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WO2025120097A1 - Stratégies à base d'arn pour la reprogrammation de cellules dendritiques et leurs utilisations - Google Patents

Stratégies à base d'arn pour la reprogrammation de cellules dendritiques et leurs utilisations Download PDF

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WO2025120097A1
WO2025120097A1 PCT/EP2024/084945 EP2024084945W WO2025120097A1 WO 2025120097 A1 WO2025120097 A1 WO 2025120097A1 EP 2024084945 W EP2024084945 W EP 2024084945W WO 2025120097 A1 WO2025120097 A1 WO 2025120097A1
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mirnas
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Fábio Alexandre Fiúza ROSA
Nejc ARH
Beatriz Lourenço VAZ
Filipe PEREIRA
Lihan XIE
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Asgard Therapeutics AB
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Definitions

  • the present invention relates to compositions and methods for reprogramming cells to type 1 conventional dendritic cells or antigen-presenting cells.
  • TF Transcription factor
  • iPSCs induced pluripotent stem cells
  • a somatic cell can also be directly converted into another specialized cell type (Pereira, Lemischka, and Moore 2012; Wang et al. 2021).
  • Direct lineage conversion has proven successful to reprogram mouse and human fibroblasts into several cell types, such as neurons, cardiomyocytes and hepatocytes, using TFs specifying the target-cell identity.
  • Direct cell conversions were also demonstrated in the hematopoietic system, where forced expression of TFs induced a macrophage fate in B cells and fibroblasts (Xie et al. 2004) and the direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors was achieved with Gata2, Gfilb, cFos and Etv6 (Pereira et al. 2013).
  • cell reprogramming experiments have relied on the use of lentiviral vectors to deliver transcription factors to target cells.
  • RNA has emerged as a promising non-viral vector for delivery of transcription factors as it allows the highest cell reprogramming efficiency when compared with other non-viral non-integrative delivery systems (Bailly, Milhavet, and Lemaitre 2022).
  • modRNA linear modified mRNA
  • srRNAs or saRNAs self-replicative/self-amplifying RNAs
  • srRNAs or saRNAs self-replicating single-stranded RNA viruses
  • ECV Venezuelan equine encephalitis virus
  • taRNA transamplifying RNA
  • taRNA is composed of a split-vector derivative of saRNA combining a non-replicating mRNA encoding an alphaviral replicase and a transreplicon RNA encoding for the therapeutic gene.
  • Beside modRNA, saRNA and taRNA, circular RNA (circRNA) has also recently emerged as a gene delivery platform (Chen et al. 2023).
  • CircRNA is a single-stranded RNA in which the 5’ and 3’ ends join to form a covalently closed loop. The circular structure confers multiple functional benefits, including improved stability, longer expression duration and easier upscaling.
  • RNA-based platforms have been shown to allow delivery of therapeutics genes, only modRNA and saRNA have been used for induction of reprogramming to pluripotency (Warren et al. 2010; Mandal and Rossi 2013; Yoshioka et al. 2013; Yoshioka and Dowdy 2017), and only modRNA has been shown to allow direct cell fate reprograming (Preskey et al. 2016; Murthy et al. 2015; Van Pham et al. 2017; Connor et al. 2018).
  • DCs antigen-presenting dendritic cells
  • the DC compartment can be divided in two functionally different DC subsets: conventional DCs (eDCs), which are professional antigen-presenting cells (APCs), and plasmacytoid DCs (pDCs) (Wculek et al. 2019). While pDCs are professional producers of type I interferons during viral infection, eDCs drive antigen- specific immune responses by presenting exogenous antigens to T cells.
  • the eDC subset can be further divided in myeloid/conventional DC type 1 (cDC1 or DC1s) and myeloid/conventional DC type 2 (cDC2).
  • cDC1 While cDC1 are specialized in antigen crosspresentation and priming of antigen-specific cytotoxic CD8+ T cells, cDC2s excel on their ability to present antigens to CD4+ T cells, priming antigen-specific T helper 2 (Th2), Th17 and regulatory T cell responses. This expands the flexibility of the immune system to react appropriately to a wide range of different pathogens and danger signals.
  • Human cDC1s characterized by surface expression of CD141 , CLEC9A, XCR1 and CD226 (Wculek et al. 2019; Heidkamp et al. 2016), are defined functionally by secreting immune-modulatory cytokines, including IL-12, and interferons (IFN), and chemokines such as CXCL10, and by cross-presenting antigens to CD8+ T cells (Lauterbach et al. 2010; Poulin et al. 2010). In the context of anti-tumor immunity, Batf3-/- animals lacking cDC1s fail to reject immunogenic tumours (Hildner et al. 2008).
