC-seq peak. Functional enrichment analysis for the differential ATAC-seq peaks was performed by Great software 68 using GO bioprocess ontology.

Evaluation of the Effect of epigenetic modulators in reprogramming efficiency

Transduction of cancer cell lines with PIB-IRES-EGFP lentiviral particles or EGFP as control and cultivation in the presence or absence of VPA from day 1 to day 4 of reprogramming and according to CD45 ⁺ And MHC-II or HLA-DR, in a living EGFP ⁺ On day 9 of reprogramming in cells, the reprogramming efficiency was quantified by flow cytometry. The reprogrammed cells were then analyzed as previously described.

Results

To map the kinetics of reprogramming of human cancer cells at transcriptional and epigenetic levels, mRNA sequencing and transposase accessible chromatin (ATAC) sequencing were used, reprogramming along a time course (CD 45 ⁺ HLA-DR ⁺ ) And partially reprogrammed (CD 45-HLA-DR ⁺ ) T98G cells were profiled (fig. 14A). PCA isolated all reprogramming phases (days 3, 5, 7, and 9) from the parental cells, with day 7 and day 9 mapping closest to native cDC1, indicating a stepwise acquisition of the cDC1 transcription program (fig. 14B). It is thought that partially reprogrammed cells lag in time, supporting the notion that these cells are being successfully reprogrammed en route. Interestingly, reprogramming of Human Embryonic Fibroblasts (HEF) followed a similar reprogramming trajectory (fig. 14B), indicating that the reprogramming dynamics were conserved across malignant and non-cancerous primary cells. PCA for differential open chromatin regions demonstrated that epigenetic remodeling occurred rapidly with significant changes (62% variance) between day 0 and day 3, followed by fine-tuning at later time points (days 3, 5, 7, 9) bringing the cells closer to the open chromatin pattern of cDC1 (FIG. 14B). To confirm these observations, we utilized tumor-APC gene characteristics and plotted the changes along the time course. In fact, the features are gradually applied at the transcriptional level and rapidly established at the chromatin level (fig. 14C). These data show hPIB-mediated reprogramming The program initiates rapid epigenetic remodeling followed by gradual rerouting of the cDC1 transcription program.

To test whether the cDC1 reprogramming efficiency was limited by epigenetic disorders, B16 and LLC cells were treated with valproic acid (VPA) and the reprogramming efficiency was evaluated on day 9. VPA treatment to CD45 ⁺ MHC-II ⁺ tumor-APC production was enhanced by-3 fold in LLC cells (45.9±25.5% versus 15.8±4.79%) and by-5 fold in B16 cells (29.9±19.3% versus 5.9±4.8%) (fig. 15A-B). The inventors also confirmed that transduced cells up-regulated MHC-I in the presence of VPA (fig. 15C), indicating that a larger cancer cell population became immunogenic. Functionally, tumor-APCs produced in the presence of VPA move towards OT-I CD8 ⁺ T cells present endogenous antigen (fig. 15D), become targets for T cell mediated cytotoxicity (fig. 15E), and elicit primary CD8 after incubation with exogenous antigen ⁺ T cells (fig. 15F). Next, the effect of VPA treatment in cDC1 reprogramming of human cancer cells was studied. VPA treatment was observed to increase reprogramming Cheng Xiaolv in all tested lines (fig. 15G).

Conclusion(s)

This data indicates that reprogramming of cancer cells can be enhanced by promoting chromatin accessibility during the ctc 1 reprogramming process.

EXAMPLE 14 role of SPIB and SPIC compensating PU.1 in cDC1 reprogramming

The human genome encodes nearly 2000 different transcription factors organized in multiple families and subfamilies. Transcription factors sharing significant homology are typically included in the same family/subfamily of transcription factors. Under certain conditions, a transcription factor may compensate for the lack of a particular transcription factor from the same family or subfamily. In this regard, the inventors hypothesized that homologs from pu.1, IRF8 and bat f3 can compensate for their role in cDC1 reprogramming. As a proof of concept, the ability of SPIB and SPIC (two pu.1 homologs) to replace pu.1 in cDC1 reprogramming was tested.

Method

The coding regions of SPIB and SPIC were cloned into pFUW-TetO plasmids, respectively. Lentiviral particles (pFUW-UbC-M2 rtTA) encoding each individual transcription factor or reverse tetracycline transactivator M2rtTA under the control of a constitutively active human ubiquitin C promoter were used for co-transduction (Rosa et al 2018). Reprogramming efficiency was assessed by flow cytometry in Clec9 a-tdmamato Mouse Embryonic Fibroblasts (MEFs) 9 days after transcription factor overexpression.

Results

The inventors observed that both SPIB and SPIC can replace pu.1 in the context of cDC1 reprogramming (fig. 16A). Importantly, SPIB and SPIC alone are unable to activate DC-specific reporter genes in transduced MEFs. Interestingly, SPIB induced a greater degree of reporter activation than pu.1 or SPIC, exhibiting about 8.14±1.16% tdmamato ⁺ Cells, whereas pu.1 and SPIC only exhibited 2.87±0.18% and 1.46±0.73%, respectively.

Next, tdTomato was analyzed ⁺ CD45 and MHC-II expression in cells. Remarkably, SPIB showed tdTomato coexpressing CD45 and MHC-II compared to PU.1 (17.15.+ -. 2.04%) ⁺ A 2-fold increase in cells (33.63 ±3.76%) (fig. 16B).

Conclusion(s)

These data suggest that SPIB and SPIC can compensate for the role of pu.1 in cDC1 reprogramming.

Example 15. Delivery of PU.1, IRF8 and BATF3 mediated by adenoviruses and adeno-associated viruses allows cDC1 reprogramming of healthy and cancerous cells.

Cell reprogramming strategies based on overexpression of cell type-specific transcription factors have traditionally relied on the use of retroviral or lentiviral vectors. However, the integrative nature of these viral vectors has raised safety concerns with respect to clinical use. The use of non-integrated viral systems is a good alternative to delivering transcription factors to target cells for therapeutic applications, bypassing these safety concerns. Here, the inventors hypothesized that pu.1, IRF8 and BATF3 delivery mediated by non-integrating adenoviruses and adeno-associated viruses (AAV) allows cDC1 reprogramming in unrelated cell types.

Method

Cell reprogramming

Mouse embryo fibroblasts isolated from Clec9a-tdTomato report mice, B2905 mouse melanoma cell line, IGR-39 melanoma and T98G glioblastoma human cell line, and 2778 human primary melanoma cells were seeded at a well density of 12 500 cells/12 well plate and incubated overnight with lentiviruses encoding PIB-GFP or GFP alone (Lenti), adenovirus (Ad 5 or Ad 5/F35) or AAV (AAV-DJ or AAV 2-QuadYF) using a multiplicity of infection of 50,000 RNA copies/cell, 5,000 infectious units/cell and 250,0000 genome copies/cell, respectively. When cells were incubated with lentivirus, the medium was supplemented with polybrene (8. Mu.g/mL). For the duration of the culture, the medium was changed every 2 days. In vivo GFP based on surface expression of CD45 and MHC-II or HLA-DR ⁺ On day 9 of reprogramming in cells, the efficiency of cDC1 reprogramming was quantified by flow cytometry.

Results

The inventors observed that adenovirus encoding PIB-GFP and AAV were able to induce activation of Clec9 a-tdbitmap reporter gene (fig. 17A) and surface expression of CD45 and MHC-II in mouse embryo fibroblasts (fig. 17B). As expected, tdmamto expression was not observed in cells transduced with GFP encoding viral vectors. Next, the inventors examined whether adenovirus and AAV vectors encoding PIB-GFP could reprogram mouse and human cancer cells. Surface expression of CD45 and MHC-II in B2905 mouse melanoma cancer cells (fig. 17C), and surface expression of CD45 and HLA-DR in human cancer cell lines (IGR-39 and T98G) and human primary melanoma cells 2778 (fig. 17D) were observed.

Conclusion(s)

These data suggest that delivery through adenovirus and AAV mediated pu.1, IRF8 and BATF3 allows cDC1 reprogramming in healthy as well as cancerous, mouse and human cell types.

Sequence overview

Description of SEQ ID NO.

1 SFFV (spleen focus forming virus) promoter polynucleotide sequence

2 MND (myeloproliferative sarcoma virus enhancer, deletion of negative control region, substitution of dl587rev primer binding site) promoter polynucleotide sequence

3 CAG (CMV early enhancer/chicken beta actin) promoter polynucleotide sequence

4. Cytomegalovirus (CMV) promoter polynucleotide sequence

5. Ubiquitin C (UbC) promoter polynucleotide sequence

6 EF-1alpha (EF-1 alpha) promoter polynucleotide sequence

7 EF-1alpha short (EF 1S) promoter polynucleotide sequence

8. EF-1alpha (EF 1 i) promoter polynucleotide sequence with intron

9. Phosphoglycerate kinase (PGK) promoter polynucleotide sequence

10. Human BATF3 (polypeptide sequences)

11. Human IRF8 isoform 1 (polypeptide sequence)

12. Human PU.1 isoform 1 (polypeptide sequence)

13. Human CCAAT/enhancer binding protein alpha (CEBP alpha) (polypeptide sequence)

14. Human BATF3 (Polynucleotide sequence)

15. Human IRF8 (Polynucleotide sequence)

16. Human PU.1 (Polynucleotide sequence)

17. Human CCAAT/enhancer binding protein alpha (CEBPA) (Polynucleotide sequence)

18. Human BATF (Polynucleotide sequence)

19. Human BATF (polypeptide sequences)

20. Human IRF7 (Polynucleotide sequence)

21. Human IRF7 (polypeptide sequence)

22. Human SPIB (Polynucleotide sequence)

23. Human SPIB (polypeptide sequence)

24. Human SPIC (Polynucleotide sequence)

25. Human SPIC (polypeptide sequence)

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Project

1. A composition comprising one or more constructs or vectors which, upon expression, encode the following transcription factors:

i) BATF3 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO:10 (BATF 3), e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100% identity to SEQ ID NO:10 (BATF 3);

ii) IRF8 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity with SEQ ID NO. 11 (IRF 8), such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID NO. 11 (IRF 8); and

iii) Pu.1 or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity to SEQ ID No. 12 (pu.1), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 12 (pu.1);

