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WO2026003067A1 - Transcription factors and reprogramming modulators for reprogramming cells to cdc1 cells, compositions and methods thereof - Google Patents

Transcription factors and reprogramming modulators for reprogramming cells to cdc1 cells, compositions and methods thereof

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Publication number
WO2026003067A1
WO2026003067A1 PCT/EP2025/067893 EP2025067893W WO2026003067A1 WO 2026003067 A1 WO2026003067 A1 WO 2026003067A1 EP 2025067893 W EP2025067893 W EP 2025067893W WO 2026003067 A1 WO2026003067 A1 WO 2026003067A1
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seq
cell
constructs
expression
sequence identity
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Inventor
Fábio Alexandre Fiúza ROSA
Cristiana Ferreira Pires
Carlos Filipe Ribeiro Lemos Pereira
Camila Vazquez ECHEGARAY
Evelyn HALITZKI
Malavika Sreekumar NAIR
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Asgard Therapeutics AB
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Asgard Therapeutics AB
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Definitions

  • the present invention relates to constructs, vectors and methods for reprogramming cells into dendritic or antigen-presenting cells, and uses thereof.
  • the present invention relates to novel transcription factors and reprogramming modulators that improve reprogramming, and methods thereof.
  • Background Cancer immunotherapies rely on the establishment of tumor antigen-specific T cell responses. T cells identify tumor antigens displayed on tumor cells' major histocompatibility complexes (MHC) and carry out their functions by killing cells and producing inflammatory cytokines.
  • MHC major histocompatibility complexes
  • TME tumor microenvironment
  • DCs dendritic cells
  • IRB immune checkpoint blockade
  • PD-1 anti–programmed cell death protein 1
  • CTL-4 anti–cytotoxic T lymphocyte– associated protein 4
  • cDC1s dendritic cells type 1
  • MHC class I and II express high levels of MHC class I and II, the co-stimulatory molecule CD40, and specific markers like XCR1 and CLEC9A (Cabeza-Cabrerizo et al.2021).
  • cDC1s are essential for recruiting and activating T cells through chemokine secretion and antigen cross-presentation, facilitating effective cancer immunity.
  • these unique functional attributes of cDC1s have not yet been fully harnessed for immunotherapy due to the lack of methods enabling the generation of a pure population of cDC1s.
  • P6783PC01 Cellular reprogramming offers a strategy for generating specific cell types in vivo by overexpression of cell type-specific transcription factors (TFs). In vivo cell fate reprogramming allows the conversion of endogenous somatic cells into different cell types within the organism, offering therapeutic potential directly at the site of disease bypassing the challenges associated with ex vivo cell manufacturing for personalized cell therapies.
  • mouse pancreatic exocrine cells were converted in situ to insulin-secreting ⁇ -cells by delivering three transcription factors to the pancreas using adenoviral vectors.
  • scar-forming cardiac fibroblasts were transformed into cardiomyocytes in mouse models of myocardial infarction, leading to improved heart function (Qian et al. 2012).
  • Glial cells were converted to functional neurons after brain injury or in models of neurodegenerative diseases (Torper et al.2015) and rod photoreceptors were generated within the retina, resulting in improved vision (Yao et al. 2018).
  • a transcription factor combination PU.1, IRF8, and BATF3 (PIB)— was identified as sufficient to reprogram fibroblasts or tumor cells into cDC1-like cells in vitro and in vivo, equipped with the essential signals for T cell activation, antigen presentation, co-stimulatory molecule expression, and chemokine/cytokine secretion (Rosa et al. 2018, Rosa et al.2022, Zimmermannova et al.2023). These studies provided proof-of-principle that cDC1 reprogramming could be harnessed to develop novel treatment modalities for cancer immunotherapy.
  • CRISPR prokaryotic clustered, regularly interspaced, short palindromic repeats
  • the CRISPR-associated (Cas)9 enzyme can be programmed with a single guide RNA (sgRNA) to generate site- specific DNA breaks, by directing the machinery with a single guide RNA (sgRNA) targeting a specific gene.
  • sgRNA single guide RNA
  • CRISPR/Cas9-based screens, as well as screening platforms based on shRNAs (Borkent et al. 2016, Moura-Alves et al. 2011, Oliver et al.
  • the present invention relates to one or more constructs, which upon expression encode: - one or more gene expression inhibitors of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the group consisting
  • a second aspect of the present invention relates to one or more constructs, which upon expression encodes at least two transcriptions factors reprogramming or inducing a cell, wherein the constructs further encode: - one or more gene expression inhibitors inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKM
  • barrier genes
  • a third aspect of the present invention provides one or more vectors comprising the one or more constructs of the first or second aspects of the present invention.
  • a fourth aspect of the present invention relates to a cell comprising the one or more constructs of the first or second aspects of the present invention or the one or more vectors of the third aspect of the present invention.
  • a fifth aspect of the present invention relates to a method of reprogramming or inducing a cell into a reprogrammed or induced cell, the method comprising the following steps: a ) transducing a cell with one or more constructs or vectors, which upon expression encodes at least two transcription factors reprogramming or inducing said cell; b ) inhibiting the expression of one or more genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAM
  • ICB anti-PD-1 and anti-CTLA-4
  • IgG2a and IgG2b isotype control antibodies
  • B Flow cytometry analysis and quantification of the number of surface MHC-I molecules per cell and
  • C Quantification of proliferating CTV low CD44 + ovalbumin-specific CD8 + T cells (OT-I) after co-culture with CD103 + bone marrow-derived dendritic cells (BM-DC), eGFP-transduced B16 cells (not expressing ovalbumin), MACS-enriched CD45 + and MHC-II + B16-derived cDC1-like cells or MACS-enriched CD45 + and CD11b + B16-derived macrophage-like cells (PC).
  • BM-DC, eGFP-transduced or reprogrammed cells were pulsed for 24 hours with full-length ovalbumin protein.
  • (B) Flow cytometry quantification of CD45 and HLA-DR expression in human cancer cell lines 3 days after transduction with adenoviral vectors serotype 5 encoding for PIB followed by WPRE or the mutated derivative mut6 (mut6WPRE or WPREmut6) at four different MOIs (1x102, 5x102, 1x103 and 5x103 IFU/cell).
  • (C) Representative flow cytometry plots showing reprogramming efficiency using a MOI of 1x10 3 IFU/cell at day3 (n 2). Mean ⁇ SD are represented. P6783PC01 Figure 3. The Rabbit beta-globin polyadenylation signal (rBGpA) Post- Transcriptional Regulatory Element enhances cDC1 reprogramming efficiency.
  • A Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding for PU.1, IRF8 and BATF3 containing different types of polyadenylation signals 3 days after transduction of melanoma A2058, sarcoma SK- LMS-1, glioblastoma T98G and head and neck Ca9-22 human cancer cell lines using four different MOIs (1x102, 5x102, 1x103 and 5x103 IFU/cell) and (B) 9 days after transduction in breast B0845 and melanoma M2778 human primary samples transduced at two different MOIs (1x10 2 and 1x10 3 IFU/cell), measured as percentage of transduced cells expressing CD45 and HLA-DR.
  • BGH Bovine Growth Hormone
  • TK Herpes Simplex Virus type 1 Thymidine kinase
  • Short synthetic based on the highly efficient polyA signal of the rabbit beta-globin gene
  • SV40late Viral Simian virus 40 late polyA terminator element
  • rBG or rbBG rabbit beta-globin
  • hGH human Growth Hormone.
  • Figure 4 Ad5-PIB vector with SFFV promoter, PIB polycistronic cassette, WPREmut6 and rbBG polyadenylation signal allows higher cDC1 reprogramming efficiency at low multiplicity of infection and superior efficacy.
  • Mean ⁇ SD are represented.
  • CRISPR/Cas9 screening identifies barriers and facilitators of cDC1 reprogramming.
  • A Experimental layout to reprogram human fibroblasts into DC1 cells. HDFs of four donors were transduced with Cas9 lentiviral particles and selected with blasticidin for 8 days. Then, cells were transduced with the KORE sgRNA library co- expressing GFP (KORE Cas9 HDFs). Later, cells were transduced with SFFV polycistronic lentiviral vector comprising PU.1, IRF8 and BATF3 (PIB) sequences. On day 9 of reprogramming, induced DC1 (iDC1) cells were analyzed by flow cytometry to assess the surface expression of CD45 and HLA-DR.
  • iDC1 induced DC1
  • Enriched genes in non-reprogrammed cells are defined as facilitators (highlighted dark dots, bottom right corner of panel D) and enriched genes in reprogrammed cells identify barriers of reprogramming (highlighted dark dots, upper leftblu e corner of panel D).
  • E Rank distribution of candidate genes for hemogenic reprogramming barriers and facilitators according to FC difference between reprogrammed and non-reprogrammed cells, using a threshold of 1.5 SD.
  • P6783PC01 Figure 6 shRNA screening identifies kinases and phosphates that facilitate or impede dendritic cell reprogramming.
  • A Experimental design to screen for kinases and phosphatases that inhibit or promote dendritic cell (DC) reprogramming.
  • Clec9a- tdTomato Clec9a Cre/+ Rosa26 tdTomato/+ ) double transgenic mouse embryonic fibroblasts (MEFs) were transduced with lentiviral particles encoding individual shRNAs for kinases (PKs) and phosphatases (PPs).
  • the library contains 206 shRNAs.
  • MEFs were transduced with FUW-tetO-PIB (PU.1+IRF8+BATF3) and FUW-M2rtTA inducible expression system.
  • GFP, tdT and MHC-ll expression was assessed by flow cytometry 6 days after the addition of Doxycycline (Dox).
  • Targets that led to a significant (p-value ⁇ 0.05) increase (termed barriers, Camkk2, Ppp4c, Gsk3a, Taok2, MikL, in upper panel, and Gsk3a, Ppp2rb1, in lower panel) or decrease (named facilitators, Melk, Spi1, Hspb8, Nuak2 in upper panel, and Pkrd2, Lats2, Prkaa2, Tnk1, Mlk4 in lower panel) of said activation and/or expression are highlighted in grey. Dotted horizontal line represents p- value of 0.05.
  • D Venn diagram showing the barriers (top) and facilitators (bottom) of DC reprogramming in terms of tdT and/or MHC-II expression.
  • E Human dermal fibroblasts (HDFs) were incubated with chemical compounds or DMSO control. Incubation started 3 days prior to PIB transduction and was maintained until day 9, when cells were analyzed by flow cytometry for the expression of the DC markers CD45, HLA- DR and CD40. Media with drugs was changed every 3 days.
  • F Total counts of CD45+HLA-DR+CD40+ cells relative to DMSO on day 9 following cell incubation with increasing concentrations of STO-609, CHIR99021 and Rapamycin. Each datapoint represents a different HDF donor.
  • Figure 7 Knock out of RYBP and SND1 enables higher cDC1 reprogramming efficiency.
  • FIG. 1 Venn diagram plot showing overlap between barriers of cell fate reprogramming towards iDC1s, iHSPCs and iPSCs.
  • RYBP is one of the 3 barriers conserved across the 3 reprogramming systems.
  • SND1 is a barrier conserved across iDC1 and iPSC reprogramming.
  • B Flow cytometry quantification of cDC1 reprogramming efficiency in human dermal fibroblasts (HDFs) knockout (KO) for RYBP P6783PC01 relative to HDFs KO for the safe harbor AAVS19 days after overexpression of PU.1, IRF8 and BATF3 (PIB).
  • (C) Flow cytometry quantification of cDC1 reprogramming efficiency in HDFs KO for SND1 and in HDFs KO for AAVS1 (control) 3, 6, 9, 12 and 16 days after overexpression of PIB.
  • (A) Flow cytometry quantification of cDC1 reprogramming efficiency measured by surface expression of CD45 and HLA-DR 9 days after transduction of primary glioblastoma cells and human cancer cell lines of melanoma, ovarian and gastric cancer with lentiviral vectors encoding PU.1, IRF8 and BATF3 (PIB).
  • Valproic acid HDAC inhibitor
  • Azacitidine Aza, DNA methylation inhibitor
  • mice were injected subcutaneously with YUMM1.7 melanoma cells 4 days after transduction with PIB-eGFP or control eGFP and mixing with parental cells (30% transduced and 70% non-treated cells) to induce tumor cell reprogramming in vivo along with tumor establishment.
  • YUMM1.7 cells were also treated with VPA. Tumor growth (upper panel) and mice survival (bottom panel) are shown.
  • AMPK AMP-activated protein kinase
  • mTOR mechanistic target of rapamycin
  • energy stress signals such as increased AMP/ATP ratio (via LKB1) and calcium signaling (via CAMKK2), which can be stimulated by TLR stimulation, increased intracellular calcium (Ca2+), and reactive oxygen species (O2 ⁇ ).
  • mTOR activity is modulated by multiple kinases including Ppp2r1b, GSK3a, and Map4k3. Key inhibitors and activators of these pathways are shown.
  • AMPK activators CHIR99021, AICAR, A769662; AMPK inhibitors: Compound C; mTOR inhibitors: Torin1, AZD2014, Rapamycin; mTOR activators: MHY1485, Salidroside; CAMKK2 inhibitor: STO-609; Metabolic modulators: Metformin (activates AMPK), 2-DG (2-deoxyglucose, inhibits glycolysis).
  • B Flow cytometry quantification of cDC1 reprogramming yield 9 days after transduction with lentiviral particles encoding PU.1, IRF8 and BATF3, and incubation with small molecules. Number of reprogrammed cells expressing CD45/HLA-DR relative to DMSO control are shown.
  • (C) Flow cytometry analysis and quantification of TNFa within reprogrammed CD45+HLA-DR+.
  • (D) ELISA-based quantification of IL-12p70 levels in the supernatant of reprogrammed cells treated with A769662 from day 0 to day 9, followed by overnight stimulation with Poly(I:C).
  • (E) Schematic representation of in vivo experiment where B16-F10 cells were transduced in vitro with PIB-encoding lentiviral vectors, mixed 1:1 ration with non-transduced cancer cells, and injected subcutaneously to allow tumor establishment and investigate in vivo efficacy. A769662 was injected intraperitoneally at days 1, 3 and 5 after initial tumor establishment.
  • biologically active variant refers herein to a biologically active variant of a genetic element such as of a regulatory element, of a transcription factor (TF), or of a reprogramming modulator, which retains at least some of the functional activity of the P6783PC01 parent genetic element, TF or reprogramming modulator.
  • the term encompasses variants at the polypeptide level, including protein isoforms, that exhibit a minimum of 90% sequence similarity to the parent sequence. These variants may differ in their efficiency of inducing or inhibiting gene expression compared to the parent TF or modulator.
  • BATF3 Basic Leucine Zipper ATF-Like Transcription Factor 3
  • IRF8 Interferon Regulatory Factor 8
  • PU.1 can act as said respective TF and induce or inhibit expression of the same genes in a cell as BATF3, IRF8, and PU.1, respectively, although the efficiency of the induction may be different, e.g. the efficiency of inducing or inhibiting genes is decreased or increased compared to the parent TF.
  • Identity and “homology”, with respect to a polynucleotide or polypeptide are defined herein as the percentage of nucleic acids or amino acids in the candidate sequence that are identical or homologous, respectively, to the residues of corresponding native nucleic acids or amino acids, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity / similarity / homology, and considering any conservative substitutions according to the NCIUB rules (hftp://www.chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985)) as part of the sequence identity.
  • reprogramming herein refers to converting or transdifferentiating any type of cell into a dendritic cell, such as a conventional type 1 dendritic cell (cDC1), or an antigen-presenting cell.
  • a dendritic cell such as a conventional type 1 dendritic cell (cDC1), or an antigen-presenting cell.
  • P6783PC01 “affiliation” as used herein refers to the classification or assignment of individual cells into specific categories or groups based on their gene expression profiles. The affiliation can be performed for instance by the methods described herein, for example involving using a support vector machine (SVM) classifier to predict or determine the identity or type of cells (such as dendritic cells) based on patterns found in their gene expression profiles as compared to known reference datasets.
  • SVM support vector machine
  • Treating refers herein to any administration or application of a therapeutic for the disclosed diseases, disorders and conditions in subject, and includes inhibiting the progression of the disease, slowing the disease or its progression, arresting its development, partially or fully relieving the disease, or partially or fully relieving one or more symptoms of a disease.
  • adenovirus is used to refer to any and all viruses that may be categorized as an adenovirus, including any adenovirus that infects a human or a non-human animal, including all groups, subgroups, and serotypes, except when required otherwise.
  • adenovirus refers to the virus itself or derivatives thereof and cover all serotypes and subtypes, naturally occurring (wild- type), modifications to be used as an adenoviral vector, e.g., a gene delivery vehicle, forms modified in ways known in the art, such as for example capsid mutations, and recombinant forms, replication-competent, conditionally replication-competent, or replication-deficient forms, except where indicated otherwise.
  • 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 may be categorized as an adeno-associated virus, including any adeno-associated virus that infects a human or a non-human animal, and covers all subtypes, serotypes and pseudotypes, and both naturally occurring, modified and recombinant forms, such as modifications to be used as an adeno-associated viral vector, e.g., a gene delivery vehicle except where required otherwise.
  • Ad in the context of a viral vector refers to an adenoviral vector and is typically followed by a number indicating the serotype of the adenovirus.
  • Ad5 refers to adenovirus serotype-5 vectors.
  • P6783PC01 The term “Ad”, when not followed by a specific number, covers any Ad suitable for the purpose may be used herein, such as but not limited to Ad from any serotype from any of the A, B, C, D, E, F, G Ad subgroups, for example Ad2, Ad5, or Ad35, avian Ad, bovine Ad, canine Ad, caprine Ad, equine Ad, primate Ad, non-primate Ad, and ovine Ad.
  • Primary Ad refers to Ad that infect primates
  • non-primate Ad refers to Ad that infect non-primate mammals
  • biovine Ad refers to Ad that infect bovine mammals.
  • AAV in the context of a viral vector refers to an adeno-associated virus and is typically followed by a number indicating the serotype of the adeno-associated virus.
  • AAV2 refers to adeno-associated virus serotype 2.
  • AAV AAV serotype 1
  • AAV2 AAV serotype 2
  • AAV3A AAV serotype 3A
  • AAV3B AAV serotype 4
  • AAV4 AAV serotype 5
  • AAV6 AAV serotype 6
  • AAV7 AAV serotype 7
  • AAV8 AAV8
  • AAV serotype 9 AAV9
  • AAV 10 AAV10
  • avian AAV bovine AAV
  • bovine AAV canine AAV, caprine AAV, equine AAV, primate AAV, non- primate AAV, and ovine AAV.
  • Prime AAV refers to AAV that infect primates
  • non- primate AAV refers to AAV that infect non-primate mammals
  • bovine AAV refers to AAV that infect bovine mammals.
  • TRs native terminal repeats
  • Rep proteins Rep proteins
  • capsid subunits capsid subunits
  • Ad5/3 and Ad5/F35 as described herein are examples of such hybrid or chimeric Ad vectors.
  • “AAV2-qYF” or “AAV2-QuadYF” as used herein refers to a quadruple tyrosine to phenylalanine mutant of AAV2.
  • P6783PC01 “AAV-DJ” as used herein refers to a hybrid capsid derived from DNA family shuffling of 8 wild type serotypes of AAV, including AAV 2, 4, 5, 8, 9, avian, bovine and caprine AAV.
  • AAV-DJ is a synthetic serotype, type 2/type 8/type 9 chimera, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids.
  • rBGpA or “rbBGpA” as used herein refer to the Rabbit beta-globin polyadenylation signal.
  • SV40late as used herein refers to the Viral Simian virus 40 late polyA terminator element.
  • WPREmut6 as used herein refers to the mutated Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element sequence carrying a mutation disturbing the expression of a truncated Woodchuck hepatitis virus X protein implicated in liver tumors, as first described in Kingsman et al.2005.
  • Reprogramming modulator refers to modulators of cell reprogramming, such as reprogramming to cDC1 cells, and comprise “barriers”, negative regulators, which reduce or prevent cell reprogramming and “facilitators”, positive regulators, which enhance or activate cell reprogramming.
  • Reprogramming modulators include, but are not limited to, chromatin regulators, epigenetic modifiers, RNA modifiers, and elements of signalling pathways and biological processes, such as enzymes, e.g. kinases and phosphatases.
  • gene expression inhibitors refer to elements reducing, preventing, or blocking the expression of genes, such as reducing, preventing, or blocking the expression of barrier genes.
  • barrier genes refer to genes negatively regulating cell reprogramming. Barrier genes may for instance be as described in Examples 18 and 19 herein, such as as identified herein using CRISPR/Cas9 screening or shRNA screening.
  • gene expression enhancers refer to elements increasing, activating, or triggering the expression of genes, such as increasing, activating, or triggering the expression of facilitator genes.
  • P6783PC01 As used herein “facilitator genes” refer to genes positively regulating cell reprogramming.
  • Facilitator genes may for instance be as described in Examples 18 and 19 herein, such as as identified herein using CRISPR/Cas9 screening or shRNA screening.
  • Identification of barrier and facilitator genes may preferably be performed at day 9 of cell reprogramming (e.g.9 days after cell transduction with vectors encoding transcription factors as described herein).
  • the inventors have identified barriers to cDC1 reprogramming and demonstrated that knock out or downregulation of said barriers enhances cDC1 reprogramming efficiency.
  • activation of the expression of the facilitator genes enhanced cDC1 reprogramming.
  • the present invention provides one or more constructs, which upon expression encode: - one or more gene expression inhibitors of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more barrier genes selected from the group consisting of: chromatin
  • GATA2 may be of the sequence set forth in SEQ ID NO 17 or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 17.
  • the facilitators gene and barriers genes are as measured by Tdt expression and/or MHC-II expression as reprogramming efficiency and/or fidelity readout, as indicated in the Examples and Figures herein.
  • CAMKK2 and/or PRKAA2 may act as facilitator based on one readout and as barrier based the other readout.
  • the skilled person will know that reprogramming mechanisms underlying the different readouts may be different.
  • GATA2 may be encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 16.
  • the present invention provides one or more constructs, which upon expression encodes at least two transcriptions factors for reprogramming or inducing a cell, wherein the constructs further encode: - one or more gene expression inhibitors inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 P6783PC01 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, P
  • said constructs are: - overexpression constructs, such as constructs overexpressing said one or more gene expression enhancers, or CRISPR activation constructs targeting said one or more facilitator genes; - knockdown constructs, such as shRNA constructs, miRNA-encoding constructs, or CRISPR interference constructs targeting said one or more barrier genes; and/or - gene editing constructs, such as CRISPR-Cas9 constructs targeting said one or more barrier genes.
  • the gene expression enhancers and/or the gene expression inhibitors are selected from the group consisting of: RNAs, proteins and/or genes. P6783PC01 Method for gene expression inhibition are available to the skilled person.
  • shRNA short hairpin RNA
  • RNAi RNA interference
  • shRNAs are typically 19-29 nucleotide-long sequences complementary to a target mRNA.
  • shRNA are transcribed in the nucleus and processed by the Dicer enzyme in the cytoplasm to produce short interfering RNA (siRNA).
  • siRNA are incorporated in the RNA-induced silencing complex (RISC), which binds to the target mRNA and activates its degradation, thereby silencing the expression of the gene product it encodes.
  • RISC RNA-induced silencing complex
  • shRNA can be delivered for instance by vectors, such as plasmids, viral vectors, or nanoparticles.
  • RNAs are shRNA, miRNA, siRNAs and/or antisense oligonucleotides (ASOs).
  • the proteins are transcription factors, epigenetic modifiers, such as histone-modifying enzymes, and/or CRISPR systems, such as CRISPR editing, interference and/or activation systems.
  • - the chromatin remodelling factor RB Binding Protein-4 (RBBP4) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 18;
  • - the splicing factor 3B subunit 6, (SF3B6) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 20;
  • CAMKK2 calcium/calmodulin-dependent protein kinase kinase 2
  • - the chromatin remodelling factor RB Binding Protein-4 comprises or consists of the polypeptide sequence set forth in SEQ ID NO 19, o r a variant thereof having at least 90% sequence identity to SEQ ID NO: 19, 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 to SEQ ID NO: 19;
  • - the splicing factor 3B subunit 6, (SF3B6) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 21, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 21, 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 to SEQ ID NO: 21;
  • - the calcium/calmodulin-dependent protein kinase kinase 2 comprises or consists of the polypeptide sequence set forth in SEQ ID NO 19, o r
  • the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and the at least two transcription factors, such as at least three transcription factors, are selected from the group consisting of: a ) PU.1, b) IRF8, and c) BATF3
  • the one or more constructs upon expression encode PU.1, or a biologically active variant thereof, at least 90% identical to SEQ ID NO: 10, 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% identical to SEQ ID NO: 10.
  • the one or more constructs upon expression encode GATA2, or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 17.
  • PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, 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 to SEQ ID NO: 9
  • IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, 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 to SEQ ID NO: 11.
  • BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13, 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 to SEQ ID NO: 13.
  • GATA2 is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 16.
  • the one or more constructs which upon expression encode at least two transcription factors selected from the group consisting of: PU.1, IRF8 and BATF3.
  • the one or more constructs, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1, IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the p osttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late).
  • SFFV spleen focus-forming virus
  • the one or more constructs encode upon expression PU.1, IRF8, BATF3 and GATA2.
  • the present disclosure shows that enforced gene activation by IRF8 and gene inactivation by BATF3 enables higher cDC1 reprogramming efficiency, such as measured by CD45 expression in the cells transduced by constructs comprising IRF8 further fused to the VP16 activation domain (transcriptional activation domain of herpes simplex virus protein VP16 (Triezenberg et al., 1988; Cousens et al., 1989), and/or comprising BATF3 fused to the KRAB repressor domain (Krüppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994).
  • VP16 activation domain transcriptional activation domain of herpes simplex virus protein VP16 (Triezenberg et al., 1988; Cousens et al., 1989)
  • KRAB repressor domain K
  • IRF8 is fused to the VP16 activation domain.
  • the VP16 activation domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 38, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 38.
  • the VP16 activation domain is encoded by the sequence set forth in SEQ ID NO: 39, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 39, 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% identical to SEQ ID NO: 39.
  • BATF3 is fused to the KRAB repressor domain.
  • the KRAB repressor domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 40, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 40.
  • the KRAB repressor domain is encoded by the sequence set forth in SEQ ID NO: 41, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 41, 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% identical to SEQ ID NO: 41.
  • Vectors encoding constructs modulating cell reprogramming In another aspect, the present invention provides one or more vectors comprising the one or more constructs of the first or second aspects of the present invention.
  • the one or more vectors comprising the one or more constructs of the first or second aspects of the present invention may otherwise be as described in the section “Vectors”.
  • Cell comprising constructs or vectors modulating cell reprogramming In another aspect, the present invention provides a cell comprising the one or more constructs of the first and second aspect of the present invention or the one or more vectors of the third aspect of the present invention.
  • the present invention provides a method of reprogramming or inducing a cell into a reprogrammed or induced cell, the method comprising the following steps: a ) transducing a cell with one or more constructs or vectors, which upon expression encodes at least two transcription factors reprogramming or inducing said cell; b ) inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, R ABGAP1L, EZ
  • - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- d ependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2); and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: Protein Polybromo-1 (PBRM1).
  • RBBP4 chromatin remodelling factor RB Binding Protein-4
  • SF3B6 splicing factor 3B subunit 6,
  • CAMKK2 calcium/calmodulin- d ependent protein kinase kinase 2
  • PRKAA2 AMP activated protein kinase-alpha2
  • PBRM1 Protein Polybromo-1
  • - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: WAC, UBE2I, EID2B, DNMT1, S UV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, and EZH2; and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, and RAD54L.