  • IFN interferons
  • BM progenitors have been used to derive CD141 + DC1s in vitro in the presence of FLT3L with SCF, GM-CSF and IL-4 (Poulin et al. 2010). More recently, FLT3L was combined with co-culture with Notch-expressing stromal cell lines to favour cDC1 differentiation (Kirkling et al. 2018; Balan et al. 2018). The generation of cDC1-, cDC2- and pDC-like cells from induced pluripotent stem cell (iPSC) cultures was also demonstrated (Sontag et al. 2017). However, these protocols are complex, require feeder layers and result in low yields as well as a mixture of different DC subsets with conflicting functions.
  • iPSC induced pluripotent stem cell
  • miRNAs are also interesting reprogramming factors due to their described role in cell differentiation and reprogramming (Pascale et al. 2022). miRNAs are short RNAs approximately 22 nucleotides long, which repress target mRNA transcripts with seed sequence complementarity through either mRNA cleavage or translational repression (O’Brien et al. 2018).
  • miRNAs can easily be delivered by viral vectors, as well as by non-viral vectors (e.g., RNA) alongside transcription factors and small molecules (Kogut et al. 2018; Mueller et al. 2020). miRNAs can allow or facilitate reprogramming by recapitulating their known roles in development (DeVeale, Swindlehurst-Chan, and Blelloch 2021 ; Ivey and Srivastava 2015), by targeting specific reprogramming barriers (Pascale et al. 2022; Abernathy et al. 2017; Lee et al. 2018) between others.
  • the biogenic pathway of miRNAs can generate non-archetypal variants called isomiRs from the same pre-miRNA strand, including 5’ isomiRs with 5’-offsets resulting in an altered seed sequence and target set (Morin et al. 2008; Wagner et al. 2024). isomiRs are increasingly recognized for their role in cancer, diabetes, and immune disorders (Wagner et al. 2024). For example, the -1 isomiR of miR-142-3p (a hematopoietic regulator (Sun et al. 2015) is often expressed at higher levels than its canonical strand (Karlsen et al. 2019; Manzano et al.
  • RNA-based strategies are necessary to further optimize and increase the translatability, including the scalability, of direct cell fate reprogramming approaches towards immune cell fates, in particular cDC1 reprogramming to generate homogeneous populations of differentiated human cDC1s in vitro and in vivo.
  • compositions and methods for reprogramming cells into dendritic or antigen-presenting cells are provided herein.
  • co-expression of PU.1 , IRF8 and BATF3 mediated by RNA allows direct reprogramming of cells and that efficiency can be significantly improved by co-transfection with miRNA mimics and by incubation with inhibitors of IFN signalling.
  • the inventors have discovered that direct reprogramming of cells by expression of transcription factors PU.1, IRF8 and BATF3 can be significantly improved by co-expression of miRNAs or miRNA mimics.
  • the present invention also provides compositions of isomiRs for use in cell reprogramming, and methods thereof.
  • a first aspect of the invention relates to a composition
  • a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of: PU.1 , IRF8, and BATF3, wherein the RNA construct is selected from the group consisting of: self-amplifying RNA (saRNA), circular RNA (circRNA), non-modified RNA, modified RNA (modRNA) and transamplifying RNA (taRNA).
  • saRNA self-amplifying RNA
  • circRNA circular RNA
  • modRNA modified RNA
  • taRNA transamplifying RNA
  • a second aspect of the invention relates to a composition comprising one or more miRNA mimics, wherein said one or more miRNA mimics are isomiRs, for use in cell reprogramming.
  • a third aspect of the invention relates to a method for reprogramming or inducing a cell, said method comprising the steps of: a. Contacting a cell with a composition comprising one or more constructs or vectors encoding a combination of at least two transcription factors; b. Expressing the at least two transcription factors; c. Contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics; whereby the cell is reprogrammed or induced.
  • a fourth aspect of the invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, the method comprising the steps of: a. Contacting a cell with a composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3; b. Expressing the at least two transcription factors; c.
  • composition comprising one or more miRNAs, or primary miRNAs (pri-miRNAs) thereof, or miRNA mimics, or a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, or miRNA mimics; whereby a dendritic cell or antigen-presenting cell is obtained.
  • a composition comprising one or more miRNAs, or primary miRNAs (pri-miRNAs) thereof, or miRNA mimics, or a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, or miRNA mimics; whereby a dendritic cell or antigen-presenting cell is obtained.
  • a fifth aspect of the invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of: a. Contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3; b. Expressing the at least two transcription
  • a sixth aspect of the invention relates to a method of improving cell reprogramming efficiency and/or fidelity of a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of the method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell as described herein, and wherein said method further comprises the steps of: a. assessing the expression of CD45, XCR1 , CD141 , CD226, HLA- DR, CD40, HLA-ABC, and/or MHC-II in the cell, after the step of expressing the at least two transcription factors; b.