2. The composition according to item 1, further comprising one or more constructs or vectors that, upon expression, encode one or more transcription factors selected from the group consisting of:

a) IRF7 or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 21 (IRF 7), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 21 (IRF 7);

b) A BATF or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID NO:19 (BATF), e.g., at least 75%, e.g., at least 80%, e.g., at least 85%, e.g., at least 90%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, e.g., 100% identity to SEQ ID NO:19 (BATF);

c) SPIB or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity with SEQ ID No. 23 (SPIB), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID No. 23 (SPIB);

d) SPIC or a biologically active variant thereof, wherein said biologically active variant has at least 70% identity to SEQ ID No. 25 (SPIC), e.g. at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 25 (SPIC);

3. The composition according to any one of items 1 or 2, wherein:

a) The SFFV promoter comprises or consists of a polynucleotide sequence having at least 80% identity to SEQ ID No. 1, e.g. having at least 85%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% identity to SEQ ID No. 1;

b) The MND promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 2, e.g. having at least 85%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% identity with SEQ ID NO. 2;

c) The CAG promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 3, for example at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID NO. 3;

d) The CMV promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 4, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identity with SEQ ID NO. 4;

e) The UbC promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID No. 5, e.g. having at least 85%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% identity with SEQ ID No. 5;

f) The EF-1 alpha promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 6, for example having at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identity with SEQ ID NO. 6;

g) The EF1S promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 7, for example having at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identity with SEQ ID NO. 7;

h) The EF1i promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID NO. 8, for example having at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identity with SEQ ID NO. 8; and

i) The PGK promoter comprises or consists of a polynucleotide sequence having at least 80% identity with SEQ ID No. 9, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identity with SEQ ID No. 9.

4. The composition according to any of the preceding items, wherein the composition comprises:

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; and a third construct or vector encoding the transcription factor pu.1 after expression;

and/or

5. The composition according to any of the preceding items, wherein the composition comprises:

and/or

6. the composition according to any of the preceding items, wherein the composition comprises:

And/or

7. a composition according to any of the preceding items, wherein the BATF3 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 14, e.g. having at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 14.

8. A composition according to any of the preceding items, wherein IRF8 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 15, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity with SEQ ID No. 15.

9. A composition according to any of the preceding items, wherein pu.1 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 16, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity with SEQ ID No. 16.

10. A composition according to any of the preceding items, wherein the bat is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 18, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity with SEQ ID No. 18.

11. A composition according to any of the preceding items, wherein IRF7 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 20, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity with SEQ ID No. 20.

12. A composition according to any of the preceding items, wherein the SPIB is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 22, e.g. having at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 22.

13. A composition according to any of the preceding items, wherein the SPIC is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 24, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity with SEQ ID No. 24.

14. The composition according to any of the preceding items, wherein the one or more constructs or vectors further encode a transcription factor CCAAT/enhancer binding protein a (cebpa) or a biologically active variant thereof after expression, wherein the biologically active variant has at least 70% identity with SEQ ID NO:13 (cebpa), e.g. at least 75%, e.g. at least 80%, e.g. at least 85%, e.g. at least 90%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% identity with SEQ ID NO: 13.

15. A composition according to any of the preceding items, wherein CEBP a is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID No. 17, e.g. having at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98%, e.g. at least 99%, e.g. 100% sequence identity to SEQ ID No. 17.

16. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a self-cleaving peptide operably linked to at least two of the at least three coding regions, thereby forming a single open reading frame.

17. The composition according to item 16, wherein the self-cleaving peptide is a 2A peptide.

18. The composition according to item 17, wherein the 2A peptide is selected from the group consisting of equine rhinitis virus (E2A), foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), and Leptospira venomous (T2A) peptides.

19. The composition according to any one of the preceding items, wherein the one or more constructs or vectors are viral vectors.

20. The composition according to any one of the preceding items, wherein the one or more constructs or vectors are viral vectors selected from the group consisting of: lentiviral vectors, retroviral vectors, adenoviral vectors, herpesviral vectors, poxviral vectors, adeno-associated viral vectors, paramyxoviridae vectors, rhabdoviral vectors, alphaviral vectors and flaviviral vectors.

21. A composition according to item 20, wherein said viral vector is a lentiviral vector

22. The composition according to item 20, wherein the adenovirus vector is selected from the group consisting of: wild-type Ad vectors, hybrid Ad vectors, and mutant Ad vectors.

23. The composition according to clause 22, wherein the wild-type Ad vector is Ad5 and wherein the hybrid Ad vector is Ad5/F35.

24. The composition according to item 20, wherein the adeno-associated viral vector is selected from the group consisting of: wild-type AAV vectors, hybrid AAV vectors, and mutant AAV vectors.

25. The composition according to item 24, wherein the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV 2-quadryf.

26. The composition according to any one of the preceding items, wherein the one or more constructs or vectors are plasmids.

27. The composition according to any of the preceding items, wherein the backbone of the one or more constructs or vectors is selected from the group consisting of: FUW, pRRL-cPPT, pRLL, pCCL, pCLL, pHAGE2, pWPXL, pLKO, pHIV, pLL, pCDH and pLenti.

28. The composition according to any of the preceding items, wherein the one or more constructs or vectors further comprise a post-transcriptional regulatory element (PRE) sequence.

29. The composition according to item 28, wherein the PRE sequence is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

30. The composition according to any one of the preceding items, wherein the one or more constructs or vectors further comprise a central polypurine tract (cPPT).

31. The composition according to any one of the preceding items, wherein the one or more constructs or vectors further comprise 5 'and 3' terminal repeats.

32. The composition of item 31, wherein at least one of the 5' and 3' terminal repeats is a lentiviral long terminal repeat or a self-inactivating (SIN) design of U3 with partial deletion of the 3' long terminal repeat.

33. The composition according to any one of the preceding items, wherein the one or more constructs or vectors further comprise a nucleocapsid protein packaging target site.

34. The composition according to item 33, wherein the protein packaging target site comprises an HIV-1psi sequence.

35. The composition according to any one of the preceding items, wherein the one or more constructs or vectors further comprise a REV protein responsive element (RRE).

36. The composition according to any one of the preceding items, further comprising one or more cytokines selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM).

37. The composition according to any of the preceding items, further comprising one or more epigenetic modifiers, such as histone deacetylase inhibitors.

38. The composition according to item 37, wherein the one or more histone deacetylase inhibitors is valproic acid.

39. The composition according to any one of the preceding items, wherein the composition is a pharmaceutical composition.

40. A cell comprising one or more constructs or vectors according to any of the preceding items.

41. The cell according to item 40, wherein the cell is a mammalian cell, e.g., a human or murine cell.

42. The cell according to any one of items 40 to 41, wherein the cell is selected from the group consisting of: stem cells, differentiated cells, and cancer cells, wherein:

a) The stem cells are selected from the following: pluripotent stem cells and multipotent stem cells, such as mesenchymal stem cells or hematopoietic stem cells;

b) The differentiated cell is any somatic cell, such as a fibroblast or a hematopoietic cell, such as a monocyte.

43. A cell according to any one of clauses 40 to 42, wherein the cell is a reprogrammed human dendritic cell or an antigen presenting cell, such as a human type 1 classical dendritic cell.

44. The cell according to any one of items 40 to 43, wherein the cell further expresses one or more surface markers selected from the surface markers of table 1.

45. The cell according to any one of items 40 to 44, wherein the cell is positive for one or more surface markers listed in table 1.

46. The cell according to any one of items 40 to 45, wherein the cell is CD226 positive.

47. A method of reprogramming or inducing cells into dendritic cells or antigen presenting cells, the method comprising the steps of:

c) Transducing a cell with a composition comprising a construct or vector according to any one of items 1 to 32.

d) Expressing the transcription factor;

thereby obtaining reprogrammed or induced cells.

48. The method according to item 47, wherein said reprogramming or induction is in vivo, in vitro, or ex vivo.

49. The method according to any one of clauses 47 to 48, wherein the method further comprises the step of culturing the transduced cells in a cell culture medium, wherein the step is performed before or after the expression of the transcription factor.

50. The method according to any one of clauses 47 to 49, wherein the method further comprises culturing the transduced cells in a cell culture medium comprising one or more cytokines selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM).

51. The method according to any one of clauses 47 to 50, wherein the method further comprises culturing the transduced cells in a cell culture medium comprising one or more epigenetic modifiers, such as histone deacetylase inhibitors.

52. The method according to clause 51, wherein the histone deacetylase inhibitor is valproic acid.

53. The method according to any one of clauses 47 to 52, wherein the cell is a mammalian cell, e.g., a human or murine cell.

54. The method according to any one of items 47 to 53, wherein the cells are selected from the group consisting of: stem cells, differentiated cells, and cancer cells, wherein:

e) The stem cells are selected from the following: pluripotent stem cells and multipotent stem cells, such as mesenchymal stem cells or hematopoietic stem cells;

f) The differentiated cell is any somatic cell, such as a fibroblast or a hematopoietic cell, such as a monocyte.

55. The method according to any one of items 47 to 54, wherein the transduced cells are cultured for a period of at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

56. The method according to any one of clauses 47 to 55, wherein the resulting reprogrammed or induced cell is a type 1 classical dendritic cell.

57. The method according to any one of clauses 47 to 56, wherein the resulting reprogrammed or induced cell is cluster of differentiation 45 (CD 45) positive.

58. A method according to any one of clauses 47 to 57, wherein the resulting reprogrammed or induced cell is X-C motif chemokine receptor 1 (XCR 1) positive.

59. The method according to any one of clauses 47 to 58, wherein the resulting reprogrammed or induced cell is cluster of differentiation 226 (CD 226) positive.