  • P6783PC01 This may be for instance the case in embodiments of the method making use of CRISPR approaches, such as making use of CRISPR approaches as gene expression enhancers (for instance CRISPR activation) and/or gene expression inhibitors (such as CRISPR interference or editing approaches).
  • CRISPR approaches such as making use of CRISPR approaches as gene expression enhancers (for instance CRISPR activation) and/or gene expression inhibitors (such as CRISPR interference or editing approaches).
  • - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: CAMKK2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, and P PP2R1B, RYBP, and SND1; and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB,
  • step a- c) together comprise transducing the cell with the one or more constructs of the first and second aspects of the present invention, or vectors comprising thereof of the third aspect of the present invention.
  • step b) of inhibiting comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression inhibitors inhibiting the expression of one or more barrier genes of the first and second aspect of the present invention, or vectors comprising thereof of the third aspect of the present invention.
  • step c) of activating comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression enhancers activating the expression of one or more facilitator genes of the first and second aspects of the present invention, or vectors comprising thereof of the third aspect of the present invention.
  • the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and the one or more constructs or vectors of step a), encode upon expression at least two transcription factors, such as at least three transcription factors, selected from the group consisting of: a ) PU.1, b) IRF8, and c) BATF3
  • step b) of inhibiting may comprise transducing the cell with the one or more constructs, which upon expression encode one or more gene expression inhibitors inhibiting the expression of one or more barrier genes of the first and second aspect of the present invention, such as RYBP and/or SND1, or vectors comprising thereof of the fifteenth aspect of the present invention.
  • RYBP is also known as RING1 And YY1 Binding Protein
  • SND1 is also known as Staphylococcal Nuclease And Six Domain Containing 1.
  • the step of inhibiting and/or activating the expression of one or more genes of steps b) and c) respectively is performed in parallel, before and/or after the step a) of transducing the cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a murine cell.
  • the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell.
  • the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a mesenchymal stem cell and a hematopoietic stem cell.
  • the differentiated cell is any somatic cell.
  • the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte.
  • the method further comprises culturing the transduced cell in a media comprising one or more epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors or methylation inhibitors, or contacting the transduced cell with one or more epigenetic modifiers such as histone deacetylase (HDAC) inhibitors.
  • HDAC histone deacetylase
  • the treatment, by culturing in a medium comprising epigenetic modifiers or contacting the transduced cells with epigenetic modifiers may be performed only during the transduction step, only after the transduction step, or both during transduction step and after the transduction step.
  • the treatment with epigenetic modifiers may preferably be performed starting from the transduction step (day 0) and prolonged for 2 days, preferably 3 days following the transduction step (day 3). In preferred embodiments, treatment with epigenetic modifiers is performed between day 0 (initiation of the transduction step) and day 3.
  • the one or more HDAC inhibitor is selected from the group consisting of: valproic acid (VPA), Vorinostat, Romidepsin, Belinostat, and Panobinostat. The skilled person will know that other suitable HDAC inhibitor known in the art may be used.
  • the one or more methylation inhibitor is Azacitidine (Aza).
  • the step of culturing or contacting the cells with valproic acid or azacitidine individually may be beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, melanoma cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells.
  • the treatment with epigenetic modifiers comprises a combination of valproic acid and azacitidine.
  • this combination is beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, ovarian cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells.
  • the inventors found that the further inhibition of PRC2 (polycomb repressive complex 2), the complex responsible for depositing H3K27me3 (the complex that has histone methyltransferase activity and primarily methylates lysine 27 on histone H3 protein) enhanced reprogramming efficiency and fidelity.
  • PRC2 polycomb repressive complex 2
  • H3K27me3 the complex that has histone methyltransferase activity and primarily methylates lysine 27 on histone H3 protein
  • the method further comprises culturing the transduced cell in a media comprising one or more PRC2 inhibitors, such as EZH2-specific inhibitors, for example Tazemetostat and/or GSK-126, or such as EED inhibitors, for example EED226.
  • PRC2 inhibitors such as EZH2-specific inhibitors, for example Tazemetostat and/or GSK-126, or such as EED inhibitors, for example EED226.
  • the step of culturing the transduced cell in a media comprising the one or more PRC2 inhibitors is performed from day 0 to day 2, wherein day 0 is the day of the cell transduction step.
  • the method further comprises culturing the transduced cell in a media comprising one or more AMPK activators, such as CHIR99021, AICAR, A769662, or Metformin.
  • the method further comprises culturing the transduced cell in a media comprising one or more mTOR activators, such as MHY1485 or Salidroside. In some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more glycolysis inhibitors, such as 2-deoxy-D-glucose (2-DG).
  • day 0 is the day of the transduction step and the step of culturing the transduced cell is performed from day 0 to day 9.
  • the step of culturing the transduced cell in the one or more AMPK activators, the one or more mTOR activators, or the one or more glycolysis inhibitors is performed from day 0 to day 9.
  • the present invention provides a reprogrammed or induced cell obtained by the method of the fifth aspect of the present invention.
  • the cell is an induced dendritic or antigen-presenting cell, such as a type 1 conventional dendritic cell.
  • the cell is positive for CD45 and/or negative for HLA-DR, preferably the cell is positive for CD45 and HLA-DR. This is in particular the case for methods herein aiming at reprogramming cells to cDC1 cells.
  • Vectors The constructs of the present invention find applications in the field of, but not limited to, gene therapy and cell therapy.
  • vectors such as vectors comprising further elements useful for gene delivery, expression, stability.
  • Techniques for producing vectors, such as adenoviral vectors comprising defined constructs are known to the skilled person, and typically involve cloning the constructs comprising the gene(s) of interest into a plasmid or cosmid vector and recombination P6783PC01 with a viral vector backbone such as an adenoviral backbone, transfection in host cells such as HEK293 cells for packaging, and amplification of adenoviral particles, followed by purification of the recombinant adenoviral particles and quality control.
  • vectors may be added to said vectors, such as multiple cloning sites, for example a multiple cloning site (MCS) inserted in place of the E3 adenoviral region, such as a 69-bp MCS sequence (SEQ. ID NO: 34) inserted in place of the E3 region between the XbaI sites located in the Ad5 E3 region, thereby deleting 1.9 kb from the genome.
  • MCS multiple cloning site
  • SEQ. ID NO: 34 69-bp MCS sequence
  • the present invention provides one or more vectors comprising the one or more constructs of the present disclosure.
  • the one or more constructs of the present invention may be polycistronic or monocistronic, for example each construct may encode 1, 2, or 3 transcription factors, such as PU.1, IRF8 and/or BATF3.
  • the one or more vectors comprising the one or more constructs of the present disclosure may be for example one vector comprising one construct encoding the transcription factors PU.1, IRF8 and BATF3, or more than one vectors together encoding PU.1, IRF8 and BATF3, such as 2 vectors, such as 3 vectors.
  • the one or more vectors is a viral vector.
  • the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retrovirus vectors, herpes virus vectors, pox virus vectors, adeno-associated virus vectors, paramyxoviridae vectors, rabdoviral vectors, alphaviral vectors, flaviral vectors, and adeno-associated viral vectors.
  • the viral vector is an adenoviral (Ad) vector.
  • the adenoviral vector is selected from the group consisting of: wild-type Ad vectors, chimeric Ad vectors, and mutant Ad vectors.
  • the wild-type Ad vector is Ad5.
  • the Ad vector is selected from the group consisting of: Ad5-RGD, Ad5/F35 and Ad5/3, preferably wherein the Ad vector is Ad5/F35, even more preferably wherein the Ad vector is Ad5 or Ad5-RGD.
  • the viral vector is a lentiviral vector.
  • the adeno-associated virus vector is selected from the group consisting of : wild-type AAV vectors, hybrid AAV vectors and mutant AAV vectors.
  • the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV2-QuadYF.
  • the vectors of the present invention may be preferable that native restriction sites present on the vector sequence and targeted by restriction enzymes are mutated, for example to facilitate proper cloning of the vector elements, such as directional cloning.
  • one or more SfiI sites have been mutated, preferably by silent mutations, even more preferably wherein said SfiI sites are in the pVII ORF and/or the adenovirus DNA-binding protein (DBP) ORF the vector.
  • the vector comprises an Ad5 wild-type fiber.
  • the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at P6783PC01 least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1.
  • the vector Ad5 wild-type fiber is replaced by the fiber of Ad35 (Ad5/F35).
  • the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 2.
  • the vector Ad5 wild-type fiber is modified by incorporating an RGD (arginine-glycine-aspartic acid) motif into the fiber knob protein of said vector (Ad5-RGD).
  • RGD arginine-glycine-aspartic acid
  • Ad5-RGD cellular internalization of Ad5-RGD vectors is mediated by the CD51 receptor (integrin ⁇ v), such as by the CD51 subunit of ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins.
  • the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 3.
  • the vector Ad5 wild-type fiber knob is replaced by an Ad3 fiber knob (Ad5/3).
  • Ad5/3 Ad3 fiber knob
  • the skilled person will appreciate that cellular internalization of Ad5/3 vectors is mediated by CD46 and desmoglein-2 (DSG-2).
  • the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 4.
  • the vectors of the present invention further comprise cosmid and /or plasmid elements, such as a lambda phage cos sequence, and/or lambda phage scrambled sequences.
  • cosmid and /or plasmid elements such as a lambda phage cos sequence, and/or lambda phage scrambled sequences.
  • mice B6.129S(C)-Batf3tm1Kmm/J (BATF3KO, The Jackson Laboratory) and C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT-I, The Jackson Laboratory) mice were bred in-house.
  • C57BL/6J, NOD.Cg-PrkdcSCIDIL2rgtm1Wjl/SzJ (NSG, The Jackson Laboratory) and NOD-PrkdcSCITIL2rgtm1/Rj (NXG, Janvier Labs) females aged 6-8 weeks were purchased from Charles River or Janvier-Labs.
  • mice were housed in a controlled temperature environment (23 ⁇ 2 °C) and a fixed 12-hour light/dark cycle, having free access to food and water. Mice were age-matched, gender-matched and within the same gender randomly assigned to treatment or control groups in all experiments. Numbers of mice for in vivo experiments were determined based on previous expertise, and power analysis was not performed. Mice were sacrificed by cervical dislocation when endpoints were reached. Investigators were not blinded during experimental procedures or the assessment of outcomes.
  • B16-F10, embryonic fibroblasts (MEFs), SKLMS1, CHL1, Ca922, OVK18, MKN74, YUMM1.7 and T98G cell lines, patient-derived cancer cells and dermal fibroblasts (HDFs) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM GlutaMAX, 1mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM complete).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • B16- F10 expressing Ovalbumin (B16-OVA) were maintained in DMEM complete supplemented with 0.4 mg/ml geneticin (Gibco).
  • Mouse Panc02, B2905, MB49, BRAFV600ECOX1/2KO cancer cell lines, mouse CD103+ bone marrow-derived dendritic cells (BM-DC), primary mouse and human T cells were cultured in RPMI 1640 P6783PC01 medium supplemented with 10% (v/v) FBS, 2 mM GlutaMAX, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol and 100 U/ml penicillin and 100 mg/ml streptomycin (RPMI complete).
  • YUMM1.7 melanoma cells were cultured in DMEM/F-12 with 10% (v/v) FBS, 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM/F-12 complete).
  • MDSCs were differentiated from monocytes obtained from PBMCs of healthy donors and cultured in RPMI complete.
  • Human pericytes were cultured in Pericyte medium (ScienCell). Fibroblasts were expanded on tissue-culture plates coated with 0.1% gelatin.
  • the inventors used the empty backbone pRRL.PPT-SFFV-MCS- IRES2 (SFFV-MCS) (Rosa et al.2022, Zimmermannova et al.2023) and inserted the coding sequence for mCherry by infusion cloning downstream the IRES sequence to generate pRRL.PPT-SFFV-MCS-IRES2-mCherry (SFFV-mCherry).
  • coding sequences of mouse PU.1 and C/EBPa (PC) separated by a T2A sequence were cloned first into the MCS of the pFUW-tetO-MCS vector (Rosa et al. 2022) followed by subcloning of the polycistronic cassette into the MCS of the pRRL.PPT-SFFV-MCS-IRES2-eGFP vector (PC-eGFP).
  • Adenoviral vectors (Ad) were cloned and produced at VectorBuilder.
  • Replication-deficient adenoviral vectors pAd5-SFFV-PU.1-P2A-IRF8-T2A-BATF3 (Ad- PIB) and pAd5-SFFV-PU.1-P2A-IRF8-T2A-BATF3-CMV-eGFP (Ad-PIB-eGFP) with an eGFP sequence under the control of constitutive cytomegalovirus (CMV) promoter were generated.
  • CMV cytomegalovirus
  • pAd5-CMV-eGFP (Ad-eGFP), pAd5-SFFV-Stuffer (Ad-Stuffer) and pAd5-SFFV-Stuffer-CMV-eGFP (Ad-Stuffer-eGFP) were cloned and used as controls.
  • the stuffer sequence was derived from the genome of E. Coli as a non-coding sequence and designed to have the same base pair length as polycistronic PIB.
  • P6783PC01 Viral production Transfer plasmids encoding PU.1, IRF8 and BATF3 followed by IRES-eGFP (PIB- eGFP), eGFP, PIB-mCherry, mCherry, mOrange and bicistronic PU.1 and C/EBP ⁇ followed by IRES-eGFP (PC-eGFP) were used to produce lentiviral vectors.
  • IRES-eGFP PIB- eGFP
  • PC-eGFP IRES-eGFP
  • HEK 293T cells were seeded in 15 cm plates to reach ⁇ 80% confluency and transfected with 7.5 ⁇ g packaging plasmid (psPAX2), 2.5 ⁇ g VSV-G-encoding envelope plasmid (pMD2), and 10 ⁇ g transfer plasmid combined with 60 ⁇ l of 1 mg/ml polyethyleneimine (PEI) in Opti- MEM.
  • Virus-containing supernatants were collected after 36, 48, and 72 hours, filtered using 0.45 ⁇ m low protein binding cellulose acetate filters and concentrated 100-fold with Lenti-X Concentrator (Takara) before storage at -80°C.
  • virus- containing supernatants were ultracentrifuged for 90 minutes at 4°C with 25,000 g in a SW 32 Ti Swinging-Bucket Rotor (Beckman Coulter). Lentiviral vector pellets were resuspended overnight in DMEM medium and stored in aliquots at -80°C. Lentiviral titers were quantified with the Lenti-X qRT-PCR titration kit (Takara) following the manufacturer’s protocol. In experiments using lentiviral vectors for in situ transduction, in vivo grade lentiviral particles were produced at VectorBuilder based on the third-generation system.
  • HEK 293T cells were transfected with eGFP encoding transfer plasmid, envelope plasmid encoding VSV-G and two packaging plasmids encoding Gag/Pol and Rev.
  • the supernatants were collected, and cell debris removed via centrifugation and filtration.
  • Lentiviral particles were subsequently concentrated using polyethylene glycol (PEG) precipitation and further purified through sucrose cushion ultracentrifugation.
  • Lentiviral titers were determined by quantifying the lentiviral p24 Gag protein using ELISA.
  • Ad vectors encoding for PIB (Ad-PIB or Ad5-PIB, Ad5-RGD-PIB, Ad5/F35- PIB, Ad5/3-PIB,) or PIB and eGFP (Ad-PIB-eGFP), eGFP (Ad-eGFP) or a non-coding stuffer sequence with or without eGFP (Ad-Stuffer, Ad-Stuffer-eGFP) were produced at VectorBuilder, Vector Biolabs or O.D.260 Inc, using O.D.260 Inc AdenoQuick 2.0 cloning system for Ad5-PIB, Ad5-RGD-PIB, Ad5/F35-PIB, Ad5/3-PIB.
  • Adenoviral vectors were packaged and amplified in HEK 293A cells.
  • adenoviral plasmids containing PIB, PIB-eGFP, Stuffer or eGFP were first linearized by restriction digestion with Pacl.
  • the linearized plasmid DNA was then transfected into HEK 293A expressing the adenovirus gene E1 to produce recombinant adenovirus.
  • Adenoviral particles P6783PC01 released into the culture medium were harvested and concentrated using cesium chloride (CsCl) gradient ultracentrifugation and/or chromatography.
  • CsCl cesium chloride
  • the viral titer was determined by spectrophotometry (OD260) to quantify the number of viral particles and measured for the number of infective units (IFU) by immunocytochemistry staining for transduced cells via the detection of adenovirus-specific hexon protein.
  • Flow cytometry and Fluorescence-activated cell sorting (FACS) Surface marker analysis was performed on dissociated cells from in vitro 2D cultures or single cell suspensions of digested tissue from spheroids or tissues.
  • Cells were stained with adequate antibodies diluted in phosphate-buffered saline (PBS) supplemented with 2% FBS (FACS buffer) at 4°C for 20-30 minutes in the presence of 1% mouse or rat serum, for human and mouse cells, respectively, to block unspecific binding. Tetramer staining was performed at room temperature for 30 minutes before surface marker staining and cell fixation using 4% paraformaldehyde (PFA, Thermo Fisher Scientific) for 20 minutes at 4°C without permeabilization. Intracellular staining for cytokines or proliferation marker Ki67 was performed using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) following the manufacturer's recommendation.
  • PBS phosphate-buffered saline
  • FACS buffer FACS buffer
  • Intranuclear transcription factor staining was performed using the True-Nuclear Transcription Factor Buffer Set (Biolegend) following the manufacturer's recommendation.
  • FVD fixable viability dye
  • DAPI ⁇ ,6-diamidino-2-phenylindole
  • 7-AAD 7-aminoactinomycin D
  • cDC1 reprogramming efficiency was measured by flow cytometry analysis of CD45 and MHC-II/HLA-DR expression within live or live eGFP+. Macrophage reprogramming efficiency was measured by CD45 and CD11b expression within live eGFP+ cells.
  • the expression of MHC-I/HLA-ABC, the co- stimulatory molecule CD40 and cDC1 markers XCR1, CLEC9A and CD226 was quantified by flow cytometry. Quantification of MHC-I/HLA-ABC surface molecules per cell was performed using the PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences) following manufacturer’s instructions.
  • Transduction with lentiviral vectors was performed in the presence of polybrene (8 P6783PC01 ⁇ g/ml, Sigma-Aldrich).
  • the MOI used for transduction and induction of reprogramming by lentivirus ranged between 5.5x10 7 and 5.0x10 8 GC per cell.
  • mice received 200 ⁇ g of anti-PD-1 (clone RMP1-14, BioXCell) and/or 200 ⁇ g of anti-CTLA-4 (clone 9H10, BioXCell) or rat 200 ⁇ g IgG2a (clone 2A3, BioXCell) and IgG2b (clone LTF-2, BioXCell) isotype control antibodies diluted in 100 ⁇ l PBS intraperitoneally at days 7, 10, and 13 after tumor establishment.
  • anti-PD-1 clone RMP1-14, BioXCell
  • CTLA-4 clone 9H10, BioXCell
  • rat 200 ⁇ g IgG2a clone 2A3, BioXCell
  • IgG2b clone LTF-2, BioXCell
  • Ad vectors were diluted in ice-cold PBS to reach a final volume of 30 ⁇ l and intratumorally injected when the size of tumors reached 30–90 mm3. Tumors that did not reach the required sizes were excluded from the experiment.
  • 1010 VPs of Ad-PIB or Ad-Stuffer were injected intratumorally at day 7, 9, 11 and 13 post tumor establishment.
  • tumors were isolated, dissociated, and reprogramming efficiency quantified by flow cytometry.
  • Statistical analysis All statistical analyses were performed using GraphPad Prism or R software.
  • wild type WPRE contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX) previously implicated in the development of liver tumors (Kingsman et al.2005), the inventors investigated whether WPRE sequence downstream the PIB tricistronic cassette is required for efficient reprogramming and whether the mut6WPRE, a version of WPRE that carries a mutation disturbing the expression of the WHX protein considered safe from the regulatory perspective (Zanta- Boussif et al. 2009), could be used instead for the gene therapy product.
  • ORF open-reading frame
  • adenoviral vectors containing WPRE sequence allowed higher reprogramming efficiency measured by surface expression of CD45 and HLA- DR mediated by Ad5 and Ad5/F35 at reprogramming day 3 when compared to adenoviral vectors without WPRE sequence (Fig.2A).
  • Fig.2A adenoviral vectors without WPRE sequence
  • the inventors detected higher frequency of reprogrammed cells co-expressing CD45 and HLA-DR using mut6WPRE when compared to native WPRE in the melanoma A2058 P6783PC01 (63.7% ⁇ 1.2 vs 30.25% ⁇ 5.5) and sarcoma SK-LMS-1 (23.5% ⁇ 2.55 vs 3.18% ⁇ 0.88) cell lines at MOI 1x10 3 IFU/cell (Fig.2B, C). Conclusion These data suggests that WPRE sequence downstream the PIB tricistronic cassette is required for higher cDC1 reprogramming efficiency and supports the selection of mut6WPRE sequence for the final gene therapy vector candidate.
  • the polyadenylation signal downstream the WPRE sequence can affect transgene expression and thus impact cDC1 reprogramming efficiency.
  • 6 polyA sequences were tested and investigated for their ability to induce cDC1 reprogramming.
  • the optimized expression cassette containing WPREmut6-rbBGpA allowed a major increase in reprogramming efficiency in human cancer cell lines and in the primary colorectal cancer samples CRC50 at low MOI (100 IFU/cell) when compared to the expression cassette containing WPRE-BGHpA.
  • Cas9-expressing fibroblasts were generated by by lentiviral transduction at a multiplicity of infection (MOI) of ⁇ 1 with the LentiCas9-Blast vector for constitutive expression spCas9 and blasticidin resistance cassette (Addgene #52962). Briefly, cells were plated at the density of 800,000 cells per 100 mm plate and were selected with blasticidin (10 ug/ml) for 9 days and maintained in blasticidin-containing media prior to library transduction. The Human KnockOut RNA regulators and Epifactors (KORE) sgRNA library was cloned into the optimized version of the lentiCRISPR v2 backbone (Addgene #52961) to enhance knockout efficiency.
  • MOI multiplicity of infection
  • KORE Human KnockOut RNA regulators and Epifactors
  • Genes targeted by the sgRNA library were derived from Epifactors database (https://epifactors.autosome.org) and literature on both chromatin and RNA modifiers.
  • the gRNAs sequences targeting the 1367 selected human genes were obtained using the VBC Score design tool (https://www.vbc-score.org), allocating 5 guides per gene, and including non targeting controls.
  • VBC Score design tool https://www.vbc-score.org
  • cells were plated at the density of 800,000 cells per 100 mm plate and 24 hours later transduced with the sgRNA library lentiviral vector to achieve a MOI of ⁇ 0.3-0.4, with at least 1000-fold representation (cells per guide) in each replicate.
  • cells were sorted for GFP expression using FACSAriaIII (BD Biosciences) and expanded for 10 days before reprogramming. Throughout the screenings, cells were split at a density to maintain a representation of at least 300 cells per sgRNA. Screens were performed in 4 independent HDF donors. One million cells were collected before reprogramming to serve as reference point for baseline sgRNA distribution (day 0). At Day 9, puromycin resistant, GFP+ and CD45/HLA-DR stained cells were sorted using the FACS AriaIII (BD Biosciences) and collected pellets were stored at -20°C. Genomic DNA was extracted using the QIAmp Blood Mini Kit P6783PC01 (Qiagen).
  • Amplification of sgRNA regions from the extracted genome was performed by a 2-step PCR reaction protocol, first using custom-made primers harboring the sgRNA region, followed by a second PCR for indexing Illumina Nextera XT adapters (Illumina). Libraries were thoroughly quantified and checked for quality control with dsDNA High-Sensitivity Qubit (Thermo Fisher) and Bioanalyzer (Agilent). The resulting diluted libraries from 3 independent replicates were spiked-in witt 20% PhiX and sequenced using the Illumina NextSeq 500/550 High Output (150 cycles) kit in a NextSeq 500 platform (Illumina) to determine sgRNA representation.
  • sgRNA read count data was mapped to the sgRNA library, allowing a maximum of one mismatch in the 20 bp spacer sequence.
  • Mapped reads was input to the CRISPR screen analysis pipeline MAGeCKFlute. MAGeCK was used to identify gene hits and downstream analysis was performed using FluteMLE, with loess normalization. Results The inventors performed knockout (KO) screenings using the optimized conditions for Cas9 expression, sgRNA library expression, and cDC1 reprogramming. Using the 7,409 guides corresponding to the 1367 genes in the library, an average coverage of 300 HDFs/sgRNA/replicate was kept.
  • Fig.5A Genomic DNA was collected from double positive and double negative (CD45+HLA- DR+, CD45–HLA-DR–) sorted cells, as well as day 0, and sgRNAs were amplified and sequenced (Fig.5C). To determine relative sgRNA abundance, raw read counts were normalized to reads per million and log2 transformed. Then, each sample was normalized to the baseline (day 0) and compared to the distribution of the log2 fold-change (Log2FC) of non- reprogrammed versus reprogrammed samples (Fig.5D, E).
  • Log2FC log2 fold-change
  • MAGeCKFlute pipeline Using the MAGeCKFlute pipeline, the inventors identified top candidate genes by ranking them and selecting those enriched in reprogrammed and non-reprogrammed samples (Fig.5D). Genes with an increased sgRNA count in the reprogrammed samples, which equates to an increase in the fold-change, were defined as reprogramming barriers, since its silencing may lead to better reprogramming efficiency. On the other hand, genes that showed increased fold-change in the non-reprogrammed population were defined as facilitators of DC1 reprogramming (Fig. 5E). Here, the inventors report a total of 117 barriers and 74 facilitators that are over the 1.5 standard deviation (SD) cut-off P6783PC01 compared to the median distribution of the genes.
  • SD standard deviation
  • top 10 barrier candidates the inventors identified ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L.
  • the top facilitator candidates include WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2.
  • shRNA screening identifies barriers and facilitators of reprogramming Background
  • the inventors investigated whether a loss-of-function screening platform with shRNAs could allow the identification of druggable targets and pathways that prevent or promote acquisition of cDC1 identity through direct cell reprogramming.
  • Methods Viral transduction and reprogramming for shRNA screen Clec9a-tdTomato MEFs and HDFs were seeded at a density of 30,000 cells per well on 0.1% gelatin-coated six-well plates.
  • MEFs P6783PC01 were initially transduced with each shRNA and after three days, they were transduced with PIB. Dox was added at day 0 to induce PIB expression and reprogrammed cells were analyzed at day 6 by flow cytometry (Fig. 6A). In addition to GFP and tdT expression, the inventors also assessed MHC-ll expression as an indication for reprogramming fidelity and antigen presentation capacity.
  • the screening includes a non- targeting shRNA (shNTC) as negative control and identified targets whose silencing reduced or improved tdT expression, which we respectively termed DC reprogramming facilitators or barrier (Fig. 18B).
  • Spi1 the gene encoding for the reprogramming transcription factor PU.1
  • Fig. 18B DC reprogramming facilitators or barrier
  • Fig. 18B Silencing of Spi1 (the gene encoding for the reprogramming transcription factor PU.1) resulted in dramatic reduction in tdTomato expression.
  • shRNAs that induced changes of tdTomato expression above or below 0.5-fold when compared to shNTC were re-tested for further validation studies, and their impact was validated in reprogramming efficiency and MHC-ll expression (Fig. 6C).
  • iPSC reprogramming cells were sorted at day 20 based on the surface markers CD13, EpCAM, SSEA4 and TRA-1-60 (Non- reprogrammed cells: CD13+ EpCAM- SSEA4- TRA-1-60-; Reprogrammed cells: CD13- EpCAM+ SSEA4+ TRA-1-60+).