  • a higher level of expression of CD45, XCR1 , CD141, CD226, HLA-DR, HLA- ABC, CD40, and/or MHC-II preferably a higher level of expression of CD45 and MHC- II, even more preferably a higher level of expression of CD45 and HLA-DR after contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, or miRNA mimics, indicates increased cell reprogramming efficiency; and, wherein a higher level of expression of CD45, XCR1 , CD141, CD226, HLA-DR, HLA- ABC, CD40, and/or MHC-II, preferably a higher level of expression of XCR1, even more preferably a higher level of expression of CD226 after contacting the cells with
  • An eighth aspect of the present invention relates to a reprogrammed or induced cell obtained by the method as described herein, preferably wherein the cell expresses CLEC9A, CD45, CD141 , XCR1, CD226, HLA-DR, HLA-ABC, CD40, and/or MHC-II.
  • a ninth aspect of the present invention relates to the composition, the cell, or the reprogrammed or induced cell as described herein, for use in medicine.
  • a tenth aspect of the present invention relates to composition, the cell, or the reprogrammed or induced cell as described herein, for use in the treatment of cancer or infectious diseases.
  • miRNA screening identifies miR-124, miR-126, miR-142 and miR-150 as enhancers of cDC1 reprogramming.
  • A 15 miRNA candidates were chosen based on their role in hematopoiesis or cell reprogramming and cloned into lentiviral vectors. miRNAs were co-transduced with PU.1 , IRF8 and BATF3 (PIB) reprogramming factors, and induced dendritic cells (iDCs) screened for Clec9a-tdT+ reporter activation, and expression of CD45, MHC-II and XCR1.
  • B Quantification of Clec9a-tdT reporter activation.
  • C Flow cytometry plots showing expression of XCR1 in Clec9a-tdT + cells.
  • A Reprogramming efficiency measured by the percentage of cells expressing the markers CD45 and HLA-DR in reprogrammed human dermal fibroblasts (HDFs) at day 9 of reprogramming, with and without human miR-124, miR-142 or the combination.
  • C Flow cytometry plots of induced cDC1 markers CD45, HLA-DR and CD226, with or without human miR-124 and miR-142. *P ⁇ 0.05, **P ⁇ 0.01 , ***P ⁇ 0.001 one-way ANOVA with Dunnett’s multiple comparison.
  • A miR-124, miR-142 or a scrambled (SCR) pri-miRNA were co-transduced with PIB TFs into HDFs.
  • Reprogrammed populations were sorted for total RNA-seq and ATAC- seq at day 3 (CD45+), day 6 (CD45+HLA-DR+ and CD45+H LA-DR-) and day 9 (CD45+HLA-DR+ and CD45+ HLA-DR-).
  • CD45+HLA-DR+ and CD45+H LA-DR- day 9
  • Partially reprogrammed CD45+ HLA-DR- (+) and fully reprogrammed CD45+ HLA-DR+ (++) populations were sorted at days 6 and 9.
  • HDFs and DC1s HLA-DR+ CD11c+ CD141+
  • DC2s HLA-DR+ CD11c+ CD1c+ CD141-
  • pDCs HLA-DR+ CD11c- CD123+
  • B Activation of a cDC1 gene expression signature measured by the percentage of genes upregulated in cDC1s when compared to HDFs.
  • D Genes downregulated by miR-124 and miR-142 and their cell type enrichment on days 3, 6 and 9.
  • miR-124 induces a permissive chromatin landscape.
  • miR-124, miR-142 or a scrambled (SCR) pri-miRNA were co-transduced with PIB TFs into HDFs.
  • Reprogrammed populations were sorted for total RNA-seq and ATAC-seq at day 3 (CD45+), day 6 (CD45+HLA-DR+ and CD45+HLA-DR-) and day 9 (CD45+HLA-DR+ and CD45+ HLA-DR-).
  • CD45+HLA-DR+ and CD45+ HLA-DR- Partially reprogrammed CD45+ HLA-DR- (+) and fully reprogrammed CD45+ HLA-DR+ (++) populations were sorted at days 6 and 9.
  • HDFs and DC1s HLA-DR+ CD11c+ CD141+
  • DC2s HLA-DR+ CD11c+ CD1c+ CD141-
  • pDCs HLA-DR+ CD11c- CD123+
  • A Percent of accessible cDC1 genes compared to HDF gene accessibility. Activation of a cDC1 chromatin accessibility signature measured by the percentage of genes with increased accessibility in cDC1 when compared to HDFs.