60. The method according to any one of clauses 47 to 59, wherein the resulting reprogrammed or induced cell is positive for the human leukocyte antigen-DR isoform (HLA-DR).

61. A reprogrammed or induced cell obtained according to the method defined in any one of items 47 to 60.

62. A reprogrammed or induced cell according to item 61, wherein said cell is a dendritic cell or an antigen presenting cell, such as a type 1 classical dendritic cell.

63. The reprogrammed or induced cell according to any of items 61 to 62, wherein the resulting reprogrammed or induced cell is positive for one or more surface markers listed in table 1.

64. The reprogrammed or induced cell according to any of items 61 to 63, wherein the resulting reprogrammed or induced cell is CD45, HLA-DR, CD141, CLEC9A, XCR1 and/or CD226 positive.

65. A composition according to any one of items 1 to 39, a cell according to any one of items 40 to 46, and/or a reprogrammed or induced cell according to any one of items 61 to 64 for use in medicine.

66. A composition according to any one of items 1 to 39, a cell according to any one of items 40 to 46, and/or a reprogrammed or induced cell according to any one of items 61 to 64 for use in the treatment of cancer or infectious disease.

67. The composition, cell and/or reprogrammed cell of clause 66, wherein said cancer is selected from the group consisting of: basal cell carcinoma, cervical atypical hyperplasia, sarcoma, germ cell tumor, retinoblastoma, glioblastoma, lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hematological cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, uterine cancer, vaginal cancer, breast cancer, head and neck cancer, gastric cancer, oral cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, pleural cancer, bladder and urothelial cancer, hollow organ cancer, esophageal cancer, gastric cancer, cholangiocarcinoma, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureteral cancer, renal cancer, liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, skin cancer, melanoma and myeloma, preferably wherein the cancer is selected from the group consisting of: melanoma, head and neck cancer, breast cancer, colorectal cancer, liver cancer, lymphoma, bladder cancer and urothelial cancer, pancreatic cancer and glioblastoma.

68. A method of treating cancer or an infectious disease, the method comprising administering to an individual in need thereof a composition according to any one of items 1 to 38, cells according to any one of items 40 to 46, a pharmaceutical composition according to item 39, and/or reprogrammed or induced cells according to any one of items 61 to 64.

69. Use of a composition according to any one of items 1 to 38, a cell according to any one of items 40 to 46, a pharmaceutical composition according to item 39, and/or a reprogrammed or induced cell according to any one of items 61 to 64 for the manufacture of a medicament for the treatment of cancer or infectious disease.

Sequence listing

<110> Alasgard treatment Co., ltd

<120> human induced dendritic cell type 1 (hiDC 1)

<130> P5481PC00

<160> 25

<170> PatentIn version 3.5

<210> 1

<211> 406

<212> DNA

<213> artificial

<220>

<223> SFFV (spleen focus forming virus) promoter sequence

<220>

<221> misc_feature

<223> SFFV (spleen focus forming virus) promoter sequence

<400> 1

gtaacgccat tttgcaaggc atggaaaaat accaaaccaa gaatagagaa gttcagatca 60

agggcgggta catgaaaata gctaacgttg ggccaaacag gatatctgcg gtgagcagtt 120

tcggccccgg cccggggcca agaacagatg gtcaccgcag tttcggcccc ggcccgaggc 180

caagaacaga tggtccccag atatggccca accctcagca gtttcttaag acccatcaga 240

tgtttccagg ctcccccaag gacctgaaat gaccctgcgc cttatttgaa ttaaccaatc 300

agcctgcttc tcgcttctgt tcgcgcgctt ctgcttcccg agctctataa aagagctcac 360

aacccctcac tcggcgcgcc agtcctccga cagactgagt cggccg 406

<210> 2

<211> 596

<212> DNA

<213> artificial

<220>

<223> MND (myeloproliferative sarcoma virus enhancer, deletion of negative control region, substitution of dl587rev primer binding site)

Promoter sequence

<220>

<221> misc_feature

Promoter sequence

<400> 2

cgggtttatt acagggacag cagagatcca gtttgggaat tagcttgatc gattagtcca 60

atttgttaaa gacaggatat cagtggtcca ggctctagtt ttgactcaac aatatcacca 120

gctgaagcct atagagtacg agccatagat agaataaaag attttattta gtctccagaa 180

aaagggggga atgaaagacc ccacctgtag gtttggcaag ctaggatcaa ggttaggaac 240

agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc 300

tcagggccaa gaacagttgg aacagcagaa tatgggccaa acaggatatc tgtggtaagc 360

agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc ccgccctcag 420

cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt 480

gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc 540

cgagctcaat aaaagagccc acaacccctc actcggcgcg atctagatct cgaatc 596

<210> 3

<211> 584

<212> DNA

<213> artificial

<220>

<223> CAG (CMV early enhancer/chicken beta actin) promoter sequence

<220>

<221> misc_feature

<223> CAG (CMV early enhancer/chicken beta actin) promoter sequence

<400> 3

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 60

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 120

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 180

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 240

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 300

ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 360

ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 420

ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 480

gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 540

ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcg 584

<210> 4

<211> 508

<212> DNA

<213> artificial

<220>

<223> Cytomegalovirus (CMV) promoter sequence

<220>

<221> misc_feature

<223> Cytomegalovirus (CMV) promoter sequence

<400> 4

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480

acggtgggag gtctatataa gcagagct 508

<210> 5

<211> 1212

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<223> ubiquitin C (UbC) promoter sequence

<400> 5

ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg 60

ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag 120

cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag 180

gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg 240

aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat 300

gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt 360

cgcggttctt gtttgtggat cgctgtgatc gtcacttggt gagtagcggg ctgctgggct 420

ggccggggct ttcgtggccg ccgggccgct cggtgggacg gaagcgtgtg gagagaccgc 480

caagggctgt agtctgggtc cgcgagcaag gttgccctga actgggggtt ggggggagcg 540

cagcaaaatg gcggctgttc ccgagtcttg aatggaagac gcttgtgagg cgggctgtga 600

ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga acccaaggtc ttgaggcctt 660

cgctaatgcg ggaaagctct tattcgggtg agatgggctg gggcaccatc tggggaccct 720

gacgtgaagt ttgtcactga ctggagaact cggtttgtcg tctgttgcgg gggcggcagt 780

tatggcggtg ccgttgggca gtgcacccgt acctttggga gcgcgcgccc tcgtcgtgtc 840

gtgacgtcac ccgttctgtt ggcttataat gcagggtggg gccacctgcc ggtaggtgtg 900

cggtaggctt ttctccgtcg caggacgcag ggttcgggcc tagggtaggc tctcctgaat 960

cgacaggcgc cggacctctg gtgaggggag ggataagtga ggcgtcagtt tctttggtcg 1020

gttttatgta cctatcttct taagtagctg aagctccggt tttgaactat gcgctcgggg 1080

ttggcgagtg tgttttgtga agttttttag gcaccttttg aaatgtaatc atttgggtca 1140

atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta aattctggcc gtttttggct 1200

tttttgttag ac 1212

<210> 6

<211> 231

<212> DNA

<213> artificial

<220>

<223> EF-1 alpha (EF-1 alpha) promoter sequence

<220>

<221> misc_feature

<223> EF-1 alpha (EF-1 alpha) promoter sequence

<400> 6

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca g 231

<210> 7

<211> 212

<212> DNA

<213> artificial

<220>

<223> EF-1 alpha short (EF 1S) promoter sequence

<220>

<221> misc_feature

<223> EF-1 alpha short (EF 1S) promoter sequence

<400> 7

gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa 60

ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120

gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180

tttttcgcaa cgggtttgcc gccagaacac ag 212

<210> 8

<211> 1179

<212> DNA

<213> artificial

<220>

<223> EF-1 alpha (EF 1 i) promoter sequence with intron

<220>

<221> misc_feature

<223> EF-1 alpha (EF 1 i) promoter sequence with intron

<400> 8

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg 360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg 420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct 480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg 540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc 600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga 660

gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct 720

ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca 780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg 840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg 900

tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat 960

tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg 1020

gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa 1080

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca 1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1179

<210> 9

<211> 505

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> phosphoglycerate kinase (PGK) promoter sequence

<400> 9

gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt 60

ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc 120

agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg 180

cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc 240

gcacgtctca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga 300

ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg 360

gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg 420

cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac 480

cgaatcaccg acctctctcc ccagg 505

<210> 10

<211> 126

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> human BATF3 (protein sequence)

<400> 10

Met Ser Gln Gly Leu Pro Ala Ala Gly Ser Val Leu Gln Arg Ser Val

1 5 10 15

Ala Ala Pro Gly Asn Gln Pro Gln Pro Gln Pro Gln Gln Gln Ser Pro

20 25 30

Glu Asp Asp Asp Arg Lys Val Arg Arg Arg Glu Lys Asn Arg Val Ala

35 40 45

Ala Gln Arg Ser Arg Lys Gln Thr Gln Lys Ala Asp Lys Leu His Glu

50 55 60

Glu Tyr Glu Ser Leu Glu Gln Glu Asn Thr Met Leu Arg Arg Glu Ile

65 70 75 80

Gly Lys Leu Thr Glu Glu Leu Lys His Leu Thr Glu Ala Leu Lys Glu

85 90 95

His Glu Lys Met Cys Pro Leu Leu Leu Cys Pro Met Asn Phe Val Pro

100 105 110

Val Pro Pro Arg Pro Asp Pro Val Ala Gly Cys Leu Pro Arg

115 120 125

<210> 11

<211> 425

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> human IRF8 isoform 1 (protein sequence)