  • induced HSPCs cells were sorted at day 15 based on the surface expression of CD9 and CD49f (Non-reprogrammed: CD9- CD49f-; reprogrammed: CD9+CD49f+). Cells were sorted using the FACS AriaIII (BD Biosciences) and collected pellets were stored at -20°C.
  • Genomic DNA was extracted using the QIAmp Blood Mini Kit (Qiagen). Amplification of sgRNA regions from the extracted genome was performed by a 2-step PCR reaction protocol, first using custom-made primers harboring the sgRNA region, followed by a second PCR for indexing Illumina Nextera XT adapters (Illumina). Libraries were thoroughly quantified and checked for quality control with dsDNA High-Sensitivity Qubit (Thermo Fisher) and Bioanalyzer (Agilent). The resulting diluted libraries from 3 independent replicates were spiked-in with 20% PhiX and sequenced using the Illumina NextSeq 500/550 High Output (150 cycles) kit in a NextSeq 500 platform (Illumina) to determine sgRNA representation.
  • sgRNA read count data was mapped to the sgRNA library, allowing a maximum of one mismatch in the 20 bp spacer sequence.
  • Mapped reads was input to the CRISPR screen analysis pipeline MAGeCKFlute. MAGeCK was used to identify gene hits and downstream analysis was performed using FluteMLE, with loess normalization. Results The inventors performed KO screenings using the optimized conditions for Cas9 expression, sgRNA library expression, and HSPC and iPSC reprogramming.
  • Cells P6783PC01 were reprogrammed to induced HSPCs with overexpression of GATA2, GFI1B and FOS (Gomes et al.2018), and to iPSCs by overexpression of OCT4, Sox2, Klf4 and c- Myc (Takahashi et al.2007), and populations were sorted at endpoint day 15 (iHSPC) or day 20 (iPSC). Genomic DNA was collected from reprogrammed and non- reprogrammed purified cells, as well as day 0, and sgRNAs were amplified and sequenced. First, the inventors identified 82 barriers and 125 facilitators of iPSC reprogramming, and 95 barriers and 111 facilitators of iHSPC reprogramming.
  • the inventors then performed a comparative analysis to explore the relationship between barriers shared in the intersections of reprogramming to cDC1s (Example 18), iHSPCs and iPSCs, and identified 3 barriers that were conserved across reprogramming systems (Fig.7A). Interestingly, KO of one of these 3 barriers (RYBP) enable enhanced cDC1 reprogramming efficiency (Fig.7B).
  • RYBP KO of one of these 3 barriers
  • Fig.7B To validate additional barriers to cDC1 reprogramming, the inventors selected 13 barriers, performed single knockouts and reprogrammed the cells to cDC1-like cells by overexpression of PU.1, IRF8 and BATF3.
  • the inventors investigated whether HDAC inhibition synergizes with inhibition of DNA methylation for enhanced cDC1 reprogramming efficiency.
  • the inventors also explore the impact of HDAC inhibition in in vivo efficacy and in reprogramming mediated by adenoviral vectors.
  • VPA valproic acid
  • P6783PC01 HDAC inhibitor valproic acid
  • Azacitidine Azacitidine
  • PIB lentiviral particles encoding PU.1, IRF8 and BATF3
  • YUMM1.7 cells were transduced with PIB-encoding lentiviral vectors, incubated or not with VPA until reprogramming day 4, mixed with non-treated YUMM1.7 cells (ratio 30% transduced to 70% non-treated cells) and injected subcutaneously in mice to profile tumor growth and survival overtime.
  • VPA treatment enabled higher in vivo efficacy associated with 90% complete tumor regression compared to 20% complete tumor regression in groups injected with transduced cells not treated with VPA (Fig.8B).
  • VPA treatment also enable higher cDC1 reprogramming efficiency in cancer cells mediated by adenoviral vectors.
  • YUMM1.7 cancer cells were transduced with PIB-encoding adenoviral vectors, treated with VPA from day 0 to day 3, and reprogramming efficiency was profiled at day 3.
  • the inventors observed that treatment with VPA also enhanced cDC1 reprogramming efficiency mediated by adenoviral vectors (Fig.8C).
  • HDAC inhibition enables higher in vivo efficacy, and enhances cDC1 reprogramming mediated by adenoviral vectors.
  • Example 10. PRC2 inhibition enhances reprogramming efficiency and fidelity. Background Given that PU.1, IRF8 and BATF3 engage Polycomb-repressed chromatin early during lineage conversion in H3K27me3-marked regions, the inventors sought to determine whether inhibition of PRC2, the complex responsible for depositing H3K27me3, would impact reprogramming efficiency and fidelity.
  • fibroblasts were treated with small molecule inhibitors targeting PRC2 components during the early reprogramming window (day 0 to day 2).
  • P6783PC01 Methods To induce cDC1 reprogramming, human dermal fibroblasts (HDFs) were seeded at 300,000 cells per gelatin-coated plate. The next day, cells were transduced overnight with lentiviral particles in DMEM Complete medium containing 8 ⁇ g/mL polybrene. After 16 hours (day 0), the medium was replaced, and cells were treated with 1 ⁇ M, 5 ⁇ M, or 10 ⁇ M of PRC2 inhibitors or DMSO as a control.
  • HDFs human dermal fibroblasts
  • Inhibitors included EZH2-specific compounds (Tazemetostat and GSK-126) and an EED inhibitor (EED226). Media was refreshed every 2–3 days. On day 9, reprogramming efficiency (% CD45+HLA-DR+ cells) and fidelity (% CD40+CD226+ cells) in live, transduced cells were assessed via flow cytometry. Results Fibroblasts were treated with small molecule inhibitors targeting PRC2 components during the early reprogramming window (day 0 to day 2). Inhibition of PRC2 led to a significant increase in reprogramming efficiency (+11.6%) and fidelity (+11.0%), as measured by the proportion of reprogrammed cells expressing CD45 and HLA-DR, and CD40 and CD226, respectively (Fig. 9).
  • CD45 expression was assessed by flow cytometry at day 9, and fold change (FC) in CD45 induction was calculated relative to wild-type (untagged) TFs.
  • expression of KRAB-fused BATF3 enhanced CD45 induction strongly, suggesting that BATF3 functions as a transcriptional repressor during reprogramming.
  • Example 7 of the present application the inventors have used small chemical inhibitors to validate targets identified in an shRNA screen to be involved in cDC1 reprogramming and observed that chemical inhibition of GSK3A with CHIR99021 and inhibition of mTOR signaling with rapamycin, respectively, increased and decreased cDC1 reprogramming yield on day 9 (Fig.6F). These data suggested that metabolism, energy sensing and AMPK-GSK3-mTOR signalling axis are important regulators of the reprogramming process.
  • the inventors transduced cancer cells in vitro with lentivirus SFFV-PIB-eGFP (expressing PU.1, IRF8, BATF3 and eGFP as a reporter of transduction) or empty lentiviral vector as a transduction control SFFV-eGFP (empty lentiviral backbone expressing eGFP) and 16 hours after transduction injected the cells into C57BL/6J mice as a mix of 1:1 transduced eGFP+ to untransduced eGFP- cells.
  • the transduction was performed in the presence of polybrene (8 ⁇ g/ml).
  • AMPK activators (CHIR99021, AICAR, A769662, Metformin), mTOR activators (MHY1485, Salidroside) and inhibitors of glycolysis (2-DG or 2-deoxyglucose) enabled higher reprogramming efficiency.
  • AMPK inhibitors Compound C
  • mTOR inhibitors Torin1, AZD2014, Rapamycin
  • CAMKK2 inhibitors STO-609 reduced reprogramming efficiency (Fig. 11B).
  • CD45+HLA-DR+ reprogrammed cells generated in the presence of A769662 also produced higher levels of TNF-a and IL- 12p70, 2 cytokines critical for antigen presentation (Fig.
  • the inventors transduced B16-F10 cancer cells in vitro with lentivirus encoding PIB, 16 hours after transduction injected the cells into C57BL/6J mice as a mix of 1:1 transduced eGFP+ to untransduced eGFP- cells, and treated mice with 3 IP injections of A769662 at days 1, 3 and 5 after tumor establishment (Fig.11E-F).
  • the inventors observed a significant increase in in vivo efficacy and delayed tumor growth when in vivo reprogramming was combined with A769662 treatment.
  • RBBP4 chromatin remodelling factor RB Binding Protein-4 (RBBP4) - polynucleotide sequence ATGGCCGACAAGGAAGCAGCCTTCGACGACGCAGTGGAAGAACGAGTGATCAAC GAGGAATACAAAATATGGAAAAAGAACACCCCTTTTCTTTATGATTTGGTGATGAC CCATGCTCTGGAGTGGCCCAGCCTAACTGCCCAGTGGCTTCCAGATGTAACCAGA CCAGAAGGGAAAGATTTCAGCATTCATCGACTTGTCCTGGGGACACACACATCGG ATGAACAAAACCATCTTGTTATAGCCAGTGTGCAGCTCCCTAATGATGATGCTCAG TTTGATGCGTCACACTACGACAGTGAGAAAGGAGAATTTGGAGGTTTTGGTTCAG TTAGTGGAAAAATTGAAATAGAAATCAAGATCAACCATGAAGGAAGTAAACAGG GCCCGTTATATGCCCCAGAACCCTTGTATCATCGCAACAAAGACTCCTTCCAGTG ATGTTCTTGTT
  • RBBP4 chromatin remodelling factor RB Binding Protein-4 (RBBP4) - polypeptide sequence
  • MADKEAAFDDAVEERVINEEYKIWKKNTPFLYDLVMTHALEWPSLTAQWLPDVTRPE GKDFSIHRLVLGTHTSDEQNHLVIASVQLPNDDAQFDASHYDSEKGEFGGFGSVSGKI EIEIKINHEGEVNRARYMPQNPCIIATKTPSSDVLVFDYTKHPSKPDPSGECNPDLRLR GHQKEGYGLSWNPNLSGHLLSASDDHTICLWDISAVPKEGKVVDAKTIFTGHTAVVE DVSWHLLHESLFGSVADDQKLMIWDTRSNNTSKPSHSVDAHTAEVNCLSFNPYSEFI LATGSADKTVALWDLRNLKLKLHSFESHKDEIFQVQWSPHNETILASSGTDRRLNVW DLSKIGEEQSPEDAEDGPPELLFIHGGHTAKISDFSWNPNEPWVICSVSEDNIMQVWQ
  • splicing factor 3B subunit 6 (SF3B6) - polynucleotide sequence ATGGCGATGCAAGCGGCCAAGAGGGCGAACATTCGACTTCCACCTGAAGTAAATC GGATATTGTATATAAGAAATTTGCCATACAAAATCACAGCTGAAGAAATGTATGATA TATTTGGGAAATATGGACCTATTCGTCAAATCAGAGTGGGGAACACACCTGAAACT AGAGGAACAGCTTATGTGGTCTATGAGGACATCTTTGATGCCAAGAATGCATGTG ATCACCTATCGGGATTCAATGTTTGTAACAGATACCTTGTGGTTTTGTACTATAATG CCAACAGGGCATTTCAGAAGATGGACACAAAGAAGAAGGAGGAACAGTTGAAGCT TCTCAAGGAGAAATATGGCATCAACACAGATCCACCAAAATAA SEQ.
  • splicing factor 3B subunit 6 (SF3B6) - polypeptide sequence MAMQAAKRANIRLPPEVNRILYIRNLPYKITAEEMYDIFGKYGPIRQIRVGNTPETRGTA YVVYEDIFDAKNACDHLSGFNVCNRYLVVLYYNANRAFQKMDTKKKEEQLKLLKEKY GINTDPPK P6783PC01 SEQ.
  • PBRM1 Protein Polybromo-1 (PBRM1) - polypeptide sequence MRRLAFRGAGCALVKLKKLDSMGSKRRRATSPSSSVSGDFDDGHHSVSTPGPSRKR RRLSNLPTVDPIAVCHELYNTIRDYKDEQGRLLCELFIRAPKRRNQPDYYEVVSQPIDL MKIQQKLKMEEYDDVNLLTADFQLLFNNAKSYYKPDSPEYKAACKLWDLYLRTRNEF VQKGEADDEDDDEDGQDNQGTVTEGSSPAYLKEILEQLLEAIVVATNPSGRLISELFQ KLPSKVQYPDYYAIIKEPIDLKTIAQRIQNGSYKSIHAMAKDIDLLAKNAKTYNEPGSQV FKDANSIKKIFYMKKAEIEHHEMAKSSLRMRTPSNLAAARLTGPSHSKGSLGEERNPT SKYYRNKRAVQGGRLSAITMALQYGSESEEDAALAAARYEEGESEAESITSFMDVSN
  • SEQ. ID NO: 34 69-bp MCS sequence AGATCTTCTAGACCCGGGAGCGGCCGCTGTCGACCTGCAGGATCCGAATTCGAT ATCACTAGTGGTACC SEQ ID NOs: 35-37 are empty sequences. SEQ.
  • KRAB polynucleotide sequence CGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGA AGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGATGCTGGAGAACTA TAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGT TGGAGAAGGGAGAAGAGCCT SEQ. ID NO: 41 P6783PC01 KRAB polypeptide sequence RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEK GEEP
  • Flavell, IL-7R licenses a population of epigenetically poised memory CD8 + T cells with superior antitumor efficacy that are critical for melanoma memory.
  • H. Mizuguchi, T. Hayakawa Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes. Gene 285, 69–77 (2002).
  • P. Moura-Alves A. Neves-Costa, H. Raquel, T. R. Pacheco, B. D’Almeida, R. Rodrigues, I. Cadima-Couto, ⁇ . Chora, M.

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Abstract

The present disclosure provides modulators of the cell reprogramming process to enhance cell reprogramming efficiency and fidelity, constructs comprising thereof, and methods thereof.

Description

P6783PC01 Transcription factors and reprogramming modulators for reprogramming cells to cDC1 cells, compositions and methods thereof Technical field The present invention relates to constructs, vectors and methods for reprogramming cells into dendritic or antigen-presenting cells, and uses thereof. In particular, the present invention relates to novel transcription factors and reprogramming modulators that improve reprogramming, and methods thereof. Background Cancer immunotherapies rely on the establishment of tumor antigen-specific T cell responses. T cells identify tumor antigens displayed on tumor cells' major histocompatibility complexes (MHC) and carry out their functions by killing cells and producing inflammatory cytokines. However, tumor cells often fail to activate T cells due to downregulation of antigen presentation pathways, the creation of an immunosuppressive tumor microenvironment (TME), and the absence or dysfunction of professional antigen presenting cells, such as dendritic cells (DCs). This presents a challenge for achieving widespread success with current cancer immunotherapy approaches. For example, immune checkpoint blockade (ICB), while transforming solid tumor treatment, yields a response rate of only 60% in melanoma patients treated with anti–programmed cell death protein 1 (PD-1) and anti–cytotoxic T lymphocyte– associated protein 4 (CTLA-4). Other less immunogenic cancer types, like breast cancer, microsatellite-stable colorectal cancer, and glioblastoma, exhibit resistance to immunotherapy, with long-term immunity induced in less than 5% of patients. Growing evidence suggests that conventional dendritic cells type 1 (cDC1s) play a crucial role in T cell-mediated tumor regression and response to ICB across various cancer types. cDC1s, a rare subset of DCs, express high levels of MHC class I and II, the co-stimulatory molecule CD40, and specific markers like XCR1 and CLEC9A (Cabeza-Cabrerizo et al.2021). Within tumors, cDC1s are essential for recruiting and activating T cells through chemokine secretion and antigen cross-presentation, facilitating effective cancer immunity. However, these unique functional attributes of cDC1s have not yet been fully harnessed for immunotherapy due to the lack of methods enabling the generation of a pure population of cDC1s. P6783PC01 Cellular reprogramming offers a strategy for generating specific cell types in vivo by overexpression of cell type-specific transcription factors (TFs). In vivo cell fate reprogramming allows the conversion of endogenous somatic cells into different cell types within the organism, offering therapeutic potential directly at the site of disease bypassing the challenges associated with ex vivo cell manufacturing for personalized cell therapies. For example, mouse pancreatic exocrine cells were converted in situ to insulin-secreting β-cells by delivering three transcription factors to the pancreas using adenoviral vectors. Similarly, scar-forming cardiac fibroblasts were transformed into cardiomyocytes in mouse models of myocardial infarction, leading to improved heart function (Qian et al. 2012). Glial cells were converted to functional neurons after brain injury or in models of neurodegenerative diseases (Torper et al.2015) and rod photoreceptors were generated within the retina, resulting in improved vision (Yao et al. 2018). Moreover, a transcription factor combination—PU.1, IRF8, and BATF3 (PIB)— was identified as sufficient to reprogram fibroblasts or tumor cells into cDC1-like cells in vitro and in vivo, equipped with the essential signals for T cell activation, antigen presentation, co-stimulatory molecule expression, and chemokine/cytokine secretion (Rosa et al. 2018, Rosa et al.2022, Zimmermannova et al.2023). These studies provided proof-of-principle that cDC1 reprogramming could be harnessed to develop novel treatment modalities for cancer immunotherapy. Recently, components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have been repurposed during the last years for use in mammalian cells (Li et al. 2023). As part of the system, the CRISPR-associated (Cas)9 enzyme can be programmed with a single guide RNA (sgRNA) to generate site- specific DNA breaks, by directing the machinery with a single guide RNA (sgRNA) targeting a specific gene. CRISPR/Cas9-based screens, as well as screening platforms based on shRNAs (Borkent et al. 2016, Moura-Alves et al. 2011, Oliver et al. 2017) have been used as an approach to identify regulators of diverse biological processes such as cancer, immune cell mechanisms, and cell fate reprogramming to pluripotency or alternative cell fates (Li et al.2023, Buquicchio et al. 2021, Bock et al. 2022). However, modulators of cell reprogramming are still in demand, and in particular modulators of cDC1 reprogramming to improve reprogramming efficiency and fidelity. The optimization of direct cell reprogramming tools and methods remains a challenge, and further improvements are needed, for example to improve efficiency of the cDC1 reprogramming process and bring the technology to wider clinical use. Constructs and methods enhancing cDC1 reprogramming, a fortiori in vivo, and medical uses thereof, P6783PC01 are still in demand. Further, methods for identifying regulators of the cell reprogramming process and targets to enhance cell reprogramming efficiency and fidelity, such as cDC1 cell reprogramming efficiency and fidelity, are lacking. Summary The present invention provides solutions to the above-mentioned challenges and needs. In one aspect, the present invention relates to one or more constructs, which upon expression encode: - one or more gene expression inhibitors of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, and FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. P6783PC01 A second aspect of the present invention relates to one or more constructs, which upon expression encodes at least two transcriptions factors reprogramming or inducing a cell, wherein the constructs further encode: - one or more gene expression inhibitors inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. A third aspect of the present invention provides one or more vectors comprising the one or more constructs of the first or second aspects of the present invention. A fourth aspect of the present invention relates to a cell comprising the one or more constructs of the first or second aspects of the present invention or the one or more vectors of the third aspect of the present invention. P6783PC01 A fifth aspect of the present invention relates to a method of reprogramming or inducing a cell into a reprogrammed or induced cell, the method comprising the following steps: a) transducing a cell with one or more constructs or vectors, which upon expression encodes at least two transcription factors reprogramming or inducing said cell; b) inhibiting the expression of one or more genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or c) activating the expression of one or more genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. Description of Drawings Figure 1. In vivo cDC1 reprogramming elicits potent antitumor immunity. (A) 88% of B16 cells were transduced with lentiviral vectors encoding PIB-eGFP (PIB: PU.1, IRF8, and BATF3) or PC-eGFP (PC: PU.1 and C/EBPa) in vitro, mixed with 12% of P6783PC01 parental B16 cells and a total of 1x105 cells per mouse were injected subcutaneously to induce tumor cell reprogramming in vivo along with tumor establishment. Injection of eGFP-transduced cells mixed with parental cells were included as controls. Additionally, cell mixtures were maintained in vitro to quantify transduced cells at day 3 and reprogramming efficiency until day 9. ICB (anti-PD-1 and anti-CTLA-4) or isotype control (IgG2a and IgG2b) antibodies were administered intraperitoneally at day 7, 10, and 13 post tumor establishment. Tumor growth (upper panel) and survival of mice (bottom panel) are shown (n=10). (B) Flow cytometry analysis and quantification of the number of surface MHC-I molecules per cell and (H) MHC-II expression in B16 cells by mean fluorescence intensity (MFI) after 3 and 9 days of in vitro reprogramming (n=3). (C) Quantification of proliferating CTVlowCD44+ ovalbumin-specific CD8+ T cells (OT-I) after co-culture with CD103+bone marrow-derived dendritic cells (BM-DC), eGFP-transduced B16 cells (not expressing ovalbumin), MACS-enriched CD45+ and MHC-II+ B16-derived cDC1-like cells or MACS-enriched CD45+ and CD11b+ B16-derived macrophage-like cells (PC). BM-DC, eGFP-transduced or reprogrammed cells were pulsed for 24 hours with full-length ovalbumin protein. To start the co-culture, ovalbumin-containing media was washed away extensively, and fresh media added together with OT-I cells and co- cultures were performed for 72 hours (n=4-7). Data in panel B and C are shown as mean ^ SD. Survival analysis in panel A was performed by log-rank Mantel-Cox test. ****p<0.0001. Figure 2. Addition of a WPREmut6 downstream the tricistronic cassette allows higher cDC1 reprogramming efficiency across human cancer cells. (A) Flow cytometry quantification of cDC1 reprogramming efficiency in T98G human cancer cells 3 days after transduction with Ad5 and Ad5/F35 adenoviral vectors encoding PU.1, IRF8 and BATF3 (PIB) in a tricistronic cassette followed by a Woodchuck Hepatitis Virus Post- Transcriptional Regulatory Element (WPRE) sequence. Adenoviral vectors encoding PIB without WPRE or encoding eGFP only were included as controls. Reprogramming efficiency was measured by the frequency of transduced GFP+ cells expressing CD45 and/or HLA-DR (n=2). Mean ± SD are represented. (B) Flow cytometry quantification of CD45 and HLA-DR expression in human cancer cell lines 3 days after transduction with adenoviral vectors serotype 5 encoding for PIB followed by WPRE or the mutated derivative mut6 (mut6WPRE or WPREmut6) at four different MOIs (1x102, 5x102, 1x103 and 5x103 IFU/cell). (C) Representative flow cytometry plots showing reprogramming efficiency using a MOI of 1x103 IFU/cell at day3 (n=2). Mean ± SD are represented. P6783PC01 Figure 3. The Rabbit beta-globin polyadenylation signal (rBGpA) Post- Transcriptional Regulatory Element enhances cDC1 reprogramming efficiency. (A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding for PU.1, IRF8 and BATF3 containing different types of polyadenylation signals 3 days after transduction of melanoma A2058, sarcoma SK- LMS-1, glioblastoma T98G and head and neck Ca9-22 human cancer cell lines using four different MOIs (1x102, 5x102, 1x103 and 5x103 IFU/cell) and (B) 9 days after transduction in breast B0845 and melanoma M2778 human primary samples transduced at two different MOIs (1x102 and 1x103 IFU/cell), measured as percentage of transduced cells expressing CD45 and HLA-DR. (n=2). Mean ± SD are represented. BGH: Bovine Growth Hormone, TK: Herpes Simplex Virus type 1 Thymidine kinase, Short synthetic: based on the highly efficient polyA signal of the rabbit beta-globin gene, SV40late: Viral Simian virus 40 late polyA terminator element, rBG or rbBG: rabbit beta-globin, hGH: human Growth Hormone. Figure 4. Ad5-PIB vector with SFFV promoter, PIB polycistronic cassette, WPREmut6 and rbBG polyadenylation signal allows higher cDC1 reprogramming efficiency at low multiplicity of infection and superior efficacy. (A) Flow cytometry quantification of cDC1 reprogramming efficiency mediated by adenoviral vectors serotype 5 encoding PU.1, IRF8 and BATF3 (PIB) followed by WPRE-BGHpA or WPREmut6-rbBGpA 3 days after transduction of glioblastoma T98G, melanoma A2058, sarcoma SK-LMS-1 and head and neck Ca9-22 human cancer cell lines, and (B) Colorectal CRC50 and head and neck ASG04 primary cancer samples using two different MOIs (100 and 1000 IFU/cell). Reprogramming efficiency was measured as percentage of transduced cells expressing CD45 and HLA-DR (n=2-11). Mean ± SD are represented. (C) Patient-derived cancer samples of melanoma (n=6 patients) were transduced with Ad5-PIB vectors with WPRE-BGHpA (Ad5-PIB) or WPREmut6-rbBGpA (AT-108) and reprogramming efficiency was evaluated by flow cytometry quantification of CD45 and HLA-DR, HLA-ABC, cDC1 markers CD141 and CLEC9A, and co- stimulatory molecules CD40, CD80 and CD86. (D) Flow cytometry quantification of CD45, HLA-DR and CD40 expression in patient-derived organoids of colorectal (n=2 patients) and head and neck (n=2 patients) cancer 3 days after transduction with Ad5- PIB or AT-108. (E) Human SK-LMS-1 cell line-derived xenografts were injected with Ad5- PIB or AT-108 at days 0, 2, 4 and 6, and in vivo reprogramming was evaluated by flow P6783PC01 cytometry quantification of CD45 and HLA-DR expression in CD44+ SK-LMS-1 cells. (F) Primary melanoma cells transduced with Ad5-PIB or AT-108 at day 8 post transduction were pulsed with long MART-1 peptide and stimulated overnight with TLR agonists (TLR3 (Poly I:C), TLR4 (LPS) and TLR7/8 (R848)). Antigen cross-presentation was evaluated 8 days after co-cultured with HLA-A2+ MART-1+ CD8+ T cells in the presence of IL-2 and IL-7 (n=3 HLA-A2+ patients). (G) Primary melanoma cells transduced with Ad5-PIB or AT-108 at day 8 post transduction were stimulated overnight with TLR agonists (TLR3 (Poly I:C), TLR4 (LPS) and TLR7/8 (R848)), and cytokines (IL-12p70, TNFa and IFNB) in culture media were quantified using cytometric bead array (CBA). (H) Ad5-PIB or AT-108 were delivered via intra-tumoral injection in B16 tumors at days 0, 2, 4 and 6, and survival was profiled overtime. Mice received immune-checkpoint blockade with aPD-1 and a-CTLA-4 antibodies at days 0, 3 and 6. Figure 5. CRISPR/Cas9 screening identifies barriers and facilitators of cDC1 reprogramming. (A) Experimental layout to reprogram human fibroblasts into DC1 cells. HDFs of four donors were transduced with Cas9 lentiviral particles and selected with blasticidin for 8 days. Then, cells were transduced with the KORE sgRNA library co- expressing GFP (KORE Cas9 HDFs). Later, cells were transduced with SFFV polycistronic lentiviral vector comprising PU.1, IRF8 and BATF3 (PIB) sequences. On day 9 of reprogramming, induced DC1 (iDC1) cells were analyzed by flow cytometry to assess the surface expression of CD45 and HLA-DR. (B) Representative images at day 9 of DC reprogramming for HDFs transduced with either the MCS control or the PIB polycistronic vector. Reprogrammed cells display a dendritic like-morphology. GFP expression from the KORE library is denoted in the lower panels. (C) Representative flow cytometry plots showing populations of interest isolated at day 9 of reprogramming by FACS containing the sgRNA library and stained for CD45 and HLA-DR. (D) Median log2 fold-change (logFC) of sgRNA representation. Using day 0 as the baseline for normalization, reprogrammed versus non-reprogrammed samples at day 9 were compared for enrichment analysis. Enriched genes in non-reprogrammed cells are defined as facilitators (highlighted dark dots, bottom right corner of panel D) and enriched genes in reprogrammed cells identify barriers of reprogramming (highlighted dark