  • B Number of differentially accessible peaks compared to scrambled (SCR) control at days 3, 6 and 9.
  • A miRNA mimics were transfected in HDFs on days 0, 3 and 6, after transduction with PIB TFs.
  • B Proportion of human dermal fibroblasts (HDFs) expressing antigen- presenting-cell markers CD45 and HLA-DR upon transfection with canonical miR-124 and miR-142 mimics.
  • C Exemplificative flow cytometry plots of CD45 and HLA-DR expression.
  • D miRNA mimics were transfected in HDFs on days -2, 0, 3 or 6, before or after transduction with PIB TFs.
  • E Fold change of reprogramming efficiency to CD45+ HLA-DR+ iDCs upon miR-124-3p transfection compared to scrambled control.
  • H qPCR quantification of the cDC1 marker XCR1 expression in human dermal fibroblasts (HDFs) reprogrammed to CD45+ HLA-DR+ iDCs upon transfection with miR-142 canonical or isomiR mimics.
  • HDFs human dermal fibroblasts
  • I Proportion of reprogrammed human embryonic fibroblasts (HEFs) expressing antigen-presenting-cell markers CD45 and HLA-DR upon transfection with canonical miR-124 and miR-142 mimics and 5’ isomiRs of miR-142. Exemplificative flow cytometry plots of CD45 and HLA-DR expression are shown on the top panel.
  • SCR scrambled control.
  • Figure 7 Pathway analysis of miR-124 and miR-142 overexpression in iDC reprogramming.
  • A Schematic representation of the approach to determine the mechanisms of miR- 124 and miR-142 in DC reprogramming.
  • GRaNIE and GRaNPA packages were used to predict transcription factors (TFs) explaining gene expression changes based on transcriptome and genome accessibility data. Then, we combined target prediction for previously validated miRNA strands, and centrality within the differentially expressed (DE) protein network to infer first-order miRNA targets responsible for the effect on iDC reprogramming.
  • B GRaNPA predictions for effector regulators behind miR-124 (left) or miR-142 overexpression. Strands of the respective miRNA targeting GRaNPA transcripts are listed to the right of each plot. KLF5-miR-124 interaction was not computationally predicted.
  • (A) PU.1 , IRF8, BATF3 and GFP ml- ⁇ P-modified mRNAs were transfected in HDFs on days 0, 2, 4 and 6.
  • HDFs Human dermal fibroblasts
  • JAK inhibitors including Ruxolitinib (rux), Baricitinib (Bari, JAK1/JAK2 inhibitor), Fedratinib (Fedra, JAK2 inhibitor), Upadacitinib (Upa, JAK1 inhibitor) and Filgotinib (Filgo, JAK 1 inhibitor), at 3 different concentrations (5, 10 or 15 uM) from day 0 to day 9.
  • Ruxolitinib rux
  • Baricitinib Bari, JAK1/JAK2 inhibitor
  • Fedratinib Fredra, JAK2 inhibitor
  • Upadacitinib Upa, JAK1 inhibitor
  • Filgotinib Filgotinib
  • SK-LMS-1 cells are transfected with an EGFP-encoding RNA on day 0 and monitored by flow cytometry from day 1 to day 8 to determine the transfection efficiency, dose-dependence, and kinetics of EGFP expression.
  • EGFP-positive cells are gated in live cells, and their frequencies are indicated.
  • MFI mean fluorescence intensity
  • G Workflow for characterizing payload expression from three different RNA platforms: linear, circular, and self-amplifying RNA, after 1 transfection at day 0 (-) or 3 transfections at day 0, 3 and 6 (+).
  • H Flow cytometry quantification of frequency and
  • I GFP MFI of eGFP+ cells.
  • SK-MS-1 human cancer cells were transfected with linear, circular and selfamplifying RNA encoding PU.1, IRF8 and BATF3, and cDC1 reprogramming efficiency was profiled at day 3.
  • B Flow cytometry quantification of cDC1 reprogramming efficiency measured as frequency of CD45+ HLA-DR+ cells gate in live cells (linear and circular RNA) or eGFP+ cells (saRNA). Doses of RNA used for transfection are shown.
  • (A) SK-LMS-1 human cancer cells were treated with one (day 0), two (day 0 and 3) or three (day 0, 3 and 6) with linear, circular and self-amplifying RNA encoding PU.1, IRF8 and BATF3, and reprogramming efficiency was profiled at day 3, 6 and 9.
  • F, G Flow cytometry quantification of (F) frequency of CD40+ cells and (G) mean fluorescence intensity (MFI) of HLA-ABC in cancer cells after transfection with the 3 RNA platforms.
  • H Flow cytometry quantification of cDC1 reprogramming efficiency measured as frequency of eGFP+ or