<400> 11

Met Cys Asp Arg Asn Gly Gly Arg Arg Leu Arg Gln Trp Leu Ile Glu

1 5 10 15

Gln Ile Asp Ser Ser Met Tyr Pro Gly Leu Ile Trp Glu Asn Glu Glu

20 25 30

Lys Ser Met Phe Arg Ile Pro Trp Lys His Ala Gly Lys Gln Asp Tyr

35 40 45

Asn Gln Glu Val Asp Ala Ser Ile Phe Lys Ala Trp Ala Val Phe Lys

50 55 60

Gly Lys Phe Lys Glu Gly Asp Lys Ala Glu Pro Ala Thr Trp Lys Thr

65 70 75 80

Arg Leu Arg Cys Ala Leu Asn Lys Ser Pro Asp Phe Glu Glu Val Thr

85 90 95

Asp Arg Ser Gln Leu Asp Ile Ser Glu Pro Tyr Lys Val Tyr Arg Ile

100 105 110

Val Pro Glu Glu Glu Gln Lys Cys Lys Leu Gly Val Ala Thr Ala Gly

115 120 125

Cys Val Asn Glu Val Thr Glu Met Glu Cys Gly Arg Ser Glu Ile Asp

130 135 140

Glu Leu Ile Lys Glu Pro Ser Val Asp Asp Tyr Met Gly Met Ile Lys

145 150 155 160

Arg Ser Pro Ser Pro Pro Glu Ala Cys Arg Ser Gln Leu Leu Pro Asp

165 170 175

Trp Trp Ala Gln Gln Pro Ser Thr Gly Val Pro Leu Val Thr Gly Tyr

180 185 190

Thr Thr Tyr Asp Ala His His Ser Ala Phe Ser Gln Met Val Ile Ser

195 200 205

Phe Tyr Tyr Gly Gly Lys Leu Val Gly Gln Ala Thr Thr Thr Cys Pro

210 215 220

Glu Gly Cys Arg Leu Ser Leu Ser Gln Pro Gly Leu Pro Gly Thr Lys

225 230 235 240

Leu Tyr Gly Pro Glu Gly Leu Glu Leu Val Arg Phe Pro Pro Ala Asp

245 250 255

Ala Ile Pro Ser Glu Arg Gln Arg Gln Val Thr Arg Lys Leu Phe Gly

260 265 270

His Leu Glu Arg Gly Val Leu Leu His Ser Ser Arg Gln Gly Val Phe

275 280 285

Val Lys Arg Leu Cys Gln Gly Arg Val Phe Cys Ser Gly Asn Ala Val

290 295 300

Val Cys Lys Gly Arg Pro Asn Lys Leu Glu Arg Asp Glu Val Val Val

305 310 315 320

Phe Asp Thr Ser Gln Phe Phe Arg Glu Leu Gln Gln Phe Tyr Asn Ser

325 330 335

Gln Gly Arg Leu Pro Asp Gly Arg Val Val Leu Cys Phe Gly Glu Glu

340 345 350

Phe Pro Asp Met Ala Pro Leu Arg Ser Lys Leu Ile Leu Val Gln Ile

355 360 365

Glu Gln Leu Tyr Val Arg Gln Leu Ala Glu Glu Ala Gly Lys Ser Cys

370 375 380

Gly Ala Gly Ser Val Met Gln Ala Pro Glu Glu Pro Pro Pro Asp Gln

385 390 395 400

Val Phe Arg Met Phe Pro Asp Ile Cys Ala Ser His Gln Arg Ser Phe

405 410 415

Phe Arg Glu Asn Gln Gln Ile Thr Val

420 425

<210> 12

<211> 271

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> human PU.1 isoform 1 (protein sequence)

<400> 12

Met Leu Gln Ala Cys Lys Met Glu Gly Phe Pro Leu Val Pro Pro Gln

1 5 10 15

Pro Ser Glu Asp Leu Val Pro Tyr Asp Thr Asp Leu Tyr Gln Arg Gln

20 25 30

Thr His Glu Tyr Tyr Pro Tyr Leu Ser Ser Asp Gly Glu Ser His Ser

35 40 45

Asp His Tyr Trp Asp Phe His Pro His His Val His Ser Glu Phe Glu

50 55 60

Ser Phe Ala Glu Asn Asn Phe Thr Glu Leu Gln Ser Val Gln Pro Pro

65 70 75 80

Gln Leu Gln Gln Leu Tyr Arg His Met Glu Leu Glu Gln Met His Val

85 90 95

Leu Asp Thr Pro Met Val Pro Pro His Pro Ser Leu Gly His Gln Val

100 105 110

Ser Tyr Leu Pro Arg Met Cys Leu Gln Tyr Pro Ser Leu Ser Pro Ala

115 120 125

Gln Pro Ser Ser Asp Glu Glu Glu Gly Glu Arg Gln Ser Pro Pro Leu

130 135 140

Glu Val Ser Asp Gly Glu Ala Asp Gly Leu Glu Pro Gly Pro Gly Leu

145 150 155 160

Leu Pro Gly Glu Thr Gly Ser Lys Lys Lys Ile Arg Leu Tyr Gln Phe

165 170 175

Leu Leu Asp Leu Leu Arg Ser Gly Asp Met Lys Asp Ser Ile Trp Trp

180 185 190

Val Asp Lys Asp Lys Gly Thr Phe Gln Phe Ser Ser Lys His Lys Glu

195 200 205

Ala Leu Ala His Arg Trp Gly Ile Gln Lys Gly Asn Arg Lys Lys Met

210 215 220

Thr Tyr Gln Lys Met Ala Arg Ala Leu Arg Asn Tyr Gly Lys Thr Gly

225 230 235 240

Glu Val Lys Lys Val Lys Lys Lys Leu Thr Tyr Gln Phe Ser Gly Glu

245 250 255

Val Leu Gly Arg Gly Gly Leu Ala Glu Arg Arg His Pro Pro His

260 265 270

<210> 13

<211> 239

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> human CCAAT/enhancer binding protein alpha (CEBP alpha) (protein sequence)

<400> 13

Met Pro Gly Gly Ala His Gly Pro Pro Pro Gly Tyr Gly Cys Ala Ala

1 5 10 15

Ala Gly Tyr Leu Asp Gly Arg Leu Glu Pro Leu Tyr Glu Arg Val Gly

20 25 30

Ala Pro Ala Leu Arg Pro Leu Val Ile Lys Gln Glu Pro Arg Glu Glu

35 40 45

Asp Glu Ala Lys Gln Leu Ala Leu Ala Gly Leu Phe Pro Tyr Gln Pro

50 55 60

Pro Pro Pro Pro Pro Pro Ser His Pro His Pro His Pro Pro Pro Ala

65 70 75 80

His Leu Ala Ala Pro His Leu Gln Phe Gln Ile Ala His Cys Gly Gln

85 90 95

Thr Thr Met His Leu Gln Pro Gly His Pro Thr Pro Pro Pro Thr Pro

100 105 110

Val Pro Ser Pro His Pro Ala Pro Ala Leu Gly Ala Ala Gly Leu Pro

115 120 125

Gly Pro Gly Ser Ala Leu Lys Gly Leu Gly Ala Ala His Pro Asp Leu

130 135 140

Arg Ala Ser Gly Gly Ser Gly Ala Gly Lys Ala Lys Lys Ser Val Asp

145 150 155 160

Lys Asn Ser Asn Glu Tyr Arg Val Arg Arg Glu Arg Asn Asn Ile Ala

165 170 175

Val Arg Lys Ser Arg Asp Lys Ala Lys Gln Arg Asn Val Glu Thr Gln

180 185 190

Gln Lys Val Leu Glu Leu Thr Ser Asp Asn Asp Arg Leu Arg Lys Arg

195 200 205

Val Glu Gln Leu Ser Arg Glu Leu Asp Thr Leu Arg Gly Ile Phe Arg

210 215 220

Gln Leu Pro Glu Ser Ser Leu Val Lys Ala Met Gly Asn Cys Ala

225 230 235

<210> 14

<211> 384

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> human BATF3 (DNA sequence)

<400> 14

atgtcgcaag ggctcccggc cgccggcagc gtcctgcaga ggagcgtcgc ggcgcccggg 60

aaccagccgc agccgcagcc gcagcagcag agccctgagg atgatgacag gaaggtccga 120

aggagagaaa aaaaccgagt tgctgctcag agaagtcgga agaagcagac ccagaaggct 180

gacaagctcc atgaggaata tgagagcctg gagcaagaaa acaccatgct gcggagagag 240

atcgggaagc tgacagagga gctgaagcac ctgacagagg cactgaagga gcacgagaag 300

atgtgcccgc tgctgctctg ccctatgaac tttgtgccag tgcctccccg gccggaccct 360

gtggccggct gcttgccccg atga 384

<210> 15

<211> 1287

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> human IRF8 (DNA sequence)

<400> 15

atgtgtgacc ggaatggtgg tcggcggctt cgacagtggc tgatcgagca gattgacagt 60

agcatgtatc caggactgat ttgggagaat gaggagaaga gcatgttccg gatcccttgg 120

aaacacgctg gcaagcaaga ttataatcag gaagtggatg cctccatttt taaggcctgg 180

gcagttttta aagggaagtt taaagaaggg gacaaagctg aaccagccac ttggaagacg 240

aggttacgct gtgctttgaa taagagccca gattttgagg aagtgacgga ccggtcccaa 300

ctggacattt ccgagccata caaagtttac cgaattgttc ctgaggaaga gcaaaaatgc 360

aaactaggcg tggcaactgc tggctgcgtg aatgaagtta cagagatgga gtgcggtcgc 420

tctgaaatcg acgagctgat caaggagcct tctgtggacg attacatggg gatgatcaaa 480

aggagccctt ccccgccgga ggcctgtcgg agtcagctcc ttccagactg gtgggcgcag 540

cagcccagca caggcgtgcc gctggtgacg gggtacacca cctacgacgc gcaccattca 600

gcattctccc agatggtgat cagcttctac tatgggggca agctggtggg ccaggccacc 660

accacctgcc ccgagggctg ccgcctgtcc ctgagccagc ctgggctgcc cggcaccaag 720

ctgtatgggc ccgagggcct ggagctggtg cgcttcccgc cggccgacgc catccccagc 780

gagcgacaga ggcaggtgac gcggaagctg ttcgggcacc tggagcgcgg ggtgctgctg 840

cacagcagcc ggcagggcgt gttcgtcaag cggctgtgcc agggccgcgt gttctgcagc 900

ggcaacgccg tggtgtgcaa aggcaggccc aacaagctgg agcgtgatga ggtggtccag 960

gtcttcgaca ccagccagtt cttccgagag ctgcagcagt tctataacag ccagggccgg 1020

cttcctgacg gcagggtggt gctgtgcttt ggggaagagt ttccggatat ggcccccttg 1080

cgctccaaac tcattctcgt gcagattgag cagctgtatg tccggcaact ggcagaagag 1140

gctgggaaga gctgtggagc cggctctgtg atgcaggccc ccgaggagcc gccgccagac 1200

caggtcttcc ggatgtttcc agatatttgt gcctcacacc agagatcatt tttcagagaa 1260

aaccaacaga tcaccgtcgg ctccggc 1287

<210> 16

<211> 819

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> human PU.1 (DNA sequence)

<400> 16

atgttacagg cgtgcaaaat ggaagggttt cccctcgtcc cccctccatc agaagacctg 60

gtgccctatg acacggatct ataccaacgc caaacgcacg agtattaccc ctatctcagc 120

agtgatgggg agagccatag cgaccattac tgggacttcc acccccacca cgtgcacagc 180

gagttcgaga gcttcgccga gaacaacttc acggagctcc agagcgtgca gcccccgcag 240

ctgcagcagc tctaccgcca catggagctg gagcagatgc acgtcctcga tacccccatg 300

gtgccacccc atcccagtct tggccaccag gtctcctacc tgccccggat gtgcctccag 360

tacccatccc tgtccccagc ccagcccagc tcagatgagg aggagggcga gcggcagagc 420

cccccactgg aggtgtctga cggcgaggcg gatggcctgg agcccgggcc tgggctcctg 480

cctggggaga caggcagcaa gaagaagatc cgcctgtacc agttcctgtt ggacctgctc 540

cgcagcggcg acatgaagga cagcatctgg tgggtggaca aggacaaggg caccttccag 600

ttctcgtcca agcacaagga ggcgctggcg caccgctggg gcatccagaa gggcaaccgc 660

aagaagatga cctaccagaa gatggcgcgc gcgctgcgca actacggcaa gacgggcgag 720

gtcaagaagg tgaagaagaa gctcacctac cagttcagcg gcgaagtgct gggccgcggg 780

ggcctggccg agcggcgcca cccgccccac ggcagcggc 819

<210> 17

<211> 720

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> human CCAAT/enhancer binding protein alpha (CEBPA) (DNA sequence)

<400> 17

atgcccgggg gagcgcacgg gcccccgccc ggctacggct gcgcggccgc cggctacctg 60

gacggcaggc tggagcccct gtacgagcgc gtcggggcgc cggcgctgcg gccgctggtg 120

atcaagcagg agccccgcga ggaggatgaa gccaagcagc tggcgctggc cggcctcttc 180

ccttaccagc cgccgccgcc gccgccgccc tcgcacccgc acccgcaccc gccgcccgcg 240

cacctggccg ccccgcacct gcagttccag atcgcgcact gcggccagac caccatgcac 300

ctgcagcccg gtcaccccac gccgccgccc acgcccgtgc ccagcccgca ccccgcgccc 360

gcgctcggtg ccgccggcct gccgggccct ggcagcgcgc tcaaggggct gggcgccgcg 420

caccccgacc tccgcgcgag tggcggcagc ggcgcgggca aggccaagaa gtcggtggac 480

aagaacagca acgagtaccg ggtgcggcgc gagcgcaaca acatcgcggt gcgcaagagc 540

cgcgacaagg ccaagcagcg caacgtggag acgcagcaga aggtgctgga gctgaccagt 600

gacaatgacc gcctgcgcaa gcgggtggaa cagctgagcc gcgaactgga cacgctgcgg 660

ggcatcttcc gccagctgcc agagagctcc ttggtcaagg ccatgggcaa ctgcgcgtga 720

<210> 18

<211> 913

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> Chile alkaline leucine zipper ATF-like transcription factor (BATF)

<400> 18

aaagcgagcg acatgtccct ttggggagca gtccctctgc accccagagt gaggaggacg 60

caggggtcag aggtggctac agggcaggca gaggaggcac ctgtaggggg tggtgggctg 120

gtggcccagg agaagtcagg aagggagccc agctggtgac aagagagccc agaggtgcct 180

ggggctgagt gtgagagccc ggaagatttc agccatgcct cacagctccg acagcagtga 240

ctccagcttc agccgctctc ctccccctgg caaacaggac tcatctgatg atgtgagaag 300

agttcagagg agggagaaaa atcgtattgc cgcccagaag agccgacaga ggcagacaca 360

gaaggccgac accctgcacc tggagagcga agacctggag aaacagaacg cggctctacg 420

caaggagatc aagcagctca cagaggaact gaagtacttc acgtcggtgc tgaacagcca 480

cgagcccctg tgctcggtgc tggccgccag cacgccctcg ccccccgagg tggtgtacag 540

cgcccacgca ttccaccaac ctcatgtcag ctccccgcgc ttccagccct gagcttccga 600

tgcggggaga gcagagcctc gggaggggca cacagactgt ggcagagctg cgcccatccc 660

gcagaggccc ctgtccacct ggagacccgg agacagaggc ctggacaagg agtgaacacg 720

ggaactgtca cgactggaag ggcgtgaggc ctcccagcag tgccgcagcg tttcgagggg 780

cgtgtgctgg accccaccac tgtgggttgc aggcccaatg cagaagagta ttaagaaaga 840

tgctcaagtc ccatggcaca gagcaaggcg ggcagggaac ggttattttt ctaaataaat 900

gctttaaaag aaa 913

<210> 19

<211> 125

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> basic leucine zipper transcription factor ATF-like [ Chinesian ], peptide sequence

<400> 19

Met Pro His Ser Ser Asp Ser Ser Asp Ser Ser Phe Ser Arg Ser Pro

1 5 10 15

Pro Pro Gly Lys Gln Asp Ser Ser Asp Asp Val Arg Arg Val Gln Arg

20 25 30

Arg Glu Lys Asn Arg Ile Ala Ala Gln Lys Ser Arg Gln Arg Gln Thr

35 40 45

Gln Lys Ala Asp Thr Leu His Leu Glu Ser Glu Asp Leu Glu Lys Gln

50 55 60

Asn Ala Ala Leu Arg Lys Glu Ile Lys Gln Leu Thr Glu Glu Leu Lys

65 70 75 80

Tyr Phe Thr Ser Val Leu Asn Ser His Glu Pro Leu Cys Ser Val Leu

85 90 95

Ala Ala Ser Thr Pro Ser Pro Pro Glu Val Val Tyr Ser Ala His Ala

100 105 110

Phe His Gln Pro His Val Ser Ser Pro Arg Phe Gln Pro

115 120 125

<210> 20

<211> 2008

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> Chile interferon regulatory factor 7 (IRF 7)

<400> 20

gagacgaaac ttcccgtccc ggcggctctg gcacccaggg tccggcctgc gccttcccgc 60

caggcctgga cactggttca acacctgtga cttcatgtgt gcgcgccggc cacacctgca 120

gtcacacctg tagccccctc tgccaagaga tccataccga ggcagcgtcg gtggctacaa 180

gccctcagtc cacacctgtg gacacctgtg acacctggcc acacgacctg tggccgcggc 240

ctggcgtctg ctgcgacagg agcccttacc tcccctgtta taacacctga ccgccaccta 300

actgcccctg cagaaggagc aatggccttg gctcctgaga ggtaagagcc cggcccaccc 360

tctccagatg ccagtccccg agcgccctgc agccggccct gactctccgc ggccgggcac 420

ccgcagggca gccccacgcg tgctgttcgg agagtggctc cttggagaga tcagcagcgg 480

ctgctatgag gggctgcagt ggctggacga ggcccgcacc tgtttccgcg tgccctggaa 540

gcacttcgcg cgcaaggacc tgagcgaggc cgacgcgcgc atcttcaagg cctgggctgt 600

ggcccgcggc aggtggccgc ctagcagcag gggaggtggc ccgccccccg aggctgagac 660

tgcggagcgc gccggctgga aaaccaactt ccgctgcgca ctgcgcagca cgcgtcgctt 720

cgtgatgctg cgggataact cgggggaccc ggccgacccg cacaaggtgt acgcgctcag 780

ccgggagctg tgctggcgag aaggcccagg cacggaccag actgaggcag aggcccccgc 840

agctgtccca ccaccacagg gtgggccccc agggccattc ctggcacaca cacatgctgg 900

actccaagcc ccaggccccc tccctgcccc agctggtgac aagggggacc tcctgctcca 960

ggcagtgcaa cagagctgcc tggcagacca tctgctgaca gcgtcatggg gggcagatcc 1020

agtcccaacc aaggctcctg gagagggaca agaagggctt cccctgactg gggcctgtgc 1080

tggaggccca gggctccctg ctggggagct gtacgggtgg gcagtagaga cgacccccag 1140

ccccgggccc cagcccgcgg cactaacgac aggcgaggcc gcggccccag agtccccgca 1200

ccaggcagag ccgtacctgt caccctcccc aagcgcctgc accgcggtgc aagagcccag 1260

cccaggggcg ctggacgtga ccatcatgta caagggccgc acggtgctgc agaaggtggt 1320

gggacacccg agctgcacgt tcctatacgg ccccccagac ccagctgtcc gggccacaga 1380

cccccagcag gtagcattcc ccagccctgc cgagctcccg gaccagaagc agctgcgcta 1440

cacggaggaa ctgctgcggc acgtggcccc tgggttgcac ctggagcttc gggggccaca 1500

gctgtgggcc cggcgcatgg gcaagtgcaa ggtgtactgg gaggtgggcg gacccccagg 1560

ctccgccagc ccctccaccc cagcctgcct gctgcctcgg aactgtgaca cccccatctt 1620

cgacttcaga gtcttcttcc aagagctggt ggaattccgg gcacggcagc gccgtggctc 1680

cccacgctat accatctacc tgggcttcgg gcaggacctg tcagctggga ggcccaagga 1740

gaagagcctg gtcctggtga agctggaacc ctggctgtgc cgagtgcacc tagagggcac 1800

gcagcgtgag ggtgtgtctt ccctggatag cagcagcctc agcctctgcc tgtccagcgc 1860

caacagcctc tatgacgaca tcgagtgctt ccttatggag ctggagcagc ccgcctagaa 1920

cccagtctaa tgagaactcc agaaagctgg agcagcccac ctagagctgg ccgcggccgc 1980

ccagtctaat aaaaagaact ccagaaca 2008

<210> 21

<211> 516

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> interferon regulatory factor 7 [ Chinesian ], peptide sequence

<400> 21

Met Pro Val Pro Glu Arg Pro Ala Ala Gly Pro Asp Ser Pro Arg Pro

1 5 10 15

Gly Thr Arg Arg Ala Ala Pro Arg Val Leu Phe Gly Glu Trp Leu Leu

20 25 30

Gly Glu Ile Ser Ser Gly Cys Tyr Glu Gly Leu Gln Trp Leu Asp Glu

35 40 45

Ala Arg Thr Cys Phe Arg Val Pro Trp Lys His Phe Ala Arg Lys Asp

50 55 60

Leu Ser Glu Ala Asp Ala Arg Ile Phe Lys Ala Trp Ala Val Ala Arg

65 70 75 80

Gly Arg Trp Pro Pro Ser Ser Arg Gly Gly Gly Pro Pro Pro Glu Ala

85 90 95

Glu Thr Ala Glu Arg Ala Gly Trp Lys Thr Asn Phe Arg Cys Ala Leu

100 105 110

Arg Ser Thr Arg Arg Phe Val Met Leu Arg Asp Asn Ser Gly Asp Pro

115 120 125

Ala Asp Pro His Lys Val Tyr Ala Leu Ser Arg Glu Leu Cys Trp Arg

130 135 140

Glu Gly Pro Gly Thr Asp Gln Thr Glu Ala Glu Ala Pro Ala Ala Val

145 150 155 160

Pro Pro Pro Gln Gly Gly Pro Pro Gly Pro Phe Leu Ala His Thr His

165 170 175

Ala Gly Leu Gln Ala Pro Gly Pro Leu Pro Ala Pro Ala Gly Asp Lys

180 185 190

Gly Asp Leu Leu Leu Gln Ala Val Gln Gln Ser Cys Leu Ala Asp His

195 200 205

Leu Leu Thr Ala Ser Trp Gly Ala Asp Pro Val Pro Thr Lys Ala Pro

210 215 220

Gly Glu Gly Gln Glu Gly Leu Pro Leu Thr Gly Ala Cys Ala Gly Gly

225 230 235 240

Pro Gly Leu Pro Ala Gly Glu Leu Tyr Gly Trp Ala Val Glu Thr Thr

245 250 255

Pro Ser Pro Gly Pro Gln Pro Ala Ala Leu Thr Thr Gly Glu Ala Ala

260 265 270

Ala Pro Glu Ser Pro His Gln Ala Glu Pro Tyr Leu Ser Pro Ser Pro

275 280 285

Ser Ala Cys Thr Ala Val Gln Glu Pro Ser Pro Gly Ala Leu Asp Val

290 295 300

Thr Ile Met Tyr Lys Gly Arg Thr Val Leu Gln Lys Val Val Gly His

305 310 315 320

Pro Ser Cys Thr Phe Leu Tyr Gly Pro Pro Asp Pro Ala Val Arg Ala

325 330 335

Thr Asp Pro Gln Gln Val Ala Phe Pro Ser Pro Ala Glu Leu Pro Asp

340 345 350

Gln Lys Gln Leu Arg Tyr Thr Glu Glu Leu Leu Arg His Val Ala Pro

355 360 365

Gly Leu His Leu Glu Leu Arg Gly Pro Gln Leu Trp Ala Arg Arg Met

370 375 380

Gly Lys Cys Lys Val Tyr Trp Glu Val Gly Gly Pro Pro Gly Ser Ala

385 390 395 400

Ser Pro Ser Thr Pro Ala Cys Leu Leu Pro Arg Asn Cys Asp Thr Pro

405 410 415

Ile Phe Asp Phe Arg Val Phe Phe Gln Glu Leu Val Glu Phe Arg Ala

420 425 430

Arg Gln Arg Arg Gly Ser Pro Arg Tyr Thr Ile Tyr Leu Gly Phe Gly

435 440 445

Gln Asp Leu Ser Ala Gly Arg Pro Lys Glu Lys Ser Leu Val Leu Val

450 455 460

Lys Leu Glu Pro Trp Leu Cys Arg Val His Leu Glu Gly Thr Gln Arg

465 470 475 480

Glu Gly Val Ser Ser Leu Asp Ser Ser Ser Leu Ser Leu Cys Leu Ser

485 490 495

Ser Ala Asn Ser Leu Tyr Asp Asp Ile Glu Cys Phe Leu Met Glu Leu

500 505 510

Glu Gln Pro Ala

515

<210> 22

<211> 3792

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> Chile Spi-B transcription factor (SPIB)

<400> 22

ggcaaacagc ccgcccggca ccaccatgct cgccctggag gctgcacagc tcgacgggcc 60

acacttcagc tgtctgtacc cagatggcgt cttctatgac ctggacagct gcaagcattc 120

cagctaccct gattcagagg gggctcctga ctccctgtgg gactggactg tggccccacc 180

tgtcccagcc accccctatg aagccttcga cccggcagca gccgctttta gccaccccca 240

ggctgcccag ctctgctacg aaccccccac ctacagccct gcagggaacc tcgaactggc 300

ccccagcctg gaggccccgg ggcctggcct ccctgcatac cccacggaga acttcgctag 360

ccagaccctg gttcccccgg catatgcccc gtaccccagc cctgtgctat cagaggagga 420

agacttaccg ttggacagcc ctgccctgga ggtctcggac agcgagtcgg atgaggccct 480

cgtggctggc cccgagggga agggatccga ggcagggact cgcaagaagc tgcgcctgta 540

ccagttcctg ctggggctac tgacgcgcgg ggacatgcgt gagtgcgtgt ggtgggtgga 600

gccaggcgcc ggcgtcttcc agttctcctc caagcacaag gaactcctgg cgcgccgctg 660

gggccagcag aaggggaacc gcaagcgcat gacctaccag aagctggcgc gcgccctccg 720

aaactacgcc aagaccggcg agatccgcaa ggtcaagcgc aagctcacct accagttcga 780

cagcgcgctg ctgcctgcag tccgccgggc ctgagcacac ccgaggctcc cacctgcgga 840

gccgctgggg gacctcacgt cccagccagg atccccctgg aagaaaaagg gcgtccccac 900

actctaggtg ataggactta cgcatcccca ccttttgggg taaggggagt gctgccctgc 960

cataatcccc aagcccagcc cgggcctgtc tgggattccc cacttgtgcc tggggtcctc 1020

tgggatttct ttgtcatgta cagactccct gggatcctca tgttttgggt gacaggacct 1080

atggaccact atactcgggg aggcagggta gcagttcttc cagaatccca agagcttctc 1140

tgggattttc ttgtgatatc tgattcccca gtgaggcctg ggacgttttt aagatcgctg 1200

tgtgtctgta aaccctgaat ctcatctggg gtgggggccc tgctggcaac cctgagccct 1260

gtccaaggtt ccctcttgtc agatctgaga tttcctagtt atgtctgggg ccctctggga 1320

gctgttatca tctcagatct cttcgcccat ctatggctgt gttgtcacat ctgtcccctc 1380

atttttgaga tcccccaatt ctctggaact attctgctgc ccctttttat gtgtctggag 1440

ttccccaatc acatctaggg ctcctccaag atccttttgt catgtctgaa atcactcttg 1500

agaggtctgg ggtggaggat ggggagtcag tgaaatgtgt catgtctggg ccctgtcagg 1560

gacacccttg ttatatctgg gatcctccaa tcacatctga gacctcctag gctctccatc 1620

tgatatgccc tttcagggac cccacaaaga ctgagttctc atggggatcc tacccttcct 1680

agtgccactc cctatggcca tgctgaagac cactctggcc acgcgactga ttttgggtga 1740

tcatggcagc tccccaccca tgtcatttct aaccagaagt ctcaaggtcg tcacccccct 1800

gccccccaac cgaggccccg gtcgctggtg gtggtctctt tagtgcactg tagcacttgg 1860

tggtggaggt gtgagggatc cacattaaca gcaggccatc agctgggcaa tggctcacac 1920

ctgtaatccc agcactttgg gaggcgaggc agggggaatg gcttgaaccc aggcattcaa 1980

gaccagcctg ggcaacataa tgagacctcg tctctacaaa acataacaaa aacaattagc 2040

cgagcgtggg ggtgaacacc tgtggtccca gctgctcagg aggctgaggt gggaggatct 2100

cttgagccca ggaagtagga ggctgtagtg agctgtaatc gtgccactgc actccagcct 2160

gggcgacaga gtgagacacc gtcttaaaaa caaaaacaag gccgggcacg gtggctcatg 2220

cctgttgtcc cagcactttg ggaggccgag gcaggcggat cacgaggtcg agagatcgag 2280

accatcctgg ccaacatggt gaaaccctgt ctctactaaa aatacagaaa ttagctgggc 2340

gtggtggcac gtgcctgtag tcccagctac tcgggaggct gaggcaagag aatcgcttga 2400

acgtgggagg cagaggttgc agtgagccta gattgtgcca ctgcactcca gcctggggga 2460

cagagcgaga ctccgtctga aaataaaaac aacaaaaaca gcagaccatt caaaataggg 2520

agactttgca taatccagat ttctgccttc acttaaaact ttggacggtc tggagagagt 2580

cggccagttt tcggtggggg gtggggagct ggaacaggac agtagccttt cctaatgagg 2640

catttgttct ccaatctgcc ccagtcgctg ccatccctgg ctatctcacc ctagcagctt 2700

ctcaagcctg ttggctttag accactgtat aaacccagct ggaactgaag cctgggtgga 2760

ctatggagcc ctggttggga cccccaggga gtcaaaggct gcgggccaag aggccagagg 2820

tccttgagcc tgggtgggca ggtggatcta gggtgcatga cttgctgctt cccaacctta 2880

gtttgtccct tctgtgaaaa agggagagaa ggaggaggaa gatctcaaaa agactttcca 2940

gcccagtgcg gtggctcacg cctgtaatcc cagcactttg ggaggccgat gcaggtggat 3000

cacctgaggt aggagttcaa gaccagcctg accaacatag tgaagcccct tctctactaa 3060

aaatacaaaa ttagctgggc gtggtggcat gtgcctgtac tcccagctac ttgggaggct 3120

gaggcaggag aatcgcttga acctgggagg cggaggttgt agtgagctga gatcacacca 3180

ctgcacacca gcctgggcga caagagcgaa actccgtctc aaaaaaaaaa aactgttgca 3240

gccccgttga gcctttgaca ccgcctgaaa tccaccccac tcccaggagg aggaggagga 3300

aggaatgcca atgacctaga gacacgagaa gtccatgtgg aggcacacag cagctgatgg 3360

cagagcccag gctgggacct gcccttaaga gaatgagtgg gaagggggag ggaggaaggg 3420

caggtaaaac gtcctcccca gggccccctg caacggggaa ggtacttttt acaaaagcta 3480

tcattgtcac cctaaatgtg gaataaaata agatgcatcg acgtagacaa acctcctggg 3540

accttttgtc agggactgca atcctgcccc tccactgagg ccgctggctc tcagagacac 3600

cgtgacatca cgggtgatga tgagaggagt tcaaagagag aattatatgc tggcgcggtg 3660

gctctgtaat cccaacactt tggggggcca aggcaggagg atcgcttgag tacaggagtt 3720

tgaaaccagc ctgggcaaga tagtgagatc cccttcccac ccgtctacaa aaaaaataaa 3780

aaattagcgg gg 3792

<210> 23

<211> 262

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> transcription factor Spi-B [ Chinesemedicine ], peptide sequence

<400> 23

Met Leu Ala Leu Glu Ala Ala Gln Leu Asp Gly Pro His Phe Ser Cys

1 5 10 15

Leu Tyr Pro Asp Gly Val Phe Tyr Asp Leu Asp Ser Cys Lys His Ser

20 25 30

Ser Tyr Pro Asp Ser Glu Gly Ala Pro Asp Ser Leu Trp Asp Trp Thr

35 40 45

Val Ala Pro Pro Val Pro Ala Thr Pro Tyr Glu Ala Phe Asp Pro Ala

50 55 60

Ala Ala Ala Phe Ser His Pro Gln Ala Ala Gln Leu Cys Tyr Glu Pro

65 70 75 80

Pro Thr Tyr Ser Pro Ala Gly Asn Leu Glu Leu Ala Pro Ser Leu Glu

85 90 95

Ala Pro Gly Pro Gly Leu Pro Ala Tyr Pro Thr Glu Asn Phe Ala Ser

100 105 110

Gln Thr Leu Val Pro Pro Ala Tyr Ala Pro Tyr Pro Ser Pro Val Leu

115 120 125

Ser Glu Glu Glu Asp Leu Pro Leu Asp Ser Pro Ala Leu Glu Val Ser

130 135 140

Asp Ser Glu Ser Asp Glu Ala Leu Val Ala Gly Pro Glu Gly Lys Gly

145 150 155 160

Ser Glu Ala Gly Thr Arg Lys Lys Leu Arg Leu Tyr Gln Phe Leu Leu

165 170 175

Gly Leu Leu Thr Arg Gly Asp Met Arg Glu Cys Val Trp Trp Val Glu

180 185 190

Pro Gly Ala Gly Val Phe Gln Phe Ser Ser Lys His Lys Glu Leu Leu

195 200 205

Ala Arg Arg Trp Gly Gln Gln Lys Gly Asn Arg Lys Arg Met Thr Tyr

210 215 220

Gln Lys Leu Ala Arg Ala Leu Arg Asn Tyr Ala Lys Thr Gly Glu Ile

225 230 235 240

Arg Lys Val Lys Arg Lys Leu Thr Tyr Gln Phe Asp Ser Ala Leu Leu

245 250 255

Pro Ala Val Arg Arg Ala

260

<210> 24

<211> 1217

<212> DNA

<213> Chile person

<220>

<221> misc_feature

<223> Chile Spi-C transcription factor (SPIC)

<400> 24

gatggttatg tcttaacaat tctaattttt aaaaaaaata ttactatttt ttttcatcag 60

agccattgca ctggaaaata attttttatt ttcatgatag cctaccgttg aatttaccgt 120

tctgatatta atgaaacatc tctataaagg gttgaagtgt cttcccggat tgtcaactta 180

ttttatttta ttttcttcaa gcaacaattg ctaaggaaca gaattgtcaa tttattaatg 240

aaatatgacg tgtgttgaac aagacaagct gggtcaagca tttgaagatg cttttgaggt 300

tctgaggcaa cattcaactg gagatcttca gtactcgcca gattacagaa attacctggc 360

tttaatcaac catcgtcctc atgtcaaagg aaattccagc tgctatggag tgttgcctac 420

agaggagcct gtctataatt ggagaacggt aattaacagt gctgcggact tctattttga 480

aggaaatatt catcaatctc tgcagaacat aactgaaaac cagctggtac aacccactct 540

tctccagcaa aaggggggaa aaggcaggaa gaagctccga ctgtttgaat accttcacga 600

atccctgtat aatccggaga tggcatcttg tattcagtgg gtagataaaa ccaaaggcat 660

ctttcagttt gtatcaaaaa acaaagaaaa acttgccgag ctttggggga aaagaaaagg 720

caacaggaag accatgactt accagaaaat ggccagggca ctcagaaatt acggaagaag 780

tggggaaatt accaaaatcc ggaggaagct gacttaccag ttcagtgagg ccattctcca 840

aagactctct ccatcctatt tcctggggaa agagatcttc tattcacagt gtgttcaacc 900

tgatcaagaa tatctcagtt taaataactg gaatgcaaat tataattata catatgccaa 960

ttaccatgag ctaaatcacc atgattgcta aatatacttt catatttcat ggtttactgg 1020

catcggaaat ctctacaagt tttaatgatt tctccctccc tctctttttt tcctcctctg 1080

aagaaattta ggatttttct cttaaagcaa atactaaaga ggaaaaaaaa ttaactttat 1140

tgttgctttt atcaaagagt atgtaatcta tactaacttg ttgggaaatt ctgccaatga 1200

acaacttttt tataata 1217

<210> 25

<211> 248

<212> PRT

<213> Chile person

<220>

<221> MISC_FEATURE

<223> transcription factor Spi-C [ Chinesemedicine ], peptide sequence

<400> 25

Met Thr Cys Val Glu Gln Asp Lys Leu Gly Gln Ala Phe Glu Asp Ala

1 5 10 15

Phe Glu Val Leu Arg Gln His Ser Thr Gly Asp Leu Gln Tyr Ser Pro

20 25 30

Asp Tyr Arg Asn Tyr Leu Ala Leu Ile Asn His Arg Pro His Val Lys

35 40 45

Gly Asn Ser Ser Cys Tyr Gly Val Leu Pro Thr Glu Glu Pro Val Tyr

50 55 60

Asn Trp Arg Thr Val Ile Asn Ser Ala Ala Asp Phe Tyr Phe Glu Gly

65 70 75 80

Asn Ile His Gln Ser Leu Gln Asn Ile Thr Glu Asn Gln Leu Val Gln

85 90 95

Pro Thr Leu Leu Gln Gln Lys Gly Gly Lys Gly Arg Lys Lys Leu Arg

100 105 110

Leu Phe Glu Tyr Leu His Glu Ser Leu Tyr Asn Pro Glu Met Ala Ser

115 120 125

Cys Ile Gln Trp Val Asp Lys Thr Lys Gly Ile Phe Gln Phe Val Ser

130 135 140

Lys Asn Lys Glu Lys Leu Ala Glu Leu Trp Gly Lys Arg Lys Gly Asn

145 150 155 160

Arg Lys Thr Met Thr Tyr Gln Lys Met Ala Arg Ala Leu Arg Asn Tyr

165 170 175

Gly Arg Ser Gly Glu Ile Thr Lys Ile Arg Arg Lys Leu Thr Tyr Gln

180 185 190

Phe Ser Glu Ala Ile Leu Gln Arg Leu Ser Pro Ser Tyr Phe Leu Gly

195 200 205

Lys Glu Ile Phe Tyr Ser Gln Cys Val Gln Pro Asp Gln Glu Tyr Leu

210 215 220

Ser Leu Asn Asn Trp Asn Ala Asn Tyr Asn Tyr Thr Tyr Ala Asn Tyr

225 230 235 240

His Glu Leu Asn His His Asp Cys

245

Claims

wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of the transcription factor, wherein the promoter region comprises a Spleen Focus Forming Virus (SFFV) promoter.

2. The composition according to claim 1, further comprising one or more constructs or vectors which, upon expression, encode one or more transcription factors selected from the group consisting of:

3. The composition according to any one of claims 1 or 2, wherein the SFFV promoter comprises or consists of a polynucleotide sequence having at least 80% identity to SEQ ID No. 1, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID No. 1.

4. A composition according to any one of the preceding claims, wherein the composition comprises:

And/or

5. A composition according to any one of the preceding claims, wherein the composition comprises:

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor pu.1 after expression, and a fourth construct or vector encoding the transcription factor IRF7 after expression.

6. A composition according to any one of the preceding claims, wherein the composition comprises:

and/or

i) A first construct or vector encoding the transcription factor BATF3 after expression; a second construct or vector encoding the transcription factor IRF8 after expression; a third construct or vector encoding the transcription factor pu.1 after expression, and a fourth construct or vector encoding the transcription factor bat after expression.

7. A composition according to any of the preceding claims, wherein the BATF3 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 14, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 14.

8. A composition according to any of the preceding claims, wherein IRF8 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 15, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 15.

9. A composition according to any of the preceding claims, wherein pu.1 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 16, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 16.

10. A composition according to any of the preceding claims, wherein the BATF is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 18, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 18.

11. A composition according to any of the preceding claims, wherein IRF7 is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 20, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 20.

12. A composition according to any of the preceding claims, wherein the SPIB is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 22, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 22.

13. A composition according to any of the preceding claims, wherein the SPIC is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 24, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 24.

14. The composition according to any of the preceding claims, wherein the one or more constructs or vectors after expression further encode a transcription factor CCAAT/enhancer binding protein a (cebpa) or a biologically active variant thereof, wherein the biologically active variant has at least 70% identity with SEQ ID NO:13 (cebpa), such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity with SEQ ID NO: 13.

15. A composition according to any of the preceding claims, wherein CEBP a is encoded by a polynucleotide sequence having at least 90% sequence identity with SEQ ID No. 17, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID No. 17.

16. The composition of any one of the preceding claims, wherein the one or more constructs or vectors further comprise a self-cleaving peptide operably linked to at least two of the at least three coding regions, thereby forming a single open reading frame.

17. The composition according to claim 16, wherein the self-cleaving peptide is a 2A peptide.

18. The composition according to claim 17, wherein the 2A peptide is selected from the group consisting of equine rhinitis virus (E2A), foot and mouth disease virus (F2A), porcine teschovirus-1 (P2A), and echinococcosis minor virus (T2A) peptide.

19. A composition according to any one of the preceding claims, wherein the one or more constructs or vectors are viral vectors.

20. The composition according to any one of the preceding claims, wherein the one or more constructs or vectors are viral vectors selected from the group consisting of: lentiviral vectors, retroviral vectors, adenoviral vectors, herpesviral vectors, poxviral vectors, adeno-associated viral vectors, paramyxoviridae vectors, rhabdoviral vectors, alphaviral vectors and flaviviral vectors.

21. The composition according to claim 20, wherein the viral vector is a lentiviral vector.

22. The composition according to claim 20, wherein the adenovirus vector is selected from the group consisting of: wild-type Ad vectors, hybrid Ad vectors, and mutant Ad vectors.

23. The composition according to claim 22, wherein the wild-type Ad vector is Ad5 and wherein the hybrid Ad vector is Ad5/F35.

24. The composition according to claim 20, wherein the adeno-associated viral vector is selected from the group consisting of: wild-type AAV vectors, hybrid AAV vectors, and mutant AAV vectors.

25. A composition according to claim 24, wherein the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV 2-quadryf.

26. A composition according to any one of the preceding claims, wherein the one or more constructs or vectors are plasmids.

27. The composition according to any of the preceding claims, wherein the backbone of the one or more constructs or vectors is selected from the group consisting of: FUW, pRRL-cPPT, pRLL, pCCL, pCLL, pHAGE2, pWPXL, pLKO, pHIV, pLL, pCDH and pLenti.

28. The composition according to any of the preceding claims, wherein the one or more constructs or vectors further comprise a post-transcriptional regulatory element (PRE) sequence.

29. The composition according to claim 28, wherein said PRE sequence is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

32. The composition according to claim 31, wherein at least one of the 5' and 3' terminal repeats is a lentiviral long terminal repeat or a self-inactivating (SIN) design of U3 with partial deletion of the 3' long terminal repeat.

33. The composition according to any of the preceding claims, wherein the one or more constructs or vectors further comprise a nucleocapsid protein packaging target site.

34. The composition according to claim 33, wherein the protein packaging target site comprises HIV-1psi sequence.

35. The composition according to any of the preceding claims, wherein the one or more constructs or vectors further comprises a REV protein responsive element (RRE).

36. The composition according to any one of the preceding claims, further comprising one or more cytokines selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM).

37. The composition according to any of the preceding claims, further comprising one or more epigenetic modifiers, such as histone deacetylase inhibitors.

38. The composition according to claim 37, wherein the one or more histone deacetylase inhibitors is valproic acid.

39. The composition according to any of the preceding claims, wherein the composition is a pharmaceutical composition.

40. A cell comprising one or more constructs or vectors according to any of the preceding claims.

41. A cell according to claim 40, wherein the cell is a mammalian cell, such as a human or murine cell.

42. The cell according to any one of claims 40 to 41, wherein the cell is selected from the group consisting of: stem cells, differentiated cells, and cancer cells, wherein:

a) The stem cells are selected from the group consisting of: pluripotent stem cells and multipotent stem cells, such as mesenchymal stem cells or hematopoietic stem cells;

43. A cell according to any one of claims 40 to 42, wherein the cell is a reprogrammed human dendritic cell or an antigen presenting cell, such as a human type 1 classical dendritic cell.

44. The cell according to any one of claims 40 to 43, wherein the cell further expresses one or more surface markers selected from the surface markers of table 1.

45. The cell according to any one of claims 40 to 44, wherein the cell is positive for one or more surface markers listed in table 1.

46. The cell according to any one of claims 40 to 45, wherein the cell is CD226 positive.

a) Transducing a cell with a composition comprising a construct or vector according to any one of claims 1 to 32;

b) Expressing the transcription factor;

thereby obtaining reprogrammed or induced cells.

48. The method according to claim 47, wherein said reprogramming or induction is in vivo, in vitro, or ex vivo.

49. The method according to any one of claims 47 to 48, wherein the method further comprises the step of culturing the transduced cells in a cell culture medium, wherein the step is performed before or after expression of the transcription factor.

50. The method according to any one of claims 47 to 49, wherein the method further comprises culturing the transduced cells in a cell culture medium comprising one or more cytokines selected from the group consisting of: IFNbeta, IFNgamma, TNF alpha, IFNalpha, IL-1 beta, IL-6, CD40I, flt3I, GM-CSF, IFN-lambda 1, IFN-omega, IL-2, IL-4, IL-15, prostaglandin 2, SCF and Oncomelanin M (OM).

51. The method according to any one of claims 47 to 50, wherein the method further comprises culturing the transduced cells in a cell culture medium comprising one or more epigenetic modifiers, such as histone deacetylase inhibitors.

52. The method of claim 51, wherein the histone deacetylase inhibitor is valproic acid.

53. The method according to any one of claims 47 to 52, wherein the cell is a mammalian cell, such as a human or murine cell.

54. The method of any one of claims 47 to 53, wherein the cells are selected from the group consisting of: stem cells, differentiated cells, and cancer cells, wherein:

55. The method according to any one of claims 47 to 54, wherein the transduced cells are cultured for a period of at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

56. The method according to any one of claims 47 to 55, wherein the resulting reprogrammed or induced cell is a type 1 classical dendritic cell.

57. The method according to any one of claims 47 to 56, wherein the resulting reprogrammed or induced cells are cluster of differentiation 45 (CD 45) positive.

58. The method according to any one of claims 47 to 57, wherein the resulting reprogrammed or induced cells are cluster of differentiation 226 (CD 226) positive.

59. The method according to any one of claims 47 to 58, wherein the resulting reprogrammed or induced cell is positive for the human leukocyte antigen-DR isoform (HLA-DR).

60. A reprogrammed or induced cell obtained according to the method defined in any one of claims 47 to 59.

61. A reprogrammed or induced cell according to claim 60, wherein said cell is a dendritic cell or an antigen presenting cell, such as a classical dendritic cell type 1.

62. The reprogrammed or induced cell according to any one of claims 60 to 61, wherein the resulting reprogrammed or induced cell is positive for one or more surface markers listed in table 1.

63. The reprogrammed or induced cell according to any of claims 60 to 62, wherein the resulting reprogrammed or induced cell is CD45, HLA-DR, CD141, CLEC9A, XCR1 and/or CD226 positive.

64. A composition according to any one of claims 1 to 39, a cell according to any one of claims 40 to 46, and/or a reprogrammed or induced cell according to any one of claims 60 to 63 for use in medicine.

65. A composition according to any one of claims 1 to 39, a cell according to any one of claims 40 to 46, and/or a reprogrammed or induced cell according to any one of claims 60 to 63 for use in the treatment of cancer or infectious disease.

66. The composition, cell, and/or reprogrammed cell of claim 65, wherein the cancer is selected from the group consisting of: basal cell carcinoma, cervical atypical hyperplasia, sarcoma, germ cell tumor, retinoblastoma, glioblastoma, lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hematological cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, uterine cancer, vaginal cancer, breast cancer, head and neck cancer, gastric cancer, oral cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, pleural cancer, urothelial cancer, hollow organ cancer, esophageal cancer, gastric cancer, cholangiocarcinoma, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureter cancer, renal cancer, liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, skin cancer, melanoma, and myeloma.

67. The composition, cell, and/or reprogrammed cell of claim 66, wherein the cancer is selected from the group consisting of: melanoma, head and neck cancer, breast cancer, colorectal cancer, liver cancer, lymphoma, bladder cancer and urothelial cancer, pancreatic cancer and glioblastoma.

68. A method of treating cancer or an infectious disease, the method comprising administering to an individual in need thereof a composition according to any one of claims 1 to 38, a cell according to any one of claims 40 to 46, a pharmaceutical composition according to claim 39, and/or a reprogrammed or induced cell according to any one of claims 60 to 63.

69. Use of a composition according to any one of claims 1 to 38, a cell according to any one of claims 40 to 46, a pharmaceutical composition according to claim 39, and/or a reprogrammed or induced cell according to any one of claims 60 to 63 for the manufacture of a medicament for the treatment of cancer or infectious disease.