dots, upper leftblu e corner of panel D). (E) Rank distribution of candidate genes for hemogenic reprogramming barriers and facilitators according to FC difference between reprogrammed and non-reprogrammed cells, using a threshold of 1.5 SD. P6783PC01 Figure 6: shRNA screening identifies kinases and phosphates that facilitate or impede dendritic cell reprogramming. (A) Experimental design to screen for kinases and phosphatases that inhibit or promote dendritic cell (DC) reprogramming. Clec9a- tdTomato (Clec9aCre/+Rosa26tdTomato/+) double transgenic mouse embryonic fibroblasts (MEFs) were transduced with lentiviral particles encoding individual shRNAs for kinases (PKs) and phosphatases (PPs). The library contains 206 shRNAs. After 3 days, MEFs were transduced with FUW-tetO-PIB (PU.1+IRF8+BATF3) and FUW-M2rtTA inducible expression system. GFP, tdT and MHC-ll expression was assessed by flow cytometry 6 days after the addition of Doxycycline (Dox). (B) 206 shRNAs and 1 shRNA for PU.1 (purple, positive reprogramming facilitator control) were tested in a primary screen. Results shown as the percentage of tdT+ in GFP+ relative to non-targeting control shRNA (shNTC). shRNAs that led to an increase in tdT activation above or below 0.5- fold were selected for further validation (N=1). (C) Volcano plots displaying the impact of each shRNA in tdT activation (upper panel) and MHC-ll expression (bottom panel) compared with the shNTC (N=3-4). Targets that led to a significant (p-value < 0.05) increase (termed barriers, Camkk2, Ppp4c, Gsk3a, Taok2, MikL, in upper panel, and Gsk3a, Ppp2rb1, in lower panel) or decrease (named facilitators, Melk, Spi1, Hspb8, Nuak2 in upper panel, and Pkrd2, Lats2, Prkaa2, Tnk1, Mlk4 in lower panel) of said activation and/or expression are highlighted in grey. Dotted horizontal line represents p- value of 0.05. (D) Venn diagram showing the barriers (top) and facilitators (bottom) of DC reprogramming in terms of tdT and/or MHC-II expression. (E) Human dermal fibroblasts (HDFs) were incubated with chemical compounds or DMSO control. Incubation started 3 days prior to PIB transduction and was maintained until day 9, when cells were analyzed by flow cytometry for the expression of the DC markers CD45, HLA- DR and CD40. Media with drugs was changed every 3 days. (F) Total counts of CD45+HLA-DR+CD40+ cells relative to DMSO on day 9 following cell incubation with increasing concentrations of STO-609, CHIR99021 and Rapamycin. Each datapoint represents a different HDF donor. Figure 7: Knock out of RYBP and SND1 enables higher cDC1 reprogramming efficiency. (A) Venn diagram plot showing overlap between barriers of cell fate reprogramming towards iDC1s, iHSPCs and iPSCs. RYBP is one of the 3 barriers conserved across the 3 reprogramming systems. SND1 is a barrier conserved across iDC1 and iPSC reprogramming. (B) Flow cytometry quantification of cDC1 reprogramming efficiency in human dermal fibroblasts (HDFs) knockout (KO) for RYBP P6783PC01 relative to HDFs KO for the safe harbor AAVS19 days after overexpression of PU.1, IRF8 and BATF3 (PIB). (C) Flow cytometry quantification of cDC1 reprogramming efficiency in HDFs KO for SND1 and in HDFs KO for AAVS1 (control) 3, 6, 9, 12 and 16 days after overexpression of PIB. Figure 8: HDAC inhibition enables higher in vivo efficacy. (A) Flow cytometry quantification of cDC1 reprogramming efficiency measured by surface expression of CD45 and HLA-DR 9 days after transduction of primary glioblastoma cells and human cancer cell lines of melanoma, ovarian and gastric cancer with lentiviral vectors encoding PU.1, IRF8 and BATF3 (PIB). Valproic acid (VPA, HDAC inhibitor) and/or Azacitidine (Aza, DNA methylation inhibitor) were added to the cell culture media between day 0 and day 4. (B) Mice were injected subcutaneously with YUMM1.7 melanoma cells 4 days after transduction with PIB-eGFP or control eGFP and mixing with parental cells (30% transduced and 70% non-treated cells) to induce tumor cell reprogramming in vivo along with tumor establishment. YUMM1.7 cells were also treated with VPA. Tumor growth (upper panel) and mice survival (bottom panel) are shown. (C) Flow cytometry quantification of cDC1 reprogramming efficiency measured by surface expression of CD45 and/or MHC-II 3 days after transduction of YUMM1.7 melanoma cancer cells with adenoviral vectors encoding PU.1, IRF8 and BATF3 (PIB). Valproic acid (VPA, HDAC inhibitor) and/or Azacitidine (Aza, DNA methylation inhibitor) were added to the cell culture media between day 0 and day 3. Figure 9. PRC2 inhibition increases reprogramming efficiency and fidelity. Flow cytometry analysis of reprogramming efficiency (expression of CD45 and HLA-DR, top panel) and fidelity (expression of CD40 and CD226, lower panel) at day 9 in HDFs transduced with PIB-encoding lentiviral vectors and treated with either DMSO or various concentrations (0.5 µM, 1 µM, 5 µM, 10 µM) of PRC2 inhibitors (GSK126, EPZ-6438, EED226) between day 0 and day 2. DMSO is shown as average across concentrations. Percentages of positive cells are shown (n = 3 biological and 3 technical replicates; mean ± SD). Figure 10. Enforced gene activation by IRF8 and gene inactivation by BATF3 enables higher cDC1 reprograming efficiency. Flow cytometry analysis of CD45 expression on day 9 in HDFs transduced with lentiviral particles carrying bicistronic cassettes (biC) encoding two reprogramming factors in combination with the third P6783PC01 transcription factor fused to either the VP16 activation domain or KRAB repressor domain. Fold change (FC) in the percentage of CD45-positive cells is shown relative to untagged TFs (n = 2 biological replicates, each with 2 technical replicates; mean ± SD). Figure 11. Regulatory network of AMPK/mTOR signalling in cellular metabolism and cDC1 reprogramming. (A) Schematic representation of the upstream activators and downstream effects of AMPK (AMP-activated protein kinase) and mTOR (mechanistic target of rapamycin) pathways. AMPK is activated in response to energy stress signals such as increased AMP/ATP ratio (via LKB1) and calcium signaling (via CAMKK2), which can be stimulated by TLR stimulation, increased intracellular calcium (Ca²⁺), and reactive oxygen species (O₂⁻). mTOR activity is modulated by multiple kinases including Ppp2r1b, GSK3a, and Map4k3. Key inhibitors and activators of these pathways are shown. AMPK activators: CHIR99021, AICAR, A769662; AMPK inhibitors: Compound C; mTOR inhibitors: Torin1, AZD2014, Rapamycin; mTOR activators: MHY1485, Salidroside; CAMKK2 inhibitor: STO-609; Metabolic modulators: Metformin (activates AMPK), 2-DG (2-deoxyglucose, inhibits glycolysis). (B) Flow cytometry quantification of cDC1 reprogramming yield 9 days after transduction with lentiviral particles encoding PU.1, IRF8 and BATF3, and incubation with small molecules. Number of reprogrammed cells expressing CD45/HLA-DR relative to DMSO control are shown. (C) Flow cytometry analysis and quantification of TNFa within reprogrammed CD45+HLA-DR+. (D) ELISA-based quantification of IL-12p70 levels in the supernatant of reprogrammed cells treated with A769662 from day 0 to day 9, followed by overnight stimulation with Poly(I:C). (E) Schematic representation of in vivo experiment where B16-F10 cells were transduced in vitro with PIB-encoding lentiviral vectors, mixed 1:1 ration with non-transduced cancer cells, and injected subcutaneously to allow tumor establishment and investigate in vivo efficacy. A769662 was injected intraperitoneally at days 1, 3 and 5 after initial tumor establishment. (F) Tumor growth curves for the 4 treatment groups are shown. Detailed description Definitions “biologically active variant” refers herein to a biologically active variant of a genetic element such as of a regulatory element, of a transcription factor (TF), or of a reprogramming modulator, which retains at least some of the functional activity of the P6783PC01 parent genetic element, TF or reprogramming modulator. The term encompasses variants at the polypeptide level, including protein isoforms, that exhibit a minimum of 90% sequence similarity to the parent sequence. These variants may differ in their efficiency of inducing or inhibiting gene expression compared to the parent TF or modulator. For example, a biologically active variant of Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF3), Interferon Regulatory Factor 8 (IRF8), and PU.1 can act as said respective TF and induce or inhibit expression of the same genes in a cell as BATF3, IRF8, and PU.1, respectively, although the efficiency of the induction may be different, e.g. the efficiency of inducing or inhibiting genes is decreased or increased compared to the parent TF. “Identity” and “homology”, with respect to a polynucleotide or polypeptide, are defined herein as the percentage of nucleic acids or amino acids in the candidate sequence that are identical or homologous, respectively, to the residues of corresponding native nucleic acids or amino acids, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity / similarity / homology, and considering any conservative substitutions according to the NCIUB rules (hftp://www.chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Generally, a given homology between two sequences implies that the identity between these sequences is at least equal to the homology; for example, if two sequences are 70% homologous to one another, they cannot be less than 70% identical to one another – but could be sharing 80% identity. "Murine" refers herein to any and all members of the family Muridae, including rats and mice. “Reprogramming” refers herein to the process of converting of differentiating cells from one cell type into another. In particular, reprogramming herein refers to converting or transdifferentiating any type of cell into a dendritic cell, such as a conventional type 1 dendritic cell (cDC1), or an antigen-presenting cell. P6783PC01 “affiliation” as used herein refers to the classification or assignment of individual cells into specific categories or groups based on their gene expression profiles. The affiliation can be performed for instance by the methods described herein, for example involving using a support vector machine (SVM) classifier to predict or determine the identity or type of cells (such as dendritic cells) based on patterns found in their gene expression profiles as compared to known reference datasets. “Treating,” or “Treatment,” refers herein to any administration or application of a therapeutic for the disclosed diseases, disorders and conditions in subject, and includes inhibiting the progression of the disease, slowing the disease or its progression, arresting its development, partially or fully relieving the disease, or partially or fully relieving one or more symptoms of a disease. As used herein, the term "adenovirus" is used to refer to any and all viruses that may be categorized as an adenovirus, including any adenovirus that infects a human or a non-human animal, including all groups, subgroups, and serotypes, except when required otherwise. Thus, as used herein, "adenovirus" refers to the virus itself or derivatives thereof and cover all serotypes and subtypes, naturally occurring (wild- type), modifications to be used as an adenoviral vector, e.g., a gene delivery vehicle, forms modified in ways known in the art, such as for example capsid mutations, and recombinant forms, replication-competent, conditionally replication-competent, or replication-deficient forms, except where indicated otherwise. 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 may be categorized as an adeno-associated virus, including any adeno-associated virus that infects a human or a non-human animal, and covers all subtypes, serotypes and pseudotypes, and both naturally occurring, modified and recombinant forms, such as modifications to be used as an adeno-associated viral vector, e.g., a gene delivery vehicle except where required otherwise. As used herein, unless otherwise specified e.g. in figure legends, the abbreviation "Ad" in the context of a viral vector refers to an adenoviral vector and is typically followed by a number indicating the serotype of the adenovirus. For example, "Ad5" refers to adenovirus serotype-5 vectors. P6783PC01 The term “Ad”, when not followed by a specific number, covers any Ad suitable for the purpose may be used herein, such as but not limited to Ad from any serotype from any of the A, B, C, D, E, F, G Ad subgroups, for example 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 Ad that infect primates, "non-primate Ad" refers to Ad that infect non-primate mammals, "bovine Ad” refers to Ad that infect bovine mammals. The genomic sequences of various serotypes of Ad, as well as the sequences of the native terminal repeats (TRs) and capsid subunits are known in the art. As used herein, the abbreviation “AAV” in the context of a viral vector refers to an 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. The term covers any suitable AAV, such as but not limited to AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3A (AAV3A), AAV serotype 3B (AAV3B), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non- primate AAV, and ovine AAV. "Primate AAV refers to AAV that infect primates, "non- primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV refers to AAV that infect bovine mammals. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. “Hybrid” or “chimeric” Ad or AAV vectors as used herein refers to vectors based on Ads or AAVs engineered in a way that the Ad or AAV vectors contains proteins derived from two or more different Ad or AAV serotypes. Ad5/3 and Ad5/F35 as described herein are examples of such hybrid or chimeric Ad vectors. “AAV2-qYF” or “AAV2-QuadYF” as used herein refers to a quadruple tyrosine to phenylalanine mutant of AAV2. P6783PC01 “AAV-DJ” as used herein refers to a hybrid capsid derived from DNA family shuffling of 8 wild type serotypes of AAV, including AAV 2, 4, 5, 8, 9, avian, bovine and caprine AAV. AAV-DJ is a synthetic serotype, type 2/type 8/type 9 chimera, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. “rBGpA” or “rbBGpA” as used herein refer to the Rabbit beta-globin polyadenylation signal. “SV40late” as used herein refers to the Viral Simian virus 40 late polyA terminator element. “WPREmut6” as used herein refers to the mutated Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element sequence carrying a mutation disturbing the expression of a truncated Woodchuck hepatitis virus X protein implicated in liver tumors, as first described in Kingsman et al.2005. Modulators of cell reprogramming “Reprogramming modulator” as used herein refers to modulators of cell reprogramming, such as reprogramming to cDC1 cells, and comprise “barriers”, negative regulators, which reduce or prevent cell reprogramming and “facilitators”, positive regulators, which enhance or activate cell reprogramming. Reprogramming modulators include, but are not limited to, chromatin regulators, epigenetic modifiers, RNA modifiers, and elements of signalling pathways and biological processes, such as enzymes, e.g. kinases and phosphatases. As used herein “gene expression inhibitors” refer to elements reducing, preventing, or blocking the expression of genes, such as reducing, preventing, or blocking the expression of barrier genes. As used herein “barrier genes” refer to genes negatively regulating cell reprogramming. Barrier genes may for instance be as described in Examples 18 and 19 herein, such as as identified herein using CRISPR/Cas9 screening or shRNA screening. As used herein “gene expression enhancers” refer to elements increasing, activating, or triggering the expression of genes, such as increasing, activating, or triggering the expression of facilitator genes. P6783PC01 As used herein “facilitator genes” refer to genes positively regulating cell reprogramming. Facilitator genes may for instance be as described in Examples 18 and 19 herein, such as as identified herein using CRISPR/Cas9 screening or shRNA screening. Identification of barrier and facilitator genes may preferably be performed at day 9 of cell reprogramming (e.g.9 days after cell transduction with vectors encoding transcription factors as described herein). In the present disclosure, the inventors have identified barriers to cDC1 reprogramming and demonstrated that knock out or downregulation of said barriers enhances cDC1 reprogramming efficiency. On the other hand, activation of the expression of the facilitator genes enhanced cDC1 reprogramming. Constructs modulating cell reprogramming In an aspect, the present invention provides one or more constructs, which upon expression encode: - one or more gene expression inhibitors of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, P6783PC01 IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, and FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. Unless otherwise indicated, the details of the nucleotide sequences and other characteristics of the barrier and facilitator genes disclosed herein, can be obtained from the HGNC (HUGO Gene Nomenclature Committee) or NCBI (National Center for Biotechnology Information) using their official aliases used herein. In some embodiments, GATA2 may be of the sequence set forth in SEQ ID NO 17 or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 17. In some embodiments, the facilitators gene and barriers genes are as measured by Tdt expression and/or MHC-II expression as reprogramming efficiency and/or fidelity readout, as indicated in the Examples and Figures herein. For example, CAMKK2 and/or PRKAA2 may act as facilitator based on one readout and as barrier based the other readout. The skilled person will know that reprogramming mechanisms underlying the different readouts may be different. In other embodiments, GATA2 may be encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 16. In a further aspect, the present invention provides one or more constructs, which upon expression encodes at least two transcriptions factors for reprogramming or inducing a cell, wherein the constructs further encode: - one or more gene expression inhibitors inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 P6783PC01 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, and FOXN2 , HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2.. In some embodiments, said constructs are: - overexpression constructs, such as constructs overexpressing said one or more gene expression enhancers, or CRISPR activation constructs targeting said one or more facilitator genes; - knockdown constructs, such as shRNA constructs, miRNA-encoding constructs, or CRISPR interference constructs targeting said one or more barrier genes; and/or - gene editing constructs, such as CRISPR-Cas9 constructs targeting said one or more barrier genes. In preferred embodiments, the gene expression enhancers and/or the gene expression inhibitors are selected from the group consisting of: RNAs, proteins and/or genes. P6783PC01 Method for gene expression inhibition are available to the skilled person. For example, short hairpin RNA (shRNA) is used for gene silencing using RNA interference (RNAi). shRNAs are typically 19-29 nucleotide-long sequences complementary to a target mRNA. shRNA are transcribed in the nucleus and processed by the Dicer enzyme in the cytoplasm to produce short interfering RNA (siRNA). siRNA are incorporated in the RNA-induced silencing complex (RISC), which binds to the target mRNA and activates its degradation, thereby silencing the expression of the gene product it encodes. shRNA can be delivered for instance by vectors, such as plasmids, viral vectors, or nanoparticles. Other methods such as direct delivery of siRNA, such as synthetic siRNA, delivery of miRNAs (or pri-miRNAs) complementary to target mRNA or short synthetic single-stranded antisense oligonucleotides (ASOs) DNA or RNA molecules complementary to target mRNA are known in the art. Thus, in yet other embodiments, the RNAs are shRNA, miRNA, siRNAs and/or antisense oligonucleotides (ASOs). In some embodiments, the proteins are transcription factors, epigenetic modifiers, such as histone-modifying enzymes, and/or CRISPR systems, such as CRISPR editing, interference and/or activation systems. In preferred embodiments of the one or more constructs of the present invention: - the chromatin remodelling factor RB Binding Protein-4 (RBBP4) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 18; - the splicing factor 3B subunit 6, (SF3B6) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 20; - the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 22, such as at least 95%, such as at least 96%, such as at least P6783PC01 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 22; - the AMP activated protein kinase-alpha2 (PRKAA2) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 24; and - the Protein Polybromo-1 (PBRM1) is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 26, 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 to SEQ ID NO: 26. In some embodiments of the one or more constructs of the present invention: - the chromatin remodelling factor RB Binding Protein-4 (RBBP4) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 19, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 19, 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 to SEQ ID NO: 19; - the splicing factor 3B subunit 6, (SF3B6) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 21, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 21, 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 to SEQ ID NO: 21; - the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 23, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 23, 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 to SEQ ID NO: 23; - the AMP activated protein kinase-alpha2 (PRKAA2) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 25, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 25, such as at P6783PC01 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 to SEQ ID NO: 25; and - the Protein Polybromo-1 (PBRM1) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 27, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 27, 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 to SEQ ID NO: 27. In other embodiments of the one or more constructs of the present invention, the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and the at least two transcription factors, such as at least three transcription factors, are selected from the group consisting of: a) PU.1, b) IRF8, and c) BATF3 In some embodiments, the one or more constructs upon expression encode PU.1, or a biologically active variant thereof, at least 90% identical to SEQ ID NO: 10, 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% identical to SEQ ID NO: 10. In other embodiments, the one or more constructs upon expression encode IRF8, or a biologically active variant thereof, at least 90% identical to SEQ ID NO: 12, 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% identical to SEQ ID NO: 12. In further embodiments, the one or more constructs upon expression encode BATF3, or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 14. In other embodiments, the one or more constructs upon expression encode GATA2, or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 17. P6783PC01 In preferred embodiments, PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, 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 to SEQ ID NO: 9 In other preferred embodiments, IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, 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 to SEQ ID NO: 11. In yet other preferred embodiments, BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13, 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 to SEQ ID NO: 13. In other embodiments, GATA2 is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 16. In preferred embodiments the one or more constructs which upon expression encode at least two transcription factors selected from the group consisting of: PU.1, IRF8 and BATF3. In some embodiments, the one or more constructs, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1, IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late). P6783PC01 In other embodiments, the one or more constructs encode upon expression PU.1, IRF8, BATF3 and GATA2. The present disclosure shows that enforced gene activation by IRF8 and gene inactivation by BATF3 enables higher cDC1 reprogramming efficiency, such as measured by CD45 expression in the cells transduced by constructs comprising IRF8 further fused to the VP16 activation domain (transcriptional activation domain of herpes simplex virus protein VP16 (Triezenberg et al., 1988; Cousens et al., 1989), and/or comprising BATF3 fused to the KRAB repressor domain (Krüppel-associated box (KRAB) transcriptional repression domain from the human zinc finger protein ZNF10 (Margolin et al., 1994). Thus, in some embodiments of the one or more constructs described herein, IRF8 is fused to the VP16 activation domain. In preferred embodiments, the VP16 activation domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 38, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 38. In other embodiments, the VP16 activation domain is encoded by the sequence set forth in SEQ ID NO: 39, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 39, 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% identical to SEQ ID NO: 39. In yet other embodiments of the one or more constructs as described herein, BATF3 is fused to the KRAB repressor domain. In preferred embodiments, the KRAB repressor domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 40, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 40. P6783PC01 In other preferred embodiments, the KRAB repressor domain is encoded by the sequence set forth in SEQ ID NO: 41, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 41, 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% identical to SEQ ID NO: 41. Vectors encoding constructs modulating cell reprogramming In another aspect, the present invention provides one or more vectors comprising the one or more constructs of the first or second aspects of the present invention. In some embodiments, the one or more vectors comprising the one or more constructs of the first or second aspects of the present invention, may otherwise be as described in the section “Vectors”. Cell comprising constructs or vectors modulating cell reprogramming In another aspect, the present invention provides a cell comprising the one or more constructs of the first and second aspect of the present invention or the one or more vectors of the third aspect of the present invention. Method of reprogramming or inducing a cell comprising modulators inhibition and/or activation In a further aspect, the present invention provides a method of reprogramming or inducing a cell into a reprogrammed or induced cell, the method comprising the following steps: a) transducing a cell with one or more constructs or vectors, which upon expression encodes at least two transcription factors reprogramming or inducing said cell; b) inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, P6783PC01 STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or c) activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, and FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1preferably activating the expression of GATA2. In some embodiments of the method of the fifth aspect of the present invention: - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin- dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2); and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: Protein Polybromo-1 (PBRM1). In some embodiments of the method of the fifth aspect of the present invention: - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, and EZH2; and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, and RAD54L. P6783PC01 This may be for instance the case in embodiments of the method making use of CRISPR approaches, such as making use of CRISPR approaches as gene expression enhancers (for instance CRISPR activation) and/or gene expression inhibitors (such as CRISPR interference or editing approaches). In other embodiments of the method of the fifth aspect of the present invention: - step b) comprises inhibiting the expression of one or more genes selected from the group consisting of: CAMKK2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, and PPP2R1B, RYBP, and SND1; and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1. This may be for instance the case in embodiments of the methods making use of shRNA, such as making use of shRNA as gene expression inhibitors. In some embodiments of the method of the fifth aspect of the present invention step a- c) together comprise transducing the cell with the one or more constructs of the first and second aspects of the present invention, or vectors comprising thereof of the third aspect of the present invention. In some embodiments of the method of the fifth aspect of the present invention step b) of inhibiting comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression inhibitors inhibiting the expression of one or more barrier genes of the first and second aspect of the present invention, or vectors comprising thereof of the third aspect of the present invention. In some embodiments of the method of the fifth aspect of the present invention step c) of activating comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression enhancers activating the expression of one or more facilitator genes of the first and second aspects of the present invention, or vectors comprising thereof of the third aspect of the present invention. P6783PC01 In some embodiments of the method of the fifth aspect of the present invention the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and the one or more constructs or vectors of step a), encode upon expression at least two transcription factors, such as at least three transcription factors, selected from the group consisting of: a) PU.1, b) IRF8, and c) BATF3 For example, in embodiments wherein the method is for reprogramming to cDC1 cells, step b) of inhibiting may comprise transducing the cell with the one or more constructs, which upon expression encode one or more gene expression inhibitors inhibiting the expression of one or more barrier genes of the first and second aspect of the present invention, such as RYBP and/or SND1, or vectors comprising thereof of the fifteenth aspect of the present invention. RYBP is also known as RING1 And YY1 Binding Protein, SND1 is also known as Staphylococcal Nuclease And Tudor Domain Containing 1. In some embodiments of the method of the fifth aspect of the present invention the step of inhibiting and/or activating the expression of one or more genes of steps b) and c) respectively is performed in parallel, before and/or after the step a) of transducing the cell. In some embodiments of the method of the fifth aspect of the present invention the cell is a mammalian cell. In some embodiments of the method of the fifth aspect of the present invention the cell is a human cell. In some embodiments of the method of the fifth aspect of the present invention the cell is a murine cell. P6783PC01 In some embodiments of the method of the fifth aspect of the present invention the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell. In some embodiments of the method of the fifth aspect of the present invention the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a mesenchymal stem cell and a hematopoietic stem cell. In some embodiments of the method of the fifth aspect of the present invention the differentiated cell is any somatic cell. In some embodiments of the method of the fifth aspect of the present invention the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte. The inventors showed in the present disclosure that HDAC and methylation inhibition enhances cell reprogramming such as cDC1 reprogramming. Thus, in some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors or methylation inhibitors, or contacting the transduced cell with one or more epigenetic modifiers such as histone deacetylase (HDAC) inhibitors. The treatment, by culturing in a medium comprising epigenetic modifiers or contacting the transduced cells with epigenetic modifiers may be performed only during the transduction step, only after the transduction step, or both during transduction step and after the transduction step. The treatment with epigenetic modifiers may preferably be performed starting from the transduction step (day 0) and prolonged for 2 days, preferably 3 days following the transduction step (day 3). In preferred embodiments, treatment with epigenetic modifiers is performed between day 0 (initiation of the transduction step) and day 3. In preferred embodiments, the one or more HDAC inhibitor is selected from the group consisting of: valproic acid (VPA), Vorinostat, Romidepsin, Belinostat, and Panobinostat. The skilled person will know that other suitable HDAC inhibitor known in the art may be used. P6783PC01 In other embodiments, the one or more methylation inhibitor is Azacitidine (Aza). For example, the step of culturing or contacting the cells with valproic acid or azacitidine individually may be beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, melanoma cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells. In some preferred embodiments, the treatment with epigenetic modifiers comprises a combination of valproic acid and azacitidine. For example, this combination is beneficial for the reprogramming of, but not limited to, brain cancer cells such as glioblastoma, ovarian cancer cells, and gastrointestinal cancer cells such as gastric carcinoma cells. In the present disclosure, the inventors found that the further inhibition of PRC2 (polycomb repressive complex 2), the complex responsible for depositing H3K27me3 (the complex that has histone methyltransferase activity and primarily methylates lysine 27 on histone H3 protein) enhanced reprogramming efficiency and fidelity. In particular, the present disclosure shows that inhibition of PRC2 using significantly enhances both efficiency (%CD45+HLA-DR+ cells) and fidelity (%CD40+CD226+ cells) in cDC1 reprogramming. Thus, in some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more PRC2 inhibitors, such as EZH2-specific inhibitors, for example Tazemetostat and/or GSK-126, or such as EED inhibitors, for example EED226. In some embodiments, the step of culturing the transduced cell in a media comprising the one or more PRC2 inhibitors is performed from day 0 to day 2, wherein day 0 is the day of the cell transduction step. The inventors found in the present disclosure that AMPK activators (CHIR99021, AICAR, A769662, Metformin), mTOR activators (MHY1485, Salidroside) and/or inhibitors of glycolysis (2-DG or 2-deoxyglucose) enabled higher cell reprogramming efficiency, such as higher cDC1 reprogramming efficiency. P6783PC01 Thus, in some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more AMPK activators, such as CHIR99021, AICAR, A769662, or Metformin. In other embodiments, the method further comprises culturing the transduced cell in a media comprising one or more mTOR activators, such as MHY1485 or Salidroside. In some embodiments, the method further comprises culturing the transduced cell in a media comprising one or more glycolysis inhibitors, such as 2-deoxy-D-glucose (2-DG). In preferred embodiments, day 0 is the day of the transduction step and the step of culturing the transduced cell is performed from day 0 to day 9. For example the step of culturing the transduced cell in the one or more AMPK activators, the one or more mTOR activators, or the one or more glycolysis inhibitors is performed from day 0 to day 9. Reprogrammed or induced cells obtained using modulators of cell reprogramming In another aspect, the present invention provides a reprogrammed or induced cell obtained by the method of the fifth aspect of the present invention. In some embodiments, the cell is an induced dendritic or antigen-presenting cell, such as a type 1 conventional dendritic cell. In other embodiments, the cell is positive for CD45 and/or negative for HLA-DR, preferably the cell is positive for CD45 and HLA-DR. This is in particular the case for methods herein aiming at reprogramming cells to cDC1 cells. Vectors The constructs of the present invention find applications in the field of, but not limited to, gene therapy and cell therapy. For these applications, and other relevant applications, it may be beneficial that said constructs are integrated in vectors, such as vectors comprising further elements useful for gene delivery, expression, stability. Techniques for producing vectors, such as adenoviral vectors comprising defined constructs are known to the skilled person, and typically involve cloning the constructs comprising the gene(s) of interest into a plasmid or cosmid vector and recombination P6783PC01 with a viral vector backbone such as an adenoviral backbone, transfection in host cells such as HEK293 cells for packaging, and amplification of adenoviral particles, followed by purification of the recombinant adenoviral particles and quality control. The skilled person will appreciate that further elements known in the art to facilitate vector manufacturing may be added to said vectors, such as multiple cloning sites, for example a multiple cloning site (MCS) inserted in place of the E3 adenoviral region, such as a 69-bp MCS sequence (SEQ. ID NO: 34) inserted in place of the E3 region between the XbaI sites located in the Ad5 E3 region, thereby deleting 1.9 kb from the genome. The skilled person will appreciate that the identification of optimized combinations of construct and vector elements, such as for cell reprogramming applications, is not straightforward due to the complex interplay of between the components of the construct (promoter, transcription factors, regulatory elements) with the vector, and host cell environment. This is a fortiori also the case for in vivo applications of said vectors. In another aspect, the present invention provides one or more vectors comprising the one or more constructs of the present disclosure. As mentioned in the section “Constructs” above, the one or more constructs of the present invention may be polycistronic or monocistronic, for example each construct may encode 1, 2, or 3 transcription factors, such as PU.1, IRF8 and/or BATF3. Therefore in some embodiments, the one or more vectors comprising the one or more constructs of the present disclosure may be for example one vector comprising one construct encoding the transcription factors PU.1, IRF8 and BATF3, or more than one vectors together encoding PU.1, IRF8 and BATF3, such as 2 vectors, such as 3 vectors. In some embodiments, the one or more vectors is a viral vector. In other embodiments, the viral vector is selected from the group consisting of: adenoviral vectors, lentiviral vectors, retrovirus vectors, herpes virus vectors, pox virus vectors, adeno-associated virus vectors, paramyxoviridae vectors, rabdoviral vectors, alphaviral vectors, flaviral vectors, and adeno-associated viral vectors. P6783PC01 In preferred embodiments, the viral vector is an adenoviral (Ad) vector. In other embodiments, the adenoviral vector is selected from the group consisting of: wild-type Ad vectors, chimeric Ad vectors, and mutant Ad vectors. In further embodiments, the wild-type Ad vector is Ad5. In other preferred embodiments, the Ad vector is selected from the group consisting of: Ad5-RGD, Ad5/F35 and Ad5/3, preferably wherein the Ad vector is Ad5/F35, even more preferably wherein the Ad vector is Ad5 or Ad5-RGD. In other embodiments, the viral vector is a lentiviral vector. In further embodiments, the adeno-associated virus vector is selected from the group consisting of : wild-type AAV vectors, hybrid AAV vectors and mutant AAV vectors. In some embodiments, the hybrid AAV vector is AAV-DJ and wherein the mutant AAV vector is AAV2-QuadYF. Similarly to restriction sites present within the construct sequences, for some applications, it may be preferable that native restriction sites present on the vector sequence and targeted by restriction enzymes are mutated, for example to facilitate proper cloning of the vector elements, such as directional cloning. Thus in some embodiments of the vectors of the present invention, one or more SfiI sites have been mutated, preferably by silent mutations, even more preferably wherein said SfiI sites are in the pVII ORF and/or the adenovirus DNA-binding protein (DBP) ORF the vector. In preferred embodiments of the one or more vectors of the present invention, the vector comprises an Ad5 wild-type fiber. The skilled person will appreciate that cellular internalization of vectors comprising an Ad5 wild-type fiber is mediated by the cell surface coxsackievirus and adenovirus receptor (CAR). Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity, such as at least 95%, such as at P6783PC01 least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1. In other preferred embodiments, the vector Ad5 wild-type fiber is replaced by the fiber of Ad35 (Ad5/F35). The skilled person will appreciate that cellular internalization of chimeric Ad5/F35 vectors is mediated by the CD46 receptor. Ad5/F35 and Ad5/35 are used herein as synonyms. Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 2. In other preferred embodiments, the vector Ad5 wild-type fiber is modified by incorporating an RGD (arginine-glycine-aspartic acid) motif into the fiber knob protein of said vector (Ad5-RGD). The skilled person will appreciate that cellular internalization of Ad5-RGD vectors is mediated by the CD51 receptor (integrin αv), such as by the CD51 subunit of αvβ3 and αvβ5 integrins. Thus, in some embodiments, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 3. In yet other preferred embodiments, the vector Ad5 wild-type fiber knob is replaced by an Ad3 fiber knob (Ad5/3). The skilled person will appreciate that cellular internalization of Ad5/3 vectors is mediated by CD46 and desmoglein-2 (DSG-2). Thus in some embodiments of the one or more vectors of the present invention, the one or more vectors of the present invention is encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 4. P6783PC01 The skilled person will appreciate that intermediate vectors, such as plasmid or cosmid vectors are typically required for the production of adenoviral vector final products, Thus in some embodiments, the vectors of the present invention further comprise cosmid and /or plasmid elements, such as a lambda phage cos sequence, and/or lambda phage scrambled sequences. Examples Example 1. General methods and materials. Mice Animal care and experimental procedures were performed in accordance with the Swedish federal regulations after approval from the Swedish Board of Agriculture. B6.129S(C)-Batf3tm1Kmm/J (BATF3KO, The Jackson Laboratory) and C57BL/6- Tg(TcraTcrb)1100Mjb/J (OT-I, The Jackson Laboratory) mice were bred in-house. C57BL/6J, NOD.Cg-PrkdcSCIDIL2rgtm1Wjl/SzJ (NSG, The Jackson Laboratory) and NOD-PrkdcSCITIL2rgtm1/Rj (NXG, Janvier Labs) females aged 6-8 weeks were purchased from Charles River or Janvier-Labs. Animals were housed in a controlled temperature environment (23±2 °C) and a fixed 12-hour light/dark cycle, having free access to food and water. Mice were age-matched, gender-matched and within the same gender randomly assigned to treatment or control groups in all experiments. Numbers of mice for in vivo experiments were determined based on previous expertise, and power analysis was not performed. Mice were sacrificed by cervical dislocation when endpoints were reached. Investigators were not blinded during experimental procedures or the assessment of outcomes. Cell culture B16-F10, embryonic fibroblasts (MEFs), SKLMS1, CHL1, Ca922, OVK18, MKN74, YUMM1.7 and T98G cell lines, patient-derived cancer cells and dermal fibroblasts (HDFs) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM GlutaMAX, 1mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM complete). B16- F10 expressing Ovalbumin (B16-OVA) were maintained in DMEM complete supplemented with 0.4 mg/ml geneticin (Gibco). Mouse Panc02, B2905, MB49, BRAFV600ECOX1/2KO cancer cell lines, mouse CD103+ bone marrow-derived dendritic cells (BM-DC), primary mouse and human T cells were cultured in RPMI 1640 P6783PC01 medium supplemented with 10% (v/v) FBS, 2 mM GlutaMAX, 1 mM sodium pyruvate, 50 mM 2-mercaptoethanol and 100 U/ml penicillin and 100 mg/ml streptomycin (RPMI complete). YUMM1.7 melanoma cells were cultured in DMEM/F-12 with 10% (v/v) FBS, 2 mM GlutaMAX, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 100 U/ml penicillin and 100 mg/ml streptomycin (DMEM/F-12 complete). MDSCs were differentiated from monocytes obtained from PBMCs of healthy donors and cultured in RPMI complete. Human pericytes were cultured in Pericyte medium (ScienCell). Fibroblasts were expanded on tissue-culture plates coated with 0.1% gelatin. All cells were dissociated from tissue-culture plates using TrypLE Express for 5-10 minutes at 37°C, split at 80% confluency and maintained in a humid environment at 37°C and 5% CO2. Reagents used for cell culture were purchased from Thermo Fisher Scientific, STEMCELL Technologies, and Nordic Biolabs. Molecular cloning Polycistronic lentiviral vector expressing the mouse or human transcription factors PU.1, IRF8 and BATF3 separated by 2A self-cleaving peptide sequences under the control of a constitutive SFFV promoter, followed by IRES2-eGFP was cloned previously (Rosa et al. 2022, Zimmermannova et al. 2023). To generate mCherry expressing vectors, the inventors used the empty backbone pRRL.PPT-SFFV-MCS- IRES2 (SFFV-MCS) (Rosa et al.2022, Zimmermannova et al.2023) and inserted the coding sequence for mCherry by infusion cloning downstream the IRES sequence to generate pRRL.PPT-SFFV-MCS-IRES2-mCherry (SFFV-mCherry). To generate a lentiviral polycistronic construct for myeloid reprogramming, the coding sequences of mouse PU.1 and C/EBPa (PC) separated by a T2A sequence were cloned first into the MCS of the pFUW-tetO-MCS vector (Rosa et al. 2022) followed by subcloning of the polycistronic cassette into the MCS of the pRRL.PPT-SFFV-MCS-IRES2-eGFP vector (PC-eGFP). Adenoviral vectors (Ad) were cloned and produced at VectorBuilder. Replication-deficient adenoviral vectors pAd5-SFFV-PU.1-P2A-IRF8-T2A-BATF3 (Ad- PIB) and pAd5-SFFV-PU.1-P2A-IRF8-T2A-BATF3-CMV-eGFP (Ad-PIB-eGFP) with an eGFP sequence under the control of constitutive cytomegalovirus (CMV) promoter were generated. pAd5-CMV-eGFP (Ad-eGFP), pAd5-SFFV-Stuffer (Ad-Stuffer) and pAd5-SFFV-Stuffer-CMV-eGFP (Ad-Stuffer-eGFP) were cloned and used as controls. The stuffer sequence was derived from the genome of E. Coli as a non-coding sequence and designed to have the same base pair length as polycistronic PIB. P6783PC01 Viral production Transfer plasmids encoding PU.1, IRF8 and BATF3 followed by IRES-eGFP (PIB- eGFP), eGFP, PIB-mCherry, mCherry, mOrange and bicistronic PU.1 and C/EBP^ followed by IRES-eGFP (PC-eGFP) were used to produce lentiviral vectors. In experiments using lentiviral vectors for in vitro transduction, lentivirus was produced using the second-generation system as previously described (Zimmermannova et al. 2023, Ferreira et al.2023). In brief, human embryonic kidney (HEK) 293T cells were seeded in 15 cm plates to reach ~80% confluency and transfected with 7.5 µg packaging plasmid (psPAX2), 2.5 µg VSV-G-encoding envelope plasmid (pMD2), and 10 µg transfer plasmid combined with 60 µl of 1 mg/ml polyethyleneimine (PEI) in Opti- MEM. Virus-containing supernatants were collected after 36, 48, and 72 hours, filtered using 0.45 µm low protein binding cellulose acetate filters and concentrated 100-fold with Lenti-X Concentrator (Takara) before storage at -80°C. Alternatively, virus- containing supernatants were ultracentrifuged for 90 minutes at 4°C with 25,000 g in a SW 32 Ti Swinging-Bucket Rotor (Beckman Coulter). Lentiviral vector pellets were resuspended overnight in DMEM medium and stored in aliquots at -80°C. Lentiviral titers were quantified with the Lenti-X qRT-PCR titration kit (Takara) following the manufacturer’s protocol. In experiments using lentiviral vectors for in situ transduction, in vivo grade lentiviral particles were produced at VectorBuilder based on the third-generation system. In brief, HEK 293T cells were transfected with eGFP encoding transfer plasmid, envelope plasmid encoding VSV-G and two packaging plasmids encoding Gag/Pol and Rev. The supernatants were collected, and cell debris removed via centrifugation and filtration. Lentiviral particles were subsequently concentrated using polyethylene glycol (PEG) precipitation and further purified through sucrose cushion ultracentrifugation. Lentiviral titers were determined by quantifying the lentiviral p24 Gag protein using ELISA. Adenoviral (Ad) vectors encoding for PIB (Ad-PIB or Ad5-PIB, Ad5-RGD-PIB, Ad5/F35- PIB, Ad5/3-PIB,) or PIB and eGFP (Ad-PIB-eGFP), eGFP (Ad-eGFP) or a non-coding stuffer sequence with or without eGFP (Ad-Stuffer, Ad-Stuffer-eGFP) were produced at VectorBuilder, Vector Biolabs or O.D.260 Inc, using O.D.260 Inc AdenoQuick 2.0 cloning system for Ad5-PIB, Ad5-RGD-PIB, Ad5/F35-PIB, Ad5/3-PIB. Adenoviral vectors were packaged and amplified in HEK 293A cells. In brief, adenoviral plasmids containing PIB, PIB-eGFP, Stuffer or eGFP were first linearized by restriction digestion with Pacl. The linearized plasmid DNA was then transfected into HEK 293A expressing the adenovirus gene E1 to produce recombinant adenovirus. Adenoviral particles P6783PC01 released into the culture medium were harvested and concentrated using cesium chloride (CsCl) gradient ultracentrifugation and/or chromatography. The viral titer was determined by spectrophotometry (OD260) to quantify the number of viral particles and measured for the number of infective units (IFU) by immunocytochemistry staining for transduced cells via the detection of adenovirus-specific hexon protein. Flow cytometry and Fluorescence-activated cell sorting (FACS) Surface marker analysis was performed on dissociated cells from in vitro 2D cultures or single cell suspensions of digested tissue from spheroids or tissues. Cells were stained with adequate antibodies diluted in phosphate-buffered saline (PBS) supplemented with 2% FBS (FACS buffer) at 4°C for 20-30 minutes in the presence of 1% mouse or rat serum, for human and mouse cells, respectively, to block unspecific binding. Tetramer staining was performed at room temperature for 30 minutes before surface marker staining and cell fixation using 4% paraformaldehyde (PFA, Thermo Fisher Scientific) for 20 minutes at 4°C without permeabilization. Intracellular staining for cytokines or proliferation marker Ki67 was performed using the Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) following the manufacturer's recommendation. Intranuclear transcription factor staining was performed using the True-Nuclear Transcription Factor Buffer Set (Biolegend) following the manufacturer's recommendation. For flow cytometry analysis requiring cell fixation, cells were stained with fixable viability dye (FVD) 450 or 520 (Thermo Fisher Scientific) before surface marker staining and fixation. For analysis without cell fixation, dead cells were excluded by addition of 4´,6-diamidino-2-phenylindole (DAPI) or 7-aminoactinomycin D (7-AAD) to the cell suspension after surface marker staining and before acquisition. Flow cytometry analysis was performed on LSR Fortessa, LSR Fortessa X20, FACSymphony A1 and Beckman Coulter Life Sciences CytoFlex Benchtop flow cytometers. FACS-sorting was performed on a BD FACS Aria III or on a FACSymphony S6 sorter, using a 100 μm nozzle. FACS data were analyzed using FlowJo v.10.0.7 (FlowJoLLC). Gates were determined according to fluorescence minus one (FMO) controls. In vitro transduction and reprogramming of tumor cells In vitro reprogramming mediated by lentiviral and adenoviral vectors was performed as previously described (Zimmermannova et al. 2023, Ferreira et al.2023). In brief, 0.5- 1x106 cells were plated per tissue culture 6-well or 10-cm plate and incubated overnight P6783PC01 with 5.5x107 and 5.0x108 genomic copies (GC) per cell in the presence of 8 µg/ml polybrene. Transduction was performed in 5 ml in a 10-cm plate, 1 ml per well in a 6- well plate, or 0.5 ml per well in a 12-well plate. After 16h, virus-containing medium was replaced with fresh medium and cells were maintained until experimental endpoint in culture with regular medium changes every 2-3 days and splitted 1:6 when 80% confluency was reached. Transduction efficiency was measured by flow cytometry using eGFP expression. cDC1 reprogramming efficiency was measured by flow cytometry analysis of CD45 and MHC-II/HLA-DR expression within live or live eGFP+. Macrophage reprogramming efficiency was measured by CD45 and CD11b expression within live eGFP+ cells. In addition, the expression of MHC-I/HLA-ABC, the co- stimulatory molecule CD40 and cDC1 markers XCR1, CLEC9A and CD226 was quantified by flow cytometry. Quantification of MHC-I/HLA-ABC surface molecules per cell was performed using the PE Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences) following manufacturer’s instructions. Tumor establishment To establish tumors, cancer cells were harvested with TrypLE Express, live cells counted by Trypan blue staining using an automated hemocytometer and injected subcutaneously into the right flanks of recipient mice in 100 µl of ice-cold PBS. Before injection, mice were anesthetized by an intraperitoneal injection of ketamine (135 mg/kg) and xylazine (3 mg/kg). For tumor growth and survival experiments, 1x105 B16- F10 in C57BL/6J mice were used. C57BL/6J mice were 6-12-week-old age-matched females. Tumor volumes were monitored with a digital caliper and calculated using the formula V = L*W*H/2. Survival was determined by predefined endpoints such as tumor size reaching 1500 mm3, tumor ulceration, or signs of animal suffering. Animals were randomized for tumor establishment and again before treatment. In vivo reprogramming of tumor cells To evaluate the immunogenicity of in vivo reprogrammed cells in syngeneic mouse melanoma models, the inventors transduced cancer cells in vitro with lentiviral (PIB, PC), and 16 hours post-transduction mixed with untransduced parental cancer cells in defined ratios and injected subcutaneously into the right flank of mice. Unless stated otherwise, cells were mixed at a 1:1 ratio of transduced and untransduced parental cancer cells. As controls, empty viral vectors (lentivirus control: eGFP) were used. Transduction with lentiviral vectors was performed in the presence of polybrene (8 P6783PC01 µg/ml, Sigma-Aldrich). The MOI used for transduction and induction of reprogramming by lentivirus ranged between 5.5x107 and 5.0x108 GC per cell. Immune checkpoint blockade treatment For single or combinatorial treatment with ICB, mice received 200 μg of anti-PD-1 (clone RMP1-14, BioXCell) and/or 200 μg of anti-CTLA-4 (clone 9H10, BioXCell) or rat 200 µg IgG2a (clone 2A3, BioXCell) and IgG2b (clone LTF-2, BioXCell) isotype control antibodies diluted in 100 µl PBS intraperitoneally at days 7, 10, and 13 after tumor establishment. In vivo delivery of viral vectors To deliver viral vectors to tumors in situ, Ad vectors were diluted in ice-cold PBS to reach a final volume of 30 µl and intratumorally injected when the size of tumors reached 30–90 mm3. Tumors that did not reach the required sizes were excluded from the experiment. To assess in situ reprogramming efficiency in human xenograft models, 1010 VPs of Ad-PIB or Ad-Stuffer were injected intratumorally at day 7, 9, 11 and 13 post tumor establishment. At day 16 after human xenograft establishment in NXG mice, tumors were isolated, dissociated, and reprogramming efficiency quantified by flow cytometry. Statistical analysis All statistical analyses were performed using GraphPad Prism or R software. Data was subjected to a normality test before using ANOVA, two-way ANOVA, Kruskal-Wallis or Mann-Whitney test and t-test. Statistical significance of two groups was determined using an unpaired two-tailed Mann-Whitney test or t-test. Group comparisons were performed using ANOVA and corrected by Dunn's or Tukey's multiple comparison test. To estimate statistically significant differences in the survival in multiple groups the log- rank Mantel-Cox test was used. Unless stated otherwise in the figure legends, data are shown as mean ± SD and n represents the total number of animals in in vivo experiments or biological replicates in in vitro experiments. Randomization was performed using the Microsoft Office Excel function (=RANDBETWEEN). Sample sizes were based on previous experience. Significance was considered with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. P6783PC01 Example 2. In vivo cDC1 reprograming induces antitumor immunity and allows higher in vivo efficacy when compared to in vivo myeloid reprogramming. Background The inventors previously identified the combination of transcription factors composed by PU.1, IRF8, and BATF3 (PIB) as sufficient to reprogram fibroblasts or tumor cells into cDC1-like cells in vitro endowed with the three signals required to activate T cells, including antigen presentation on MHC class I and II, co-stimulatory molecule expression and chemokine/cytokine secretion (Rosa et al. 2018, Rosa et al.2022, Zimmermannova et al.2023). Here, the inventors hypothesized that PIB mediate the reprogramming of tumor cells into immunogenic cDC1-like cells entirely in vivo within the TME and benchmarked antitumor efficacy of in vivo cDC1 reprogramming mediated by PIB benchmarked to myeloid reprogramming mediated by PU.1 and CEBP/a. Methods Magnetic-activated cell sorting (MACS) enrichment To purify in vitro reprogrammed mouse cancer cells by magnetic-activated cell sorting (MACS, Miltenyi) with high yields, a protocol was followed as previously described (Ferreira et al.2023). Briefly, cells were resuspended in cold FACS buffer to reach a concentration of 107 cells/ml and incubated with rat serum for 15 minutes, followed by 5 minutes incubation with biotinylated antibodies. To purify cDC1-like cancer cells, biotinylated CD45 and MHC-II antibodies were used. To purify macrophage-like cancer cells, biotinylated CD45 and CD11b antibodies were used. Cells were washed twice with FACS buffer before incubation with magnetic anti-biotin microbeads for 15 minutes. All incubations were performed on ice and labelled cells were purified using LS columns (Miltenyi) according to the manufacturer's recommendations. To purify naïve ovalbumin-specific CD8+ T cells from spleens of OT-I mice, spleens were isolated and homogenized by plunging against a 40 μm cell strainer. Red blood cells were lysed with Pharm lyse lysing buffer (BD Biosciences) for 8 minutes protected from light at room temperature and naïve CD8+ T cells were purified by MACS using the naïve mouse CD8+ T cell isolation kit (Miltenyi Biotec). Generation of bone marrow-derived CD103+ dendritic cells Mouse CD103+ bone marrow-derived dendritic cells (BM-DCs) were generated as previously described (53). In brief, bone marrow (BM) cells were harvested from long P6783PC01 bones of the leg (tibias and femurs) by crushing. Cells were then harvested in PBS supplemented with 2% FBS, filtered with a 40 μm cell strainer and plated in Petri dishes at a density of 1.5x106 cells per mL of RPMI complete medium supplemented with 5 ng/ml GM-CSF (Miltenyi) and 200 ng/ml Fltl3L (Miltenyi) for 16 days (Mayer et al. 2014). Cross-presentation assay Naïve ovalbumin-specific CD8+ T cells from spleens of OT-I mice were enriched using a naïve mouse CD8+ T cell isolation kit (Miltenyi Biotec). Enriched CD8+ T cells were labeled with CellTrace Violet (CTV, Thermo Fisher Scientific) according to manufacturer’s protocol. B16 cells were transduced with lentiviral particles encoding PIB-eGFP or PC-eGFP and reprogrammed for 9 days into cDC1-like or macrophage- like cancer cells, respectively. MACS-purified reprogrammed cancer cells, eGFP- transduced cancer cells, and CD103+ BM-DCs were incubated overnight at 37°C with ovalbumin protein (100 µg/ml) in the presence of P(I:C) (10 µg/ml) and extensively washed. Then, 1x104 cells were incubated with 1x105 naïve CTV-labeled OT-I CD8+ T cells in 96-well U-bottom plate. After 3 days of co-culture, T cells were collected, stained, and analyzed by flow cytometry. T cell proliferation was determined by dilution of CTV staining and upregulation of CD44 expression. The threshold for data plotting was fixed at 100 events within live CD8+ T cell gating. Results To test in vivo cDC1 reprogramming the inventors subcutaneously implanted a mixture of 88% PIB-eGFP-transduced B16 cells and 12% untransduced parental cells, or mixtures of eGFP-transduced and parental cells as a control, 16 hours after transduction (Figure 1A). This strategy allowed to separate delivery of the transcription factors from the in vivo reprogramming process. The inventors observed complete responses (CR) in 30% of animals and delayed tumor growth in the other animals, thereby extending median survival (MS, 43 vs.19 days, p<0.0001). When combined with anti-PD-1 and anti-CTLA-4, tumor regression was observed in all animals. To dissect whether cDC1' functional properties are critical for the observed potent antitumor immunity the inventors compared cDC1 reprogramming with myeloid reprogramming mediated by PU.1 and C/EBPα (PB) to induce macrophage-like cells (Linde et al. 2023) (Fig.1A). In vivo, cDC1 reprogramming extended MS when compared to macrophage reprogramming (43 vs.29.5 days, p<0.0001), especially P6783PC01 when combined with ICB (p=0.0003), which resulted in 100% CRs (Figure 1A). This effect is consistent with the selective induction of high levels of MHC-I and MHC-II (Fig. 1B) and cross-presentation capacity by PIB (Fig.1C). Conclusion These data suggests that in vivo cDC1 reprogramming induces effective antitumor immunity as monotherapy and in combination with immune checkpoint blockade therapy, and highlights the superior in vivo efficacy induced by cDC1 reprogramming mediated by PU.1, IRF8 and BATF3 when compared to myeloid reprogramming mediated by PU.1, and C/EBPα. Example 3. The mutated derivative mut6 of Woodchuck Hepatitis Virus Post- Transcriptional Regulatory Element (mut6WPRE) allows higher cDC1 reprogramming efficiency. Background The Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is typically included in the 3´UTR (untranslated region) in the expression cassette of gene therapy vectors to enhance mRNA stability and protein yield. Because wild type WPRE contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX) previously implicated in the development of liver tumors (Kingsman et al.2005), the inventors investigated whether WPRE sequence downstream the PIB tricistronic cassette is required for efficient reprogramming and whether the mut6WPRE, a version of WPRE that carries a mutation disturbing the expression of the WHX protein considered safe from the regulatory perspective (Zanta- Boussif et al. 2009), could be used instead for the gene therapy product. Results The inventors observed that adenoviral vectors containing WPRE sequence allowed higher reprogramming efficiency measured by surface expression of CD45 and HLA- DR mediated by Ad5 and Ad5/F35 at reprogramming day 3 when compared to adenoviral vectors without WPRE sequence (Fig.2A). Next, the impact of replacing the wild type WPRE sequence by the mut6WPRE was investigated. The inventors detected higher frequency of reprogrammed cells co-expressing CD45 and HLA-DR using mut6WPRE when compared to native WPRE in the melanoma A2058 P6783PC01 (63.7%±1.2 vs 30.25%±5.5) and sarcoma SK-LMS-1 (23.5%±2.55 vs 3.18%±0.88) cell lines at MOI 1x103 IFU/cell (Fig.2B, C). Conclusion These data suggests that WPRE sequence downstream the PIB tricistronic cassette is required for higher cDC1 reprogramming efficiency and supports the selection of mut6WPRE sequence for the final gene therapy vector candidate. Example 4. The Rabbit beta-globin polyadenylation signal (rBGpA) Post- Transcriptional Regulatory Element enhances cDC1 reprogramming efficiency. Background The polyadenylation signal downstream the WPRE sequence can affect transgene expression and thus impact cDC1 reprogramming efficiency. To identify the optimal polyadenylation signal for the final gene therapy vector, 6 polyA sequences were tested and investigated for their ability to induce cDC1 reprogramming. Results The inventors observed that all polyA tested were at least as efficient as the BGHpA across cancer cell lines and that rabbit Beta-Globin polyadenylation signal (rBGpA) was able to increase the percentage of reprogrammed cells at low MOI even in the more resistant to reprogram cell line Ca9-22 (Fig.3A). It was also observed that rBGpA allows high cDC1 reprogramming in both breast and melanoma samples (Fig. 3B). Conclusion These data suggest rBGpA as the optimal polyA signal for efficient cDC1 reprogramming and supports the selection of rBGpA for the final gene therapy candidate. Example 5. Optimized PIB-encoding expression cassette allows high reprogramming efficiency. Background P6783PC01 The inventors have previously tested different promoters, tricistronic cassettes encoding the reprogramming factors PU.1, IRF8 and BATF3 in different orders, WPRE and pA sequences, and have selected the regulatory elements 1) Spleen focus forming viral (SFFV) promoter, 2) tricistronic cassette PU.1-P2A-IRF8-T2A-BATF3, 3) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), and 3) rabbit beta-globin polyadenylation signal (rbBGpA) for the final gene therapy vector. As these elements were tested one-by-one, the inventors decided to generate adenoviral vectors encoding the full optimized expression cassette and investigate its reprogramming capacity when compared to the expression cassette previously used for in vitro efficacy studies. Results The inventors have observed that, not only the single elements tested, but also the full optimized expression cassette (WPREmut6-rbBGpA or AT-108) allows high reprogramming efficiency across human cancer cell lines and primary samples in monolayer and in organoids compared to the previous version (WPRE-BGHpA or Ad5- PIB) (Fig.4 A, B, C, D). Interestingly, the optimized expression cassette containing WPREmut6-rbBGpA allowed a major increase in reprogramming efficiency in human cancer cell lines and in the primary colorectal cancer samples CRC50 at low MOI (100 IFU/cell) when compared to the expression cassette containing WPRE-BGHpA. The inventors observed that the optimized expression cassette allowed superior in vivo reprogramming yield when delivered via intra-tumoral injection (Fig.4 E), and that reprogrammed primary cancer cells generated with the optimized cassette acquired higher antigen cross-presentation capacity in the absence of TLR stimulation (Fig.4 F), acquired cytokine secretion capacity (Fig.4 G) and ultimately enabled superior in vivo efficacy and animal survival in B16 model compared to the previous version (Fig.4 H). Conclusion These data shows that the optimized expression cassette SFFV-PIB-WPREmut6- rbBGpA allows higher reprogramming efficiency, higher functionality of reprogrammed cancer cells and superior in vivo efficacy when compared to previous versions of the expression cassette and supports its selection for the final gene therapy vector candidate. P6783PC01 Example 6. CRISPR/Cas9 screening identifies barriers and facilitators of reprogramming Background Here, the inventors investigated whether CRISPR/Cas9 screening platforms could also prove useful in investigating the regulators of DC reprogramming, enabling the understanding of principles governing cDC1 reprogramming and the identification of novel genes that could enhance or block the outcome. Methods CRISPR screenings Cas9-expressing fibroblasts were generated by by lentiviral transduction at a multiplicity of infection (MOI) of ~1 with the LentiCas9-Blast vector for constitutive expression spCas9 and blasticidin resistance cassette (Addgene #52962). Briefly, cells were plated at the density of 800,000 cells per 100 mm plate and were selected with blasticidin (10 ug/ml) for 9 days and maintained in blasticidin-containing media prior to library transduction. The Human KnockOut RNA regulators and Epifactors (KORE) sgRNA library was cloned into the optimized version of the lentiCRISPR v2 backbone (Addgene #52961) to enhance knockout efficiency. Genes targeted by the sgRNA library were derived from Epifactors database (https://epifactors.autosome.org) and literature on both chromatin and RNA modifiers. The gRNAs sequences targeting the 1367 selected human genes were obtained using the VBC Score design tool (https://www.vbc-score.org), allocating 5 guides per gene, and including non targeting controls. For library transduction, cells were plated at the density of 800,000 cells per 100 mm plate and 24 hours later transduced with the sgRNA library lentiviral vector to achieve a MOI of ~0.3-0.4, with at least 1000-fold representation (cells per guide) in each replicate. After 72h, cells were sorted for GFP expression using FACSAriaIII (BD Biosciences) and expanded for 10 days before reprogramming. Throughout the screenings, cells were split at a density to maintain a representation of at least 300 cells per sgRNA. Screens were performed in 4 independent HDF donors. One million cells were collected before reprogramming to serve as reference point for baseline sgRNA distribution (day 0). At Day 9, puromycin resistant, GFP+ and CD45/HLA-DR stained cells were sorted using the FACS AriaIII (BD Biosciences) and collected pellets were stored at -20°C. Genomic DNA was extracted using the QIAmp Blood Mini Kit P6783PC01 (Qiagen). Amplification of sgRNA regions from the extracted genome was performed by a 2-step PCR reaction protocol, first using custom-made primers harboring the sgRNA region, followed by a second PCR for indexing Illumina Nextera XT adapters (Illumina). Libraries were thoroughly quantified and checked for quality control with dsDNA High-Sensitivity Qubit (Thermo Fisher) and Bioanalyzer (Agilent). The resulting diluted libraries from 3 independent replicates were spiked-in witt 20% PhiX and sequenced using the Illumina NextSeq 500/550 High Output (150 cycles) kit in a NextSeq 500 platform (Illumina) to determine sgRNA representation. After demultiplexing, sgRNA read count data was mapped to the sgRNA library, allowing a maximum of one mismatch in the 20 bp spacer sequence. Mapped reads was input to the CRISPR screen analysis pipeline MAGeCKFlute. MAGeCK was used to identify gene hits and downstream analysis was performed using FluteMLE, with loess normalization. Results The inventors performed knockout (KO) screenings using the optimized conditions for Cas9 expression, sgRNA library expression, and cDC1 reprogramming. Using the 7,409 guides corresponding to the 1367 genes in the library, an average coverage of 300 HDFs/sgRNA/replicate was kept. Cells were reprogrammed with overexpression of PU.1, IRF8 and BATF3, and populations were sorted at endpoint day 9 (Fig.5A, B). Genomic DNA was collected from double positive and double negative (CD45+HLA- DR+, CD45–HLA-DR–) sorted cells, as well as day 0, and sgRNAs were amplified and sequenced (Fig.5C). To determine relative sgRNA abundance, raw read counts were normalized to reads per million and log2 transformed. Then, each sample was normalized to the baseline (day 0) and compared to the distribution of the log2 fold-change (Log2FC) of non- reprogrammed versus reprogrammed samples (Fig.5D, E). Using the MAGeCKFlute pipeline, the inventors identified top candidate genes by ranking them and selecting those enriched in reprogrammed and non-reprogrammed samples (Fig.5D). Genes with an increased sgRNA count in the reprogrammed samples, which equates to an increase in the fold-change, were defined as reprogramming barriers, since its silencing may lead to better reprogramming efficiency. On the other hand, genes that showed increased fold-change in the non-reprogrammed population were defined as facilitators of DC1 reprogramming (Fig. 5E). Here, the inventors report a total of 117 barriers and 74 facilitators that are over the 1.5 standard deviation (SD) cut-off P6783PC01 compared to the median distribution of the genes. As top 10 barrier candidates the inventors identified ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L. On the other hand, the top facilitator candidates include WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2. Conclusion Together, the inventors developed a CRISPR/Cas9 screening platform to investigate regulators of cDC1 reprogramming and identified multiple facilitators and barriers of the process. These can be manipulated to the optimization cDC1 reprogramming efficiency in human cells and enable more effective immunotherapies. Example 7. shRNA screening identifies barriers and facilitators of reprogramming Background Here, the inventors investigated whether a loss-of-function screening platform with shRNAs could allow the identification of druggable targets and pathways that prevent or promote acquisition of cDC1 identity through direct cell reprogramming. Methods Viral transduction and reprogramming for shRNA screen Clec9a-tdTomato MEFs and HDFs were seeded at a density of 30,000 cells per well on 0.1% gelatin-coated six-well plates. On the following day, cells were incubated overnight with a ratio of 1:1 TetO-PIB and M2rtTA, shRNA-GFP, SFFV-PIB-GFP, or SFFV-GFP lentiviral particles in a medium supplemented with polybrene (8 µg/ml). After the transduction, medium was replaced by a normal growth medium (d0). When using TetO- PIB, the medium was supplemented with Dox (1 µg/ml). The medium was changed every 2 to 3 days for the duration of cultures. Results For the initial screening, the inventors used a shRNA library composed of 206 shRNAs for specific kinases and phosphatases used in a previous study with embryonic stem cell (Lee et al.2012). These were cloned in the pLKO.pig plasmid which also encodes a GFP protein enabling the identification of the cells which were successfully transduced. MEFs P6783PC01 were initially transduced with each shRNA and after three days, they were transduced with PIB. Dox was added at day 0 to induce PIB expression and reprogrammed cells were analyzed at day 6 by flow cytometry (Fig. 6A). In addition to GFP and tdT expression, the inventors also assessed MHC-ll expression as an indication for reprogramming fidelity and antigen presentation capacity. The screening includes a non- targeting shRNA (shNTC) as negative control and identified targets whose silencing reduced or improved tdT expression, which we respectively termed DC reprogramming facilitators or barrier (Fig. 18B). Silencing of Spi1 (the gene encoding for the reprogramming transcription factor PU.1) resulted in dramatic reduction in tdTomato expression. Then, shRNAs that induced changes of tdTomato expression above or below 0.5-fold when compared to shNTC were re-tested for further validation studies, and their impact was validated in reprogramming efficiency and MHC-ll expression (Fig. 6C). After completion of the screen, 14 candidates (“barriers“) were prioritized that, upon silencing, resulted in a significant increase of reprogramming efficiency (Fig. 6D, upper panel), and 30 candidates (“facilitators”) that, upon silencing, led to a significant decrease in reprogramming efficiency (Fig.6D, bottom panel). We next sought to use small chemical inhibitors to further validate the targets identified to be involved in cDC1 reprogramming. Analysis of the main function of the selected targets showed common involvement in the AMPK and mTOR pathway. Incubation with small chemical inhibitors started 3 days prior to PIB transduction and was maintained until day 9, when cells were analyzed by flow cytometry for the expression of the DC markers CD45, HLA-DR and CD40 (Fig.6E). Interestingly, chemical inhibition of GSK3A with 1 µM CHIR99021 and inhibition of mTOR signaling with rapamycin, respectively, increased and decreased the number of reprogrammed induced dendritic cells on day 9 (Fig. 6F). Conclusion Together, the inventors developed and validated a shRNA screening platform to investigate regulators of cDC1 reprogramming and identified multiple facilitators and barriers of the process. These can be manipulated to the optimization cDC1 reprogramming efficiency in human cells and enable more effective immunotherapies. Example 8. Knock-out of RYBP and SND1 enhances cDC1 reprogramming efficiency Background P6783PC01 Here, the inventors investigated whether CRISPR/Cas9 screening platform could enable the investigation of common barriers of cell fate reprogramming towards DCs, hematopoietic stem and progenitor cells (HSPCs) and induced pluripotent stem cells (iPSCs) Methods CRISPR screening in HSPC and iPSC reprogramming Screens for DC reprogramming were performed according to Example 6. Screens in HSPC and iPSC reprogramming were performed in 3 independent HDF donors. One million cells were collected before reprogramming to serve as reference point for baseline sgRNA distribution (day 0). For iPSC reprogramming, cells were sorted at day 20 based on the surface markers CD13, EpCAM, SSEA4 and TRA-1-60 (Non- reprogrammed cells: CD13+ EpCAM- SSEA4- TRA-1-60-; Reprogrammed cells: CD13- EpCAM+ SSEA4+ TRA-1-60+). For induced HSPCs, cells were sorted at day 15 based on the surface expression of CD9 and CD49f (Non-reprogrammed: CD9- CD49f-; reprogrammed: CD9+CD49f+). Cells were sorted using the FACS AriaIII (BD Biosciences) and collected pellets were stored at -20°C. Genomic DNA was extracted using the QIAmp Blood Mini Kit (Qiagen). Amplification of sgRNA regions from the extracted genome was performed by a 2-step PCR reaction protocol, first using custom-made primers harboring the sgRNA region, followed by a second PCR for indexing Illumina Nextera XT adapters (Illumina). Libraries were thoroughly quantified and checked for quality control with dsDNA High-Sensitivity Qubit (Thermo Fisher) and Bioanalyzer (Agilent). The resulting diluted libraries from 3 independent replicates were spiked-in with 20% PhiX and sequenced using the Illumina NextSeq 500/550 High Output (150 cycles) kit in a NextSeq 500 platform (Illumina) to determine sgRNA representation. After demultiplexing, sgRNA read count data was mapped to the sgRNA library, allowing a maximum of one mismatch in the 20 bp spacer sequence. Mapped reads was input to the CRISPR screen analysis pipeline MAGeCKFlute. MAGeCK was used to identify gene hits and downstream analysis was performed using FluteMLE, with loess normalization. Results The inventors performed KO screenings using the optimized conditions for Cas9 expression, sgRNA library expression, and HSPC and iPSC reprogramming. Cells P6783PC01 were reprogrammed to induced HSPCs with overexpression of GATA2, GFI1B and FOS (Gomes et al.2018), and to iPSCs by overexpression of OCT4, Sox2, Klf4 and c- Myc (Takahashi et al.2007), and populations were sorted at endpoint day 15 (iHSPC) or day 20 (iPSC). Genomic DNA was collected from reprogrammed and non- reprogrammed purified cells, as well as day 0, and sgRNAs were amplified and sequenced. First, the inventors identified 82 barriers and 125 facilitators of iPSC reprogramming, and 95 barriers and 111 facilitators of iHSPC reprogramming. The inventors then performed a comparative analysis to explore the relationship between barriers shared in the intersections of reprogramming to cDC1s (Example 18), iHSPCs and iPSCs, and identified 3 barriers that were conserved across reprogramming systems (Fig.7A). Interestingly, KO of one of these 3 barriers (RYBP) enable enhanced cDC1 reprogramming efficiency (Fig.7B). To validate additional barriers to cDC1 reprogramming, the inventors selected 13 barriers, performed single knockouts and reprogrammed the cells to cDC1-like cells by overexpression of PU.1, IRF8 and BATF3. From the 13 candidates, SND1 KO was associated with higher cDC1 reprogramming efficiency measured by the frequency of cells co-expressing CD45 and HLA-DR at day 6, 9, 12 and 16 when compared to the control (Fig.7C). Conclusion Together, these data enabled the identification and validation of RYBP and SND1 as barriers to cDC1 reprogramming and demonstrated that knock out or downregulation of RYBP or SND1 enhances cDC1 reprogramming efficiency. Example 9. HDAC and methylation inhibition enhance cDC1 reprogramming Background The inventors have previously demonstrated that HDAC inhibition using valproic acid (VPA) enables higher reprogramming efficiency. Here, the inventors investigated whether HDAC inhibition synergizes with inhibition of DNA methylation for enhanced cDC1 reprogramming efficiency. The inventors also explore the impact of HDAC inhibition in in vivo efficacy and in reprogramming mediated by adenoviral vectors. Results The inventors profiled cDC1 reprogramming efficiency in one human primary cancer sample of glioblastoma (G3119) and 3 human cancer cell lines of melanoma (CHL1), ovarian (OVK18) and gastric (MKN74) cancer in the presence of valproic acid (VPA, P6783PC01 HDAC inhibitor), Azacitidine (Aza, methylation inhibitor), or both, from day 0 to day 3 after transduction with lentiviral particles encoding PU.1, IRF8 and BATF3 (PIB). At reprogramming day 9, the inventors observed that VPA and Aza individually enhanced reprogramming efficiency in glioblastoma, melanoma and gastric cancer cells (Fig. 8A). Interestingly, the inventors also observed that combination of VPA and Aza enabled higher reprogramming efficiency in glioblastoma, ovarian and gastric cancer, compared to their individual effect. Next, the inventors investigated the impact of VPA treatment in in vivo efficacy. YUMM1.7 cells were transduced with PIB-encoding lentiviral vectors, incubated or not with VPA until reprogramming day 4, mixed with non-treated YUMM1.7 cells (ratio 30% transduced to 70% non-treated cells) and injected subcutaneously in mice to profile tumor growth and survival overtime. Interestingly, the inventors observed that VPA treatment enabled higher in vivo efficacy associated with 90% complete tumor regression compared to 20% complete tumor regression in groups injected with transduced cells not treated with VPA (Fig.8B). Finally, the inventors investigated whether VPA treatment also enable higher cDC1 reprogramming efficiency in cancer cells mediated by adenoviral vectors. YUMM1.7 cancer cells were transduced with PIB-encoding adenoviral vectors, treated with VPA from day 0 to day 3, and reprogramming efficiency was profiled at day 3. The inventors observed that treatment with VPA also enhanced cDC1 reprogramming efficiency mediated by adenoviral vectors (Fig.8C). Conclusion Together, these data support that HDAC and DNA methylation inhibition enables higher cDC1 reprogramming efficiency in cancer cells. HDAC inhibition enables higher in vivo efficacy, and enhances cDC1 reprogramming mediated by adenoviral vectors. Example 10. PRC2 inhibition enhances reprogramming efficiency and fidelity. Background Given that PU.1, IRF8 and BATF3 engage Polycomb-repressed chromatin early during lineage conversion in H3K27me3-marked regions, the inventors sought to determine whether inhibition of PRC2, the complex responsible for depositing H3K27me3, would impact reprogramming efficiency and fidelity. For this, fibroblasts were treated with small molecule inhibitors targeting PRC2 components during the early reprogramming window (day 0 to day 2). P6783PC01 Methods To induce cDC1 reprogramming, human dermal fibroblasts (HDFs) were seeded at 300,000 cells per gelatin-coated plate. The next day, cells were transduced overnight with lentiviral particles in DMEM Complete medium containing 8 μg/mL polybrene. After 16 hours (day 0), the medium was replaced, and cells were treated with 1 μM, 5 μM, or 10 μM of PRC2 inhibitors or DMSO as a control. Inhibitors included EZH2-specific compounds (Tazemetostat and GSK-126) and an EED inhibitor (EED226). Media was refreshed every 2–3 days. On day 9, reprogramming efficiency (% CD45+HLA-DR+ cells) and fidelity (% CD40+CD226+ cells) in live, transduced cells were assessed via flow cytometry. Results Fibroblasts were treated with small molecule inhibitors targeting PRC2 components during the early reprogramming window (day 0 to day 2). Inhibition of PRC2 led to a significant increase in reprogramming efficiency (+11.6%) and fidelity (+11.0%), as measured by the proportion of reprogrammed cells expressing CD45 and HLA-DR, and CD40 and CD226, respectively (Fig. 9). Conclusion These findings underscore the role of PRC2-mediated H3K27me3 in limiting early TF engagement during reprogramming. Pulsed inhibition of PRC2 at the onset of lineage conversion significantly enhances both efficiency (%CD45+HLA-DR+ cells) and fidelity (%CD40+CD226+ cells) of reprogramming. Example 11. Enforced gene activation by IRF8 and gene inactivation by BATF3 enables higher cDC1 reprogramming efficiency. Background To decipher the functional role of each reprogramming factor in cDC1 reprogramming, the inventors employed transcriptional effector domain fusions by linking the TFs to either the VP16 activation domain or the KRAB repressor domain. These constructs were expressed in combination with bicistronic lentiviral vectors to allow modular functional assessment of each TF. Methods P6783PC01 HDFs were co-transduced with bicistronic lentiviral constructs encoding combinations of reprogramming factors (PU.1-IRF8 [PI], PU.1-BATF3 [PB], or IRF8-BATF3 [IB]) together with individual TFs (PU.1, IRF8, BATF3) fused to either the VP16 activation domain or KRAB repressor domain. Following transduction, cells were cultured under standard reprogramming conditions. CD45 expression was assessed by flow cytometry at day 9, and fold change (FC) in CD45 induction was calculated relative to wild-type (untagged) TFs. Results Expression of VP16-fused IRF8 led to an increase in CD45 induction, indicating a strong transcriptional activation role for IRF8 in the context of reprogramming (Fig.10). In contrast, expression of KRAB-fused BATF3 enhanced CD45 induction strongly, suggesting that BATF3 functions as a transcriptional repressor during reprogramming. Conclusion These data demonstrate divergent regulatory functions of reprogramming factors. Enforced transcriptional activation of target genes by IRF8 and enforced transcriptional inactivation of target genes by BATF3 enables higher reprogramming efficiency. Example 12. AMPK activation increases cDC1 reprogramming efficiency and in vivo efficacy Background In Example 7 of the present application, the inventors have used small chemical inhibitors to validate targets identified in an shRNA screen to be involved in cDC1 reprogramming and observed that chemical inhibition of GSK3A with CHIR99021 and inhibition of mTOR signaling with rapamycin, respectively, increased and decreased cDC1 reprogramming yield on day 9 (Fig.6F). These data suggested that metabolism, energy sensing and AMPK-GSK3-mTOR signalling axis are important regulators of the reprogramming process. To further explore this observation, the inventors have profiled the impact of selective activation or inhibition of intermediates of the AMPK-GSK3- mTOR signalling axis. Materials and method P6783PC01 HDFs were transduced with lentiviral constructs encoding a polycistronic PIB (PU.1– IRF8–BATF3). Cells were treated with either vehicle (DMSO) or small molecules from day 0 to day 9. At day 9, reprogramming efficiency was profiled by flow cytometry analysis of CD45+, HLA-DR+ and TNF-a+ cells. Secretion of IL12p70 by purified CD45+HLA-DR+ reprogrammed cancer cells was quantified by ELISA. To evaluate the tumor growth after in vivo cDC1 reprogramming of B16-F10 cells, the inventors transduced cancer cells in vitro with lentivirus SFFV-PIB-eGFP (expressing PU.1, IRF8, BATF3 and eGFP as a reporter of transduction) or empty lentiviral vector as a transduction control SFFV-eGFP (empty lentiviral backbone expressing eGFP) and 16 hours after transduction injected the cells into C57BL/6J mice as a mix of 1:1 transduced eGFP+ to untransduced eGFP- cells. The transduction was performed in the presence of polybrene (8 µg/ml). Results The inventors observed that AMPK activators (CHIR99021, AICAR, A769662, Metformin), mTOR activators (MHY1485, Salidroside) and inhibitors of glycolysis (2-DG or 2-deoxyglucose) enabled higher reprogramming efficiency. In contrast, AMPK inhibitors (Compound C), mTOR inhibitors (Torin1, AZD2014, Rapamycin) and CAMKK2 inhibitors (STO-609) reduced reprogramming efficiency (Fig. 11B). Interestingly, the inventors observed that CD45+HLA-DR+ reprogrammed cells generated in the presence of A769662 also produced higher levels of TNF-a and IL- 12p70, 2 cytokines critical for antigen presentation (Fig. 11C-D). To investigate whether AMPK activation in vivo would result in higher in vivo efficacy, the inventors transduced B16-F10 cancer cells in vitro with lentivirus encoding PIB, 16 hours after transduction injected the cells into C57BL/6J mice as a mix of 1:1 transduced eGFP+ to untransduced eGFP- cells, and treated mice with 3 IP injections of A769662 at days 1, 3 and 5 after tumor establishment (Fig.11E-F). The inventors observed a significant increase in in vivo efficacy and delayed tumor growth when in vivo reprogramming was combined with A769662 treatment. Conclusion Together, these data suggests that small molecule agonists of AMPK improve cDC1 reprogramming efficiency, functionality of reprogrammed cDC1-like cells and enable higher in vivo efficacy mediated by in vivo cDC1 reprogramming. P6783PC01 Sequence overview SEQ. ID NO: 1 AT-108: Ad5-PIB SEQ. ID NO: 2 AT-110: Ad5/F35-PIB SEQ. ID NO: 3 AT-111: Ad5-RGD-PIB SEQ. ID NO: 4 AT-112: Ad5/3-PIB SEQ. ID NO: 5 is an empty sequence. SEQ. ID NO: 6 mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6) – polynucleotide sequence AATCAACCTCTGGATTACAAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGC TTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTA TGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGC TGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG GACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTT GCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTG TCGGGGAAGGTCTGCTGAGACTCGGGGCTGCTCGCCTGTGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTT CCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG SEQ. ID NO: 7 rabbit beta-globin polyadenylation signal sequence (rbBGpA) – polynucleotide sequence P6783PC01 TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCT GGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCA TGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCA ATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAA TCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATAT GCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAG CCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTT TTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATG TTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCT CTTCTCTTATGGAGATC SEQ. ID NO: 8 late polyadenylation signal sequence of simian virus 40 (SV40late)– polynucleotide sequence CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGA AAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGG GGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA SEQ. ID NO: 9 PU.1 – polynucleotide sequence ATGTTACAGGCGTGCAAAATGGAAGGGTTTCCCCTCGTCCCCCCTCCATCAGAAG ACCTGGTGCCCTATGACACGGATCTATACCAACGCCAAACGCACGAGTATTACCC CTATCTCAGCAGTGATGGGGAGAGCCATAGCGACCATTACTGGGACTTCCACCCC CACCACGTGCACAGCGAGTTCGAGAGCTTCGCCGAGAACAACTTCACGGAGCTC CAGAGCGTGCAGCCCCCGCAGCTGCAGCAGCTCTACCGCCACATGGAGCTGGA GCAGATGCACGTCCTCGATACCCCCATGGTGCCACCCCATCCCAGTCTTGGCCA CCAGGTCTCCTACCTGCCCCGGATGTGCCTCCAGTACCCATCCCTGTCCCCAGC CCAGCCCAGCTCAGATGAGGAGGAGGGCGAGCGGCAGAGCCCCCCACTGGAGG TGTCTGACGGCGAGGCGGATGGCCTGGAGCCCGGGCCTGGGCTCCTGCCTGGG GAGACAGGCAGCAAGAAGAAGATCCGCCTGTACCAGTTCCTGTTGGACCTGCTC CGCAGCGGCGACATGAAGGACAGCATCTGGTGGGTGGACAAGGACAAGGGCAC CTTCCAGTTCTCGTCCAAGCACAAGGAGGCGCTGGCGCACCGCTGGGGCATCCA GAAGGGCAACCGCAAGAAGATGACCTACCAGAAGATGGCGCGCGCGCTGCGCAA CTACGGCAAGACGGGCGAGGTCAAGAAGGTGAAGAAGAAGCTCACCTACCAGTT P6783PC01 CAGCGGCGAAGTGCTGGGACGCGGGGGCCTGGCCGAGCGGCGCCACCCGCCC CAC SEQ. ID NO: 10 PU.1 – polypeptide sequence MLQACKMEGFPLVPPPSEDLVPYDTDLYQRQTHEYYPYLSSDGESHSDHYWDFHPH HVHSEFESFAENNFTELQSVQPPQLQQLYRHMELEQMHVLDTPMVPPHPSLGHQVS YLPRMCLQYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLLPGETGSKK KIRLYQFLLDLLRSGDMKDSIWWVDKDKGTFQFSSKHKEALAHRWGIQKGNRKKMTY QKMARALRNYGKTGEVKKVKKKLTYQFSGEVLGRGGLAERRHPPH SEQ. ID NO: 11 IRF8 – polynucleotide sequence ATGTGTGACCGGAATGGTGGTCGGCGGCTTCGACAGTGGCTGATCGAGCAGATT GACAGTAGCATGTATCCAGGACTGATTTGGGAGAATGAGGAGAAGAGCATGTTCC GGATCCCTTGGAAACACGCTGGCAAGCAAGATTATAATCAGGAAGTGGATGCCTC CATTTTTAAGGCCTGGGCAGTTTTTAAAGGGAAGTTTAAAGAAGGGGACAAAGCT GAACCAGCCACTTGGAAGACGAGGTTACGCTGTGCTTTGAATAAGAGCCCAGATT TTGAGGAAGTGACGGACCGGTCCCAACTGGACATTTCCGAGCCATACAAAGTTTA CCGAATTGTTCCTGAGGAAGAGCAAAAATGCAAACTAGGCGTGGCAACTGCTGGC TGCGTGAATGAAGTTACAGAGATGGAGTGCGGTCGCTCTGAAATCGACGAGCTGA TCAAGGAGCCTTCTGTGGACGATTACATGGGGATGATCAAAAGGAGCCCTTCCCC GCCGGAGGCCTGTCGGAGTCAGCTCCTTCCAGACTGGTGGGCGCAGCAGCCCA GCACAGGCGTGCCGCTGGTGACGGGGTACACCACCTACGACGCGCACCATTCAG CATTCTCCCAGATGGTGATCAGCTTCTACTATGGGGGCAAGCTGGTGGGCCAGG CCACCACCACCTGCCCCGAGGGCTGCCGCCTGTCCCTGAGCCAGCCTGGGCTG CCCGGCACCAAGCTGTATGGGCCCGAGGGCCTGGAGCTGGTGCGCTTCCCGCC GGCCGACGCCATCCCCAGCGAGCGACAGAGGCAGGTGACGCGGAAGCTGTTCG GGCACCTGGAGCGCGGGGTGCTGCTGCACAGCAGCCGGCAGGGCGTGTTCGTC AAGCGGCTGTGCCAGGGCCGCGTGTTCTGCAGCGGCAACGCCGTGGTGTGCAA AGGCAGGCCCAACAAGCTGGAGCGTGATGAGGTGGTCCAGGTCTTCGACACCAG CCAGTTCTTCCGAGAGCTGCAGCAGTTCTATAACAGCCAGGGCCGGCTTCCTGAC GGCAGGGTGGTGCTGTGCTTTGGGGAAGAGTTTCCGGATATGGCCCCCTTGCGC TCCAAACTCATTCTCGTGCAGATTGAGCAGCTGTATGTCCGGCAACTGGCAGAAG AGGCTGGGAAGAGCTGTGGAGCCGGCTCTGTGATGCAGGCCCCCGAGGAGCCG P6783PC01 CCGCCAGACCAGGTCTTCCGGATGTTTCCAGATATTTGTGCCTCACACCAGAGAT CATTTTTCAGAGAAAACCAACAGATCACCGTC SEQ. ID NO: 12 IRF8 – polypeptide sequence MCDRNGGRRLRQWLIEQIDSSMYPGLIWENEEKSMFRIPWKHAGKQDYNQEVDASIF KAWAVFKGKFKEGDKAEPATWKTRLRCALNKSPDFEEVTDRSQLDISEPYKVYRIVP EEEQKCKLGVATAGCVNEVTEMECGRSEIDELIKEPSVDDYMGMIKRSPSPPEACRS QLLPDWWAQQPSTGVPLVTGYTTYDAHHSAFSQMVISFYYGGKLVGQATTTCPEGC RLSLSQPGLPGTKLYGPEGLELVRFPPADAIPSERQRQVTRKLFGHLERGVLLHSSRQ GVFVKRLCQGRVFCSGNAVVCKGRPNKLERDEVVQVFDTSQFFRELQQFYNSQGRL PDGRVVLCFGEEFPDMAPLRSKLILVQIEQLYVRQLAEEAGKSCGAGSVMQAPEEPP PDQVFRMFPDICASHQRSFFRENQQITV SEQ. ID NO: 13 BATF3 – polynucleotide sequence ATGTCGCAAGGGCTCCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCGTCGCGGC GCCCGGGAACCAGCCGCAGCCGCAGCCGCAGCAGCAGAGCCCTGAGGATGATG ACAGGAAGGTCCGAAGGAGAGAAAAAAACCGAGTTGCTGCTCAGAGAAGTCGGA AGAAGCAGACCCAGAAGGCTGACAAGCTCCATGAGGAATATGAGAGCCTGGAGC AAGAAAACACCATGCTGCGGAGAGAGATCGGGAAGCTGACAGAGGAGCTGAAGC ACCTGACAGAGGCACTGAAGGAGCACGAGAAGATGTGCCCGCTGCTGCTCTGCC CTATGAACTTTGTGCCAGTGCCTCCCCGGCCGGACCCTGTGGCCGGCTGCTTGC CCCGA SEQ. ID NO: 14 BATF3 – polypeptide sequence MSQGLPAAGSVLQRSVAAPGNQPQPQPQQQSPEDDDRKVRRREKNRVAAQRSRK KQTQKADKLHEEYESLEQENTMLRREIGKLTEELKHLTEALKEHEKMCPLLLCPMNFV PVPPRPDPVAGCLPR SEQ. ID NO: 15 SFFV promoter – polynucleotide sequence CTGCAGCCCCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATG AAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTGCAGTAACGCCATTTTGCAAGG P6783PC01 CATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATG AAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCG GCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCA AGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATC AGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTA ACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATA AAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGC CCGGG SEQ. ID NO: 16 GATA2 – polynucleotide sequence ATGGAGGTGGCGCCCGAGCAGCCGCGCTGGATGGCGCACCCGGCCGTGCTGAA TGCGCAGCACCCCGACTCACACCACCCGGGCCTGGCGCACAACTACATGGAACC CGCGCAGCTGCTGCCTCCAGACGAGGTGGACGTCTTCTTCAATCACCTCGACTC GCAGGGCAACCCCTACTATGCCAACCCCGCTCACGCGCGGGCGCGCGTCTCCTA CAGCCCCGCGCACGCCCGCCTGACCGGAGGCCAGATGTGCCGCCCACACTTGTT GCACAGCCCGGGTTTGCCCTGGCTGGACGGGGGCAAAGCAGCCCTCTCTGCCG CTGCGGCCCACCACCACAACCCCTGGACCGTGAGCCCCTTCTCCAAGACGCCAC TGCACCCCTCAGCTGCTGGAGGCCCTGGAGGCCCACTCTCTGTGTACCCAGGGG CTGGGGGTGGGAGCGGGGGAGGCAGCGGGAGCTCAGTGGCCTCCCTCACCCCT ACAGCAGCCCACTCTGGCTCCCACCTTTTCGGCTTCCCACCCACGCCACCCAAAG AAGTGTCTCCTGACCCTAGCACCACGGGGGCTGCGTCTCCAGCCTCATCTTCCG CGGGGGGTAGTGCAGCCCGAGGAGAGGACAAGGACGGCGTCAAGTACCAGGTG TCACTGACGGAGAGCATGAAGATGGAAAGTGGCAGTCCCCTGCGCCCAGGCCTA GCTACTATGGGCACCCAGCCTGCTACACACCACCCCATCCCCACCTACCCCTCCT ATGTGCCGGCGGCTGCCCACGACTACAGCAGCGGACTCTTCCACCCCGGAGGCT TCCTGGGGGGACCGGCCTCCAGCTTCACCCCTAAGCAGCGCAGCAAGGCTCGTT CCTGTTCAGAAGGCCGGGAGTGTGTCAACTGTGGGGCCACAGCCACCCCTCTCT GGCGGCGGGACGGCACCGGCCACTACCTGTGCAATGCCTGTGGCCTCTACCACA AGATGAATGGGCAGAACCGACCACTCATCAAGCCCAAGCGAAGACTGTCGGCCG CCAGAAGAGCCGGCACCTGTTGTGCAAATTGTCAGACGACAACCACCACCTTATG GCGCCGAAACGCCAACGGGGACCCTGTCTGCAACGCCTGTGGCCTCTACTACAA GCTGCACAATGTTAACAGGCCACTGACCATGAAGAAGGAAGGGATCCAGACTCG GAACCGGAAGATGTCCAACAAGTCCAAGAAGAGCAAGAAAGGGGCGGAGTGCTT CGAGGAGCTGTCAAAGTGCATGCAGGAGAAGTCATCCCCCTTCAGTGCAGCTGC P6783PC01 CCTGGCTGGACACATGGCACCTGTGGGCCACCTCCCGCCCTTCAGCCACTCCGG ACACATCCTGCCCACTCCGACGCCCATCCACCCCTCCTCCAGCCTCTCCTTCGGC CACCCCCACCCGTCCAGCATGGTGACCGCCATGGGCTAG SEQ. ID NO: 17 GATA2 – polypeptide sequence MEVAPEQPRWMAHPAVLNAQHPDSHHPGLAHNYMEPAQLLPPDEVDVFFNHLDSQ GNPYYANPAHARARVSYSPAHARLTGGQMCRPHLLHSPGLPWLDGGKAALSAAAAH HHNPWTVSPFSKTPLHPSAAGGPGGPLSVYPGAGGGSGGGSGSSVASLTPTAAHS GSHLFGFPPTPPKEVSPDPSTTGAASPASSSAGGSAARGEDKDGVKYQVSLTESMK MESGSPLRPGLATMGTQPATHHPIPTYPSYVPAAAHDYSSGLFHPGGFLGGPASSFT PKQRSKARSCSEGRECVNCGATATPLWRRDGTGHYLCNACGLYHKMNGQNRPLIK PKRRLSAARRAGTCCANCQTTTTTLWRRNANGDPVCNACGLYYKLHNVNRPLTMKK EGIQTRNRKMSNKSKKSKKGAECFEELSKCMQEKSSPFSAAALAGHMAPVGHLPPF SHSGHILPTPTPIHPSSSLSFGHPHPSSMVTAMG SEQ. ID NO: 18 chromatin remodelling factor RB Binding Protein-4 (RBBP4) - polynucleotide sequence ATGGCCGACAAGGAAGCAGCCTTCGACGACGCAGTGGAAGAACGAGTGATCAAC GAGGAATACAAAATATGGAAAAAGAACACCCCTTTTCTTTATGATTTGGTGATGAC CCATGCTCTGGAGTGGCCCAGCCTAACTGCCCAGTGGCTTCCAGATGTAACCAGA CCAGAAGGGAAAGATTTCAGCATTCATCGACTTGTCCTGGGGACACACACATCGG ATGAACAAAACCATCTTGTTATAGCCAGTGTGCAGCTCCCTAATGATGATGCTCAG TTTGATGCGTCACACTACGACAGTGAGAAAGGAGAATTTGGAGGTTTTGGTTCAG TTAGTGGAAAAATTGAAATAGAAATCAAGATCAACCATGAAGGAGAAGTAAACAGG GCCCGTTATATGCCCCAGAACCCTTGTATCATCGCAACAAAGACTCCTTCCAGTG ATGTTCTTGTTTTTGACTATACAAAACATCCTTCTAAACCAGATCCTTCTGGAGAGT GCAACCCAGACTTGCGTCTCCGTGGACATCAGAAGGAAGGCTATGGGCTTTCTTG GAACCCAAATCTCAGTGGGCACTTACTTAGTGCTTCAGATGACCATACCATCTGCC TGTGGGACATCAGTGCCGTTCCAAAGGAGGGAAAAGTGGTAGATGCGAAGACCA TCTTTACAGGGCATACGGCAGTAGTAGAAGATGTTTCCTGGCATCTACTCCATGA GTCTCTGTTTGGGTCAGTTGCTGATGATCAGAAACTTATGATTTGGGATACTCGTT CAAACAATACTTCCAAACCAAGCCACTCAGTTGATGCTCACACTGCTGAAGTGAAC TGCCTTTCTTTCAATCCTTATAGTGAGTTCATTCTTGCCACAGGATCAGCTGACAA GACTGTTGCCTTGTGGGATCTGAGAAATCTGAAACTTAAGTTGCATTCCTTTGAGT P6783PC01 CACATAAGGATGAAATATTCCAGGTTCAGTGGTCACCTCACAATGAGACTATTTTA GCTTCCAGTGGTACTGATCGCAGACTGAATGTCTGGGATTTAAGTAAAATTGGAG AGGAACAATCCCCAGAAGATGCAGAAGACGGGCCACCAGAGTTGTTGTTTATTCA TGGTGGTCATACTGCCAAGATATCTGATTTCTCCTGGAATCCCAATGAACCTTGGG TGATTTGTTCTGTATCAGAAGACAATATCATGCAAGTGTGGCAAATGGCAGAGAAC ATTTATAATGATGAAGACCCTGAAGGAAGCGTGGATCCAGAAGGACAAGGGTCCT AG SEQ. ID NO: 19 chromatin remodelling factor RB Binding Protein-4 (RBBP4) - polypeptide sequence MADKEAAFDDAVEERVINEEYKIWKKNTPFLYDLVMTHALEWPSLTAQWLPDVTRPE GKDFSIHRLVLGTHTSDEQNHLVIASVQLPNDDAQFDASHYDSEKGEFGGFGSVSGKI EIEIKINHEGEVNRARYMPQNPCIIATKTPSSDVLVFDYTKHPSKPDPSGECNPDLRLR GHQKEGYGLSWNPNLSGHLLSASDDHTICLWDISAVPKEGKVVDAKTIFTGHTAVVE DVSWHLLHESLFGSVADDQKLMIWDTRSNNTSKPSHSVDAHTAEVNCLSFNPYSEFI LATGSADKTVALWDLRNLKLKLHSFESHKDEIFQVQWSPHNETILASSGTDRRLNVW DLSKIGEEQSPEDAEDGPPELLFIHGGHTAKISDFSWNPNEPWVICSVSEDNIMQVWQ MAENIYNDEDPEGSVDPEGQGS SEQ. ID NO: 20 splicing factor 3B subunit 6, (SF3B6) - polynucleotide sequence ATGGCGATGCAAGCGGCCAAGAGGGCGAACATTCGACTTCCACCTGAAGTAAATC GGATATTGTATATAAGAAATTTGCCATACAAAATCACAGCTGAAGAAATGTATGATA TATTTGGGAAATATGGACCTATTCGTCAAATCAGAGTGGGGAACACACCTGAAACT AGAGGAACAGCTTATGTGGTCTATGAGGACATCTTTGATGCCAAGAATGCATGTG ATCACCTATCGGGATTCAATGTTTGTAACAGATACCTTGTGGTTTTGTACTATAATG CCAACAGGGCATTTCAGAAGATGGACACAAAGAAGAAGGAGGAACAGTTGAAGCT TCTCAAGGAGAAATATGGCATCAACACAGATCCACCAAAATAA SEQ. ID NO: 21 splicing factor 3B subunit 6, (SF3B6) - polypeptide sequence MAMQAAKRANIRLPPEVNRILYIRNLPYKITAEEMYDIFGKYGPIRQIRVGNTPETRGTA YVVYEDIFDAKNACDHLSGFNVCNRYLVVLYYNANRAFQKMDTKKKEEQLKLLKEKY GINTDPPK P6783PC01 SEQ. ID NO: 22 calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) - polynucleotide sequence ATGTCATCATGTGTCTCTAGCCAGCCCAGCAGCAACCGGGCCGCCCCCCAGGAT GAGCTGGGGGGCAGGGGCAGCAGCAGCAGCGAAAGCCAGAAGCCCTGTGAGGC CCTGCGGGGCCTCTCATCCTTGAGCATCCACCTGGGCATGGAGTCCTTCATTGTG GTCACCGAGTGTGAGCCGGGCTGTGCTGTGGACCTCGGCTTGGCGCGGGACCG GCCCCTGGAGGCCGATGGCCAAGAGGTCCCCCTTGACACCTCCGGGTCCCAGG CCCGGCCCCACCTCTCCGGTCGCAAGCTGTCTCTGCAAGAGCGGTCCCAGGGTG GGCTGGCAGCCGGTGGCAGCCTGGACATGAACGGACGCTGCATCTGCCCGTCC CTGCCCTACTCACCCGTCAGCTCCCCGCAGTCCTCGCCTCGGCTGCCCCGGCGG CCGACAGTGGAGTCTCACCACGTCTCCATCACGGGTATGCAGGACTGTGTGCAG CTGAATCAGTATACCCTGAAGGATGAAATTGGAAAGGGCTCCTATGGTGTCGTCA AGTTGGCCTACAATGAAAATGACAATACCTACTATGCAATGAAGGTGCTGTCCAAA AAGAAGCTGATCCGGCAGGCCGGCTTTCCACGTCGCCCTCCACCCCGAGGCACC CGGCCAGCTCCTGGAGGCTGCATCCAGCCCAGGGGCCCCATTGAGCAGGTGTAC CAGGAAATTGCCATCCTCAAGAAGCTGGACCACCCCAATGTGGTGAAGCTGGTG GAGGTCCTGGATGACCCCAATGAGGACCATCTGTACATGGTGTTCGAACTGGTCA ACCAAGGGCCCGTGATGGAAGTGCCCACCCTCAAACCACTCTCTGAAGACCAGG CCCGTTTCTACTTCCAGGATCTGATCAAAGGCATCGAGTACTTACACTACCAGAAG ATCATCCACCGTGACATCAAACCTTCCAACCTCCTGGTCGGAGAAGATGGGCACA TCAAGATCGCTGACTTTGGTGTGAGCAATGAATTCAAGGGCAGTGACGCGCTCCT CTCCAACACCGTGGGCACGCCCGCCTTCATGGCACCCGAGTCGCTCTCTGAGAC CCGCAAGATCTTCTCTGGGAAGGCCTTGGATGTTTGGGCCATGGGTGTGACACTA TACTGCTTTGTCTTTGGCCAGTGCCCATTCATGGACGAGCGGATCATGTGTTTACA CAGTAAGATCAAGAGTCAGGCCCTGGAATTTCCAGACCAGCCCGACATAGCTGAG GACTTGAAGGACCTGATCACCCGTATGCTGGACAAGAACCCCGAGTCGAGGATC GTGGTGCCGGAAATCAAGCTGCACCCCTGGGTCACGAGGCATGGGGCGGAGCC GTTGCCGTCGGAGGATGAGAACTGCACGCTGGTCGAAGTGACTGAAGAGGAGGT CGAGAACTCAGTCAAACACATTCCCAGCTTGGCAACCGTGATCCTGGTGAAGACC ATGATACGTAAACGCTCCTTTGGGAACCCATTCGAGGGCAGCCGGCGGGAGGAA CGCTCACTGTCAGCGCCTGGAAACTTGCTCACCAAAAAACCAACCAGGGAATGTG AGTCCCTGTCTGAGCTCAAGGAAGCAAGGCAGCGAAGACAACCTCCAGGGCACC GACCCGCCCCCCGTGGGGGAGGAGGAAGTGCTCTTGTGAGAGGCAGTCCCTGC P6783PC01 GTGGAAAGTTGCTGGGCCCCCGCCCCCGGCTCCCCCGCACGCATGCATCCACTG CGGCCGGAGGAGGCCATGGAGCCCGAGTAG SEQ. ID NO: 23 calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) – polypeptide sequence MSSCVSSQPSSNRAAPQDELGGRGSSSSESQKPCEALRGLSSLSIHLGMESFIVVTE CEPGCAVDLGLARDRPLEADGQEVPLDTSGSQARPHLSGRKLSLQERSQGGLAAGG SLDMNGRCICPSLPYSPVSSPQSSPRLPRRPTVESHHVSITGMQDCVQLNQYTLKDEI GKGSYGVVKLAYNENDNTYYAMKVLSKKKLIRQAGFPRRPPPRGTRPAPGGCIQPRG PIEQVYQEIAILKKLDHPNVVKLVEVLDDPNEDHLYMVFELVNQGPVMEVPTLKPLSED QARFYFQDLIKGIEYLHYQKIIHRDIKPSNLLVGEDGHIKIADFGVSNEFKGSDALLSNTV GTPAFMAPESLSETRKIFSGKALDVWAMGVTLYCFVFGQCPFMDERIMCLHSKIKSQ ALEFPDQPDIAEDLKDLITRMLDKNPESRIVVPEIKLHPWVTRHGAEPLPSEDENCTLV EVTEEEVENSVKHIPSLATVILVKTMIRKRSFGNPFEGSRREERSLSAPGNLLTKKPTR ECESLSELKEARQRRQPPGHRPAPRGGGGSALVRGSPCVESCWAPAPGSPARMHP LRPEEAMEPE SEQ. ID NO: 24 AMP activated protein kinase-alpha2 (PRKAA2) - polynucleotide sequence ATGGTAATGGAATATGTGTCTGGAGGTGAATTATTTGACTACATCTGTAAGCATGG ACGGGTTGAAGAGATGGAAGCCAGGCGGCTCTTTCAGCAGATTCTGTCTGCTGTG GATTACTGTCATAGGCATATGGTTGTTCATCGAGACCTGAAACCAGAGAATGTCCT GTTGGATGCACACATGAATGCCAAGATAGCCGATTTCGGATTATCTAATATGATGT CAGATGGTGAATTTCTGAGAACTAGTTGCGGATCTCCAAATTATGCAGCACCTGAA GTCATCTCAGGCAGATTGTATGCAGGTCCTGAAGTTGATATCTGGAGCTGTGGTG TTATCTTGTATGCTCTTCTTTGTGGCACCCTCCCATTTGATGATGAGCATGTACCTA CGTTATTTAAGAAGATCCGAGGGGGTGTCTTTTATATCCCAGAATATCTCAATCGT TCTGTCGCCACTCTCCTGATGCATATGCTGCAGGTTGACCCACTGAAACGAGCAA CTATCAAAGACATAAGAGAGCATGAATGGTTTAAACAAGATTTGCCCAGTTACTTA TTTCCTGAAGACCCTTCCTATGATGCTAACGTCATTGATGATGAGGCTGTGAAAGA AGTGTGTGAAAAATTTGAATGTACAGAATCAGAAGTAATGAACAGTTTATATAGTG GTGACCCTCAAGACCAGCTTGCAGTGGCTTATCATCTTATCATTGACAATCGGAGA ATAATGAACCAAGCCAGTGAGTTCTACCTCGCCTCTAGTCCTCCATCTGGTTCTTT TATGGATGATAGTGCCATGCATATTCCCCCAGGCCTGAAACCTCATCCAGAAAGG P6783PC01 ATGCCACCTCTTATAGCAGACAGCCCCAAAGCAAGATGTCCATTGGATGCACTGA ATACGACTAAGCCCAAATCTTTAGCTGTGAAAAAAGCCAAGTGGCATCTTGGAATC CGAAGTCAGAGCAAACCGTATGACATTATGGCTGAAGTTTACCGAGCTATGAAGC AGCTGGATTTTGAATGGAAGGTAGTGAATGCATACCATCTTCGTGTAAGAAGAAAA AATCCAGTGACTGGCAATTACGTGAAAATGAGCTTACAACTTTACCTGGTTGATAA CAGGAGCTATCTTTTGGACTTTAAAAGCATTGATGATGAAGTAGTGGAGCAGAGAT CTGGTTCCTCAACACCTCAGCGTTCCTGTTCTGCTGCTGGCTTACACAGACCAAG ATCAAGTTTTGATTCCACAACTGCAGAGAGCCATTCACTTTCTGGCTCTCTCACTG GCTCTTTGACCGGAAGCACATTGTCTTCAGTTTCACCTCGCCTGGGCAGTCACAC CATGGATTTTTTTGAAATGTGTGCCAGTCTGATTACTACTTTAGCCCGTTGA SEQ. ID NO: 25 AMP activated protein kinase-alpha2 (PRKAA2) - polypeptide sequence MVMEYVSGGELFDYICKHGRVEEMEARRLFQQILSAVDYCHRHMVVHRDLKPENVLL DAHMNAKIADFGLSNMMSDGEFLRTSCGSPNYAAPEVISGRLYAGPEVDIWSCGVILY ALLCGTLPFDDEHVPTLFKKIRGGVFYIPEYLNRSVATLLMHMLQVDPLKRATIKDIREH EWFKQDLPSYLFPEDPSYDANVIDDEAVKEVCEKFECTESEVMNSLYSGDPQDQLAV AYHLIIDNRRIMNQASEFYLASSPPSGSFMDDSAMHIPPGLKPHPERMPPLIADSPKAR CPLDALNTTKPKSLAVKKAKWHLGIRSQSKPYDIMAEVYRAMKQLDFEWKVVNAYHL RVRRKNPVTGNYVKMSLQLYLVDNRSYLLDFKSIDDEVVEQRSGSSTPQRSCSAAGL HRPRSSFDSTTAESHSLSGSLTGSLTGSTLSSVSPRLGSHTMDFFEMCASLITTLAR SEQ. ID NO: 26 Protein Polybromo-1 (PBRM1) - polynucleotide sequence ATGAGGAGACTGGCTTTTCGAGGCGCTGGTTGTGCTCTGGTAAAGCTGAAGAAGT TGGATTCCATGGGTTCCAAGAGAAGAAGAGCTACCTCCCCTTCCAGCAGTGTCAG CGGGGACTTTGATGATGGGCACCATTCTGTGTCAACACCAGGCCCAAGCAGGAA AAGGAGGAGACTTTCCAATCTTCCAACTGTAGATCCTATTGCCGTGTGCCATGAAC TCTATAATACCATCCGAGACTATAAGGATGAACAGGGCAGACTTCTCTGTGAGCTC TTCATTAGGGCACCAAAGCGAAGAAATCAACCAGACTATTATGAAGTGGTTTCTCA GCCCATTGACTTGATGAAAATCCAACAGAAACTAAAAATGGAAGAGTATGATGATG TTAATTTGCTGACTGCTGACTTCCAGCTTCTTTTTAACAATGCAAAGTCCTATTATA AGCCAGATTCTCCTGAATATAAAGCCGCTTGCAAACTCTGGGATTTGTACCTTCGA ACAAGAAATGAGTTTGTTCAGAAAGGAGAAGCAGATGACGAAGATGATGATGAAG ATGGGCAAGACAATCAGGGCACAGTGACTGAAGGATCTTCTCCAGCTTACTTGAA P6783PC01 GGAGATCCTGGAGCAGCTTCTTGAAGCCATAGTTGTAGCTACAAATCCATCAGGA CGTCTCATTAGCGAACTTTTTCAGAAACTGCCTTCTAAAGTGCAATATCCAGATTAT TATGCAATAATTAAGGAGCCTATAGATCTCAAGACCATTGCCCAGAGGATACAGAA TGGAAGCTACAAAAGTATTCATGCAATGGCCAAAGATATAGATCTCCTCGCAAAAA ATGCCAAAACTTATAATGAGCCTGGCTCTCAAGTATTCAAGGATGCAAATTCAATT AAAAAAATATTTTATATGAAAAAGGCTGAAATTGAACATCATGAAATGGCTAAGTCA AGTCTTCGAATGAGGACTCCATCCAACTTGGCTGCAGCCAGACTGACAGGTCCTT CACACAGTAAAGGCAGCCTTGGTGAAGAGAGAAATCCCACTAGCAAGTATTACCG TAATAAAAGAGCAGTACAAGGAGGTCGTTTATCAGCAATTACAATGGCACTTCAAT ATGGCTCAGAAAGTGAAGAAGATGCTGCTTTAGCTGCTGCACGCTATGAAGAGGG AGAGTCAGAAGCAGAAAGCATCACTTCCTTTATGGATGTTTCAAATCCTTTTTATCA GCTTTATGACACAGTTAGGAGTTGTCGGAATAACCAAGGGCAGCTAATAGCTGAA CCTTTTTACCATTTGCCTTCAAAGAAAAAATACCCTGATTATTACCAGCAAATTAAA ATGCCCATATCACTACAACAGATCCGAACAAAACTGAAGAATCAAGAATATGAAAC TTTAGATCATTTGGAGTGTGATCTGAATTTAATGTTTGAAAATGCCAAACGCTATAA TGTGCCCAATTCAGCCATCTACAAGCGAGTTCTAAAATTGCAGCAAGTTATGCAGG CAAAGAAGAAAGAGCTTGCCAGGAGAGACGATATCGAGGACGGAGACAGCATGA TCTCTTCAGCCACCTCTGATACTGGTAGTGCCAAAAGAAAAAGGAACACTCATGAC AGTGAGATGTTGGGTCTCAGGAGGCTATCCAGTAAAAAGAACATAAGAAAGCAGC GAATGAAAATCTTATTCAATGTTGTTCTTGAAGCTCGAGAGCCAGGTTCAGGCAGA AGACTTTGTGACCTATTTATGGTTAAACCATCCAAAAAGGACTATCCTGATTATTAT AAAATCATCTTGGAGCCAATGGACTTGAAAATAATTGAGCATAACATCCGCAATGA CAAATATGCTGGTGAAGAGGGAATGATAGAAGACATGAAGCTGATGTTCCGGAAT GCCAGGCACTATAATGAGGAGGGCTCCCAGGTTTATAATGATGCACATATCCTGG AGAAGTTACTCAAGGAGAAAAGGAAAGAGCTGGGCCCACTGCCTGATGATGATGA CATGGCTTCTCCCAAACTCAAGCTGAGTAGGAAGAGTGGCATTTCTCCTAAAAAAT CAAAATACATGACTCCAATGCAGCAGAAACTAAATGAGGTCTATGAAGCTGTAAAG AACTATACTGATAAGAGGGGTCGCCGCCTCAGTGCCATATTTCTGAGGCTTCCCT CTAGATCTGAGTTGCCTGACTACTATCTGACTATTAAAAAGCCCATGGACATGGAA AAAATTCGAAGTCACATGATGGCCAACAAGTACCAAGATATTGACTCTATGGTTGA GGACTTTGTCATGATGTTTAATAATGCCTGTACATACAATGAGCCGGAGTCTTTGA TCTACAAAGATGCTCTTGTTCTACACAAAGTCCTGCTTGAAACACGCAGAGACCTG GAGGGAGATGAGGACTCTCATGTCCCAAATGTGACTTTGCTGATTCAAGAGCTTA TCCACAATCTTTTTGTGTCAGTCATGAGTCATCAGGATGATGAGGGAAGATGCTAC AGCGATTCTTTAGCAGAAATTCCTGCTGTGGATCCCAACTTTCCTAACAAACCACC P6783PC01 CCTTACATTTGACATAATTAGGAAGAATGTTGAAAATAATCGCTACCGTCGGCTTG ATTTATTTCAAGAGCATATGTTTGAAGTATTGGAACGAGCAAGAAGGATGAATCGG ACAGATTCAGAAATATATGAAGATGCAGTAGAACTTCAGCAGTTTTTTATTAAAATT CGTGATGAACTCTGCAAAAATGGAGAGATTCTTCTTTCACCGGCACTCAGCTATAC CACAAAACATTTGCATAATGATGTGGAGAAAGAGAGAAAGGAAAAATTGCCAAAAG AAATAGAGGAAGATAAACTAAAACGAGAAGAAGAAAAAAGAGAAGCTGAAAAGAG TGAAGATTCCTCTGGTGCTGCAGGCCTCTCAGGCTTACATCGCACATACAGCCAG GACTGTAGCTTTAAAAACAGCATGTACCATGTTGGAGATTACGTCTATGTGGAACC TGCAGAGGCCAACCTACAACCACATATCGTCTGTATTGAAAGACTGTGGGAGGAT TCAGCTGGTGAAAAATGGTTGTATGGCTGTTGGTTTTACCGACCAAATGAAACATT CCACCTGGCTACACGAAAATTTCTAGAAAAAGAAGTTTTTAAGAGTGACTATTACA ACAAAGTTCCAGTTAGTAAAATTCTAGGCAAGTGTGTGGTCATGTTTGTCAAGGAA TACTTTAAGTTATGCCCAGAAAACTTCCGAGATGAGGATGTTTTTGTCTGTGAATC ACGGTATTCTGCCAAAACCAAATCTTTTAAGAAAATTAAACTGTGGACCATGCCCA TCAGCTCAGTCAGGTTTGTCCCTCGGGATGTGCCTCTGCCTGTGGTTCGCGTGGC CTCTGTATTTGCAAATGCAGATAAAGGTGATGATGAGAAGAATACAGACAACTCAG AGGACAGTCGAGCTGAAGACAATTTTAACTTGGAAAAGGAAAAAGAAGATGTCCC TGTGGAAATGTCCAATGGTGAACCAGGTTGCCACTACTTTGAGCAGCTCCATTAC AATGACATGTGGCTGAAGGTTGGCGACTGTGTCTTCATCAAGTCCCATGGCCTGG TGCGTCCTCGTGTGGGCAGAATTGAAAAAGTATGGGTTCGAGATGGAGCTGCATA TTTTTATGGCCCCATCTTCATTCACCCAGAAGAAACAGAGCATGAGCCCACAAAAA TGTTCTACAAAAAAGAAGTATTTCTGAGTAATCTGGAAGAAACCTGCCCCATGACA TGTATTCTCGGAAAGTGTGCTGTGTTGTCATTCAAGGACTTCCTCTCCTGCAGGCC AACTGAAATACCAGAAAATGACATTCTGCTTTGTGAGAGCCGCTACAATGAGAGC GACAAGCAGATGAAGAAATTCAAAGGATTGAAGAGGTTTTCACTCTCTGCTAAAGT GGTAGATGATGAAATTTACTACTTCAGAAAACCAATTGTTCCTCAGAAGGAGCCAT CACCTTTGCTGGAAAAGAAGATCCAGTTGCTAGAAGCTAAATTTGCCGAGTTAGAA GGTGGAGATGATGATATTGAAGAGATGGGAGAAGAAGATAGTGAGGTCATTGAAC CTCCTTCTCTACCTCAGCTTCAGACCCCCCTGGCCAGTGAGCTGGACCTCATGCC CTACACACCCCCACAGTCTACCCCAAAGTCTGCCAAAGGCAGTGCAAAGAAGGAA GGCTCCAAACGGAAAATCAACATGAGTGGCTACATCCTGTTCAGCAGTGAGATGA GGGCTGTGATTAAGGCCCAACACCCAGACTACTCTTTCGGGGAGCTCAGCCGCC TGGTGGGGACAGAATGGAGAAATCTTGAGACAGCCAAGAAAGCAGAATATGAAG GTGTGATGAACCAAGGAGTGGCCCCTATGGTAGGGACTCCAGCACCAGGTGGAA GTCCATATGGACAACAGGTGGGAGTTTTGGGGCCTCCAGGGCAGCAGGCACCAC P6783PC01 CTCCATATCCCGGCCCACATCCAGCTGGACCCCCTGTCATACAGCAGCCAACAAC ACCCATGTTTGTAGCTCCCCCACCAAAGACCCAGCGGCTTCTTCACTCAGAGGCC TACCTGAAATACATTGAAGGACTCAGTGCGGAGTCCAACAGCATTAGCAAGTGGG ATCAGACACTGGCAGCTCGAAGACGCGACGTCCATTTGTCGAAAGAACAGGAGA GCCGCCTACCCTCTCACTGGCTGAAAAGCAAAGGGGCCCACACCACCATGGCAG ATGCCCTCTGGCGCCTTCGAGATTTGATGCTCCGGGACACCCTCAACATTCGCCA AGCATACAACCTAGAAAATGTTTAA SEQ. ID NO: 27 Protein Polybromo-1 (PBRM1) - polypeptide sequence MRRLAFRGAGCALVKLKKLDSMGSKRRRATSPSSSVSGDFDDGHHSVSTPGPSRKR RRLSNLPTVDPIAVCHELYNTIRDYKDEQGRLLCELFIRAPKRRNQPDYYEVVSQPIDL MKIQQKLKMEEYDDVNLLTADFQLLFNNAKSYYKPDSPEYKAACKLWDLYLRTRNEF VQKGEADDEDDDEDGQDNQGTVTEGSSPAYLKEILEQLLEAIVVATNPSGRLISELFQ KLPSKVQYPDYYAIIKEPIDLKTIAQRIQNGSYKSIHAMAKDIDLLAKNAKTYNEPGSQV FKDANSIKKIFYMKKAEIEHHEMAKSSLRMRTPSNLAAARLTGPSHSKGSLGEERNPT SKYYRNKRAVQGGRLSAITMALQYGSESEEDAALAAARYEEGESEAESITSFMDVSN PFYQLYDTVRSCRNNQGQLIAEPFYHLPSKKKYPDYYQQIKMPISLQQIRTKLKNQEY ETLDHLECDLNLMFENAKRYNVPNSAIYKRVLKLQQVMQAKKKELARRDDIEDGDSMI SSATSDTGSAKRKRNTHDSEMLGLRRLSSKKNIRKQRMKILFNVVLEAREPGSGRRL CDLFMVKPSKKDYPDYYKIILEPMDLKIIEHNIRNDKYAGEEGMIEDMKLMFRNARHYN EEGSQVYNDAHILEKLLKEKRKELGPLPDDDDMASPKLKLSRKSGISPKKSKYMTPMQ QKLNEVYEAVKNYTDKRGRRLSAIFLRLPSRSELPDYYLTIKKPMDMEKIRSHMMANK YQDIDSMVEDFVMMFNNACTYNEPESLIYKDALVLHKVLLETRRDLEGDEDSHVPNVT LLIQELIHNLFVSVMSHQDDEGRCYSDSLAEIPAVDPNFPNKPPLTFDIIRKNVENNRY RRLDLFQEHMFEVLERARRMNRTDSEIYEDAVELQQFFIKIRDELCKNGEILLSPALSY TTKHLHNDVEKERKEKLPKEIEEDKLKREEEKREAEKSEDSSGAAGLSGLHRTYSQD CSFKNSMYHVGDYVYVEPAEANLQPHIVCIERLWEDSAGEKWLYGCWFYRPNETFH LATRKFLEKEVFKSDYYNKVPVSKILGKCVVMFVKEYFKLCPENFRDEDVFVCESRYS AKTKSFKKIKLWTMPISSVRFVPRDVPLPVVRVASVFANADKGDDEKNTDNSEDSRAE DNFNLEKEKEDVPVEMSNGEPGCHYFEQLHYNDMWLKVGDCVFIKSHGLVRPRVGR IEKVWVRDGAAYFYGPIFIHPEETEHEPTKMFYKKEVFLSNLEETCPMTCILGKCAVLS FKDFLSCRPTEIPENDILLCESRYNESDKQMKKFKGLKRFSLSAKVVDDEIYYFRKPIV PQKEPSPLLEKKIQLLEAKFAELEGGDDDIEEMGEEDSEVIEPPSLPQLQTPLASELDL MPYTPPQSTPKSAKGSAKKEGSKRKINMSGYILFSSEMRAVIKAQHPDYSFGELSRLV P6783PC01 GTEWRNLETAKKAEYEGVMNQGVAPMVGTPAPGGSPYGQQVGVLGPPGQQAPPP YPGPHPAGPPVIQQPTTPMFVAPPPKTQRLLHSEAYLKYIEGLSAESNSISKWDQTLA ARRRDVHLSKEQESRLPSHWLKSKGAHTTMADALWRLRDLMLRDTLNIRQAYNLEN V SEQ ID NOs: 28-33 are empty sequences. SEQ. ID NO: 34 69-bp MCS sequence AGATCTTCTAGACCCGGGAGCGGCCGCTGTCGACCTGCAGGATCCGAATTCGAT ATCACTAGTGGTACC SEQ ID NOs: 35-37 are empty sequences. SEQ. ID NO: 38 VP16 polynucleotide sequence GCCCCCCCGACCGATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGA CGTGGCGATGGCGCATGCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGG GGACGGGGATTCCCCGGGTCCGGGATTTACCCCCCACGACTCCGCCCCCTACGG CGCTCTGGATATGGCCGACTTCGAGTTTGAGCAGATGTTTACCGATGCCCTTGGA ATTGACGAGTACGGTGGG SEQ. ID NO: 39 VP16 polypeptide sequence APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGAL DMADFEFEQMFTDALGIDEYGG SEQ. ID NO: 40 KRAB polynucleotide sequence CGGACACTGGTGACCTTCAAGGATGTATTTGTGGACTTCACCAGGGAGGAGTGGA AGCTGCTGGACACTGCTCAGCAGATCGTGTACAGAAATGTGATGCTGGAGAACTA TAAGAACCTGGTTTCCTTGGGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGT TGGAGAAGGGAGAAGAGCCT SEQ. ID NO: 41 P6783PC01 KRAB polypeptide sequence RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKPDVILRLEK GEEP
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Claims

P6783PC01 Claims 1. One or more constructs, which upon expression encode: - one or more gene expression inhibitors of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1, IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. 2. One or more constructs, which upon expression encodes at least two transcriptions factors for reprogramming or inducing a cell, wherein the constructs further encode: - one or more gene expression inhibitors inhibiting the expression of one or more genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, P6783PC01 RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B RYBP, and SND1; and/or - one or more gene expression enhancers activating the expression of one or more genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L,GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1,, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1 , preferably activating the expression of GATA2. 3. The one or more constructs according to any one of the preceding claims wherein said constructs are: - overexpression constructs, such as constructs overexpressing said one or more gene expression enhancers, or CRISPR activation constructs targeting said one or more facilitator genes; - knockdown constructs, such as shRNA constructs, miRNA- encoding constructs, or CRISPR interference constructs targeting said one or more barrier genes; and/or - gene editing constructs, such as CRISPR-Cas9 constructs targeting said one or more barrier genes. 4. The one or more constructs according to any one of the preceding claims, wherein the gene expression enhancers and/or the gene expression inhibitors are selected from the group consisting of: RNAs, proteins and/or genes. P6783PC01 5. The one or more constructs according to claim 4, wherein the RNAs are shRNA, miRNA, siRNAs and/or antisense oligonucleotides (ASOs). 6. The one or more constructs according to claim 4, wherein the proteins are transcription factors, epigenetic modifiers, such as histone-modifying enzymes, and/or CRISPR systems, such as CRISPR editing, interference and/or activation systems. 7. The one or more constructs according to any one of the preceding claims, wherein: - the chromatin remodelling factor RB Binding Protein-4 (RBBP4) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 18; - the splicing factor 3B subunit 6, (SF3B6) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 20; - the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 22; - the AMP activated protein kinase-alpha2 (PRKAA2) is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 24; and/or - the Protein Polybromo-1 (PBRM1) is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 26, such as at least 95%, such as at least 96%, such as at least 97%, such as at least P6783PC01 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 26. 8. The one or more constructs according to any one of the preceding claims, wherein: - the chromatin remodelling factor RB Binding Protein-4 (RBBP4) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 19, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 19, 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 to SEQ ID NO: 19; - the splicing factor 3B subunit 6, (SF3B6) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 21, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 21, 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 to SEQ ID NO: 21; - the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 23, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 23, 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 to SEQ ID NO: 23; - the AMP activated protein kinase-alpha2 (PRKAA2) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 25, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 25, 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 to SEQ ID NO: 25; and/or - the Protein Polybromo-1 (PBRM1) comprises or consists of the polypeptide sequence set forth in SEQ ID NO 27, or a variant thereof having at least 90% sequence identity to SEQ ID NO: 27, 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 to SEQ ID NO: 27. P6783PC01 9. The one or more constructs according to any one of the preceding claims, wherein the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and wherein the at least two transcription factors, such as at least three transcription factors, are selected from the group consisting of: a) PU.1, b) IRF8, and c) BATF3 10. The one or more constructs according to any one of the preceding claims, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late). 11. The one or more constructs according to any one of the preceding claims, which upon expression encode at least two transcription factors selected from the group consisting of: PU.1, IRF8 and BATF3, wherein the one or more constructs comprise: a spleen focus-forming virus (SFFV) promoter region; and one or more sequences selected from the group consisting of: the posttranscriptional regulatory element (PRE) mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6), the rabbit beta-globin polyadenylation signal sequence (rbBGpA), and the late polyadenylation signal sequence of simian virus 40 (SV40late). 12. The one or more constructs according any one of the preceding claims, wherein the one or more constructs upon expression encode PU.1, or a biologically active variant thereof, at least 90% identical to SEQ ID NO: 10, P6783PC01 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% identical to SEQ ID NO: 10. 13. The one or more constructs according to any one of the preceding claims, wherein the one or more constructs upon expression encode IRF8, or a biologically active variant thereof, at least 90% identical to SEQ ID NO: 12, 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% identical to SEQ ID NO: 12. 14. The one or more constructs according to any one of the preceding claims, wherein the one or more constructs upon expression encode BATF3, or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 14. 15. The one or more constructs according to any one of the preceding claims, wherein the one or more constructs upon expression encode GATA2, or a biologically active variant thereof, at least 90% identical to 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% identical to SEQ ID NO: 17. 16. The one or more constructs according to any one of the preceding claims, wherein PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 9, 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 to SEQ ID NO: 9 17. The one or more constructs according to any one of the preceding claims, wherein IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11, 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 to SEQ ID NO: 11. 18. The one or more constructs according to any one of the preceding claims, wherein BATF3 is encoded by a polynucleotide sequence with at least 90% P6783PC01 sequence identity to SEQ ID NO: 13, 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 to SEQ ID NO: 13. 19. The one or more constructs according to any one of the preceding claims, wherein GATA2 is encoded by a polynucleotide sequence with at least 90% sequence identity to 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 to SEQ ID NO: 16. 20. The one or more constructs according to any one of the preceding claims, which upon expression encode PU.1, IRF8, BATF3 and GATA2. 21. The one or more constructs according to any one of the preceding claims, wherein the mutated Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element sequence (WPREmut6) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 6, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 6, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 6. 22. The one or more constructs according to any one of the preceding claims, wherein the rabbit beta-globin polyadenylation signal sequence (rbBGpA) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 7, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 7, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 7. 23. The one or more constructs according to any one of the preceding claims, wherein the late polyadenylation signal sequence of simian virus 40 (SV40late) comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 8, or a biologically active variant thereof having at least 90% identity to SEQ ID NO: 8, such as at least 95% identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 8. P6783PC01 24. The one or more constructs according to any one of the preceding claims, wherein IRF8 is fused to a VP16 activation domain. 25. The one or more constructs according to any one of the preceding claims, wherein the VP16 activation domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 38, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 38. 26. The one or more constructs according to any one of the preceding claims, wherein the VP16 activation domain is encoded by the sequence set forth in SEQ ID NO: 39, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 39, 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% identical to SEQ ID NO: 39. 27. The one or more constructs according to any one of the preceding claims, wherein BATF3 is fused to a KRAB repressor domain. 28. The one or more constructs according to any one of the preceding claims, wherein the KRAB repressor domain is encoded by a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in SEQ ID NO: 40, or a biologically active variant thereof having at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 92%, such as at least 95%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 40. 29. The one or more constructs according to any one of the preceding claims, wherein the KRAB repressor domain is encoded by the sequence set forth in SEQ ID NO: 41, or a biologically active variant thereof, wherein the biologically active variant is at least 90% identical to SEQ ID NO: 41, such as at least 95%, P6783PC01 such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to SEQ ID NO: 41. 30. One or more vectors comprising the one or more constructs according to any one of the preceding claims. 31. The one or more vectors according to claim 30, where the one or more vectors are encoded by a polynucleotide sequence comprising SEQ ID NO: 1, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 1. 32. The one or more vectors according to any one of claims 30 to 31, where the one or more vectors are encoded by a polynucleotide sequence comprising SEQ ID NO: 2, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 2. 33. The one or more vectors according to any one of claims 30 to 32, where the one or more vectors are encoded by a polynucleotide sequence comprising SEQ ID NO: 3, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 3. 34. The one or more vectors according to any one of claims 30 to 33, where the one or more vectors are encoded by a polynucleotide sequence comprising SEQ ID NO: 4, or a variant thereof having at least 90% sequence identity, 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 to SEQ ID NO: 4. 35. A cell comprising the one or more constructs of any one of claims 1 to 29 or the one or more vectors of any one of claims 30 to 34. P6783PC01 36. A method of reprogramming or inducing a cell into a reprogrammed or induced cell, the method comprising the following steps: a) transducing a cell with one or more constructs or vectors, which upon expression encodes at least two transcription factors reprogramming or inducing said cell; b) inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, EZH2, RIPK3, MLKL, WEE1, CHEK1, CAMKV, STK32B, PPP4C, TAOK2, PPP4R2, GSK3A, MAP4K3, PKMYT1, PPP2R1B, RYBP, and SND1; and/or c) activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1), ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, RAD54L, GATA2, TWIST2, NFIL3, NR1H2, NFKBIB, ATF3, CBFB, ID2, CREG1, IRF7, BCL6, ZNF276, BATF, GFI1B, MYCL, NR4A3, IKZF1,IRF4, NR6A1, PDCD4, IRF2, STAT3, SNAI2, TFEC, MNDA, POU2F2, TCF4, IRF5, ARID4A, BAZ1A, PELI1, GATA3, GADD45B, MXD1, JUNC, JUNB, NFKBIA, RUNX1, TNNI2, PLEK, REL, ETV6, MIS18BP1, ZNF366, FOXN2, HSPB8, GPRK4, TYRO3, INSRR, PPP1R8, PPP1R14A, MAP4K5, MAPKAPK2, STK3, GUCY2G, NUAK2, MELK, PRKD2, BUB1, PRKCZ, PRKAA2, BUB1B, AURKA, PPP1R2, MOK, CILK1, ATRX, PPM1G, WNK3, CAMKK2, LATS2, IKBKB, MAP3K21, TNK1, and EM1, preferably activating the expression of GATA2. 37. The method according to claim 36, wherein: - step b) comprises inhibiting the expression of one or more barrier genes selected from the group consisting of: chromatin remodelling factor RB Binding Protein-4 (RBBP4), splicing factor 3B subunit 6, (SF3B6), calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and AMP activated protein kinase-alpha2 (PRKAA2), and/or P6783PC01 - step c) comprises activating the expression of one or more facilitator genes selected from the group consisting of: Protein Polybromo-1 (PBRM1). 38. The method according to any one of any one of the preceding claims, wherein: - step b) comprises inhibiting the expression of one or more barrier genes selected from the group consisting of: WAC, UBE2I, EID2B, DNMT1, SUV39H2, KAT2A, MOV10, GATAD2B, RABGAP1L, and EZH2; and/or - step c) comprises activating the expression of one or more genes selected from the group consisting of: ZBTB38, CDC6, CDK1, RPS6KA3, RPL11, UHRF2, HDAC10, USP17L2, BRD4, and RAD54L. 39. The method according to any one of any one of the preceding claims, wherein step a-c) together comprise transducing the cell with the one or more constructs of any one of claims 1 to 29, or vectors comprising thereof of any one of claims 30 to 34. 40. The method according to any one of any one of the preceding claims, wherein step b) of inhibiting comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression inhibitors inhibiting the expression of one or more barrier genes according to any one of claims 1 to 29, or vectors comprising thereof of any one of claims 30 to 34. 41. The method according to any one of the preceding claims, wherein step c) of activating comprises transducing the cell with the one or more constructs, which upon expression encode one or more gene expression enhancers activating the expression of one or more facilitator genes according to any one of claims 1 to 29, or vectors comprising thereof of any one of claims 30 to 34. 42. The method according to any one of any one of the preceding claims, wherein the reprogrammed or induced cell is an induced dendritic cell (DC), preferably an induced Conventional type 1 dendritic cell (cDC1), and wherein the one or more constructs or vectors of step a), encode upon expression at least two P6783PC01 transcription factors, such as at least three transcription factors, selected from the group consisting of: a) PU.1, b) IRF8, and c) BATF3 43. The method according to any one of any one of the preceding claims, wherein the step of inhibiting and/or activating the expression of one or more genes of steps b) and c) respectively is performed in parallel, before and/or after the step a) of transducing the cell. 44. The method according to any one of any one of the preceding claims, wherein the cell is a mammalian cell. 45. The method according to any one of any one of the preceding claims, wherein the cell is a human cell. 46. The method according to claim 45, wherein the cell is a murine cell. 47. The method according to any one of any one of the preceding claims, wherein the cell is selected from the group consisting of: a stem cell, a differentiated cell and a cancer cell. 48. The method according to claim 47, wherein the stem cell is selected from the group consisting of: a pluripotent stem cell and a multipotent stem cell, such as a hematopoietic stem cell. 49. The method according to claim 47, wherein the differentiated cell is any somatic cell. 50. The method according to claim 49, wherein the somatic cell is selected from the group consisting of: a fibroblast and a hematopoietic cell, such as a monocyte. P6783PC01 51. A reprogrammed or induced cell obtained by the method defined in any one of any one of the preceding claims. 52. The reprogrammed or induced cell according to claim 51, wherein the cell is an induced dendritic or antigen-presenting cell, such as a type 1 conventional dendritic cell. 53. The reprogrammed or induced cell according to any one of claims 51 to 52, wherein the cell is positive for CD45 and/or negative for HLA-DR, preferably wherein the cell is positive for CD45 and HLA-DR. 54. The method according to any one of any one of the preceding claims, wherein the method further comprises culturing the transduced cell in a media comprising one or more epigenetic modifiers, such as histone deacetylase (HDAC) inhibitors, or methylation inhibitors, preferably wherein the step of culturing the transduced cell in a media comprising one or more epigenetic modifiers is performed during and/or after the transducing step. 55. The method according to claim 54, wherein the one or more HDAC inhibitors is selected from the group consisting of: valproic acid, Vorinostat, Romidepsin, Belinostat, and Panobinostat. 56. The method according to any one of claims 54 to 55, wherein the one or more methylation inhibitor is Azacitidine. 57. The method according to any one of claims 37 to 56, wherein the method further comprises culturing the transduced cell in a media comprising valproic acid and Azacitidine. 58. The method according to any one of claims 37 to 57, wherein the step of culturing the transduced cell is performed from day 0 to day 3, and wherein day 0 is the day of the transduction step. 59. The method according to any one of claims 37 to 58, further comprising culturing the transduced cell in a media comprising one or more PRC2 P6783PC01 inhibitors, such as EZH2-specific inhibitors, for example Tazemetostat and/or GSK-126, or such as EED inhibitors, for example EED226. 60. The method according to any one of claims 37 to 59, wherein the step of culturing the transduced cell in a media comprising the one or more PRC2 inhibitors is performed from day 0 to day 2, wherein day 0 is the day of the cell transduction step. 61. The method according to any one of claims 37 to 60, wherein the method further comprises culturing the transduced cell in a media comprising one or more AMPK activators, such as CHIR99021, AICAR, A769662, or Metformin. 62. The method according to any one of claims 37 to 61, wherein the method further comprises culturing the transduced cell in a media comprising one or more mTOR activators, such as MHY1485 or Salidroside. 63. The method according to any one of claims 37 to 62, wherein the method further comprises culturing the transduced cell in a media comprising one or more glycolysis inhibitors, such as 2-deoxy-D-glucose (2-DG). 64. The method according to any one of claims 37 to 63, wherein the step of culturing the transduced cell is performed from day 0 to day 9, and wherein day 0 is the day of the transduction step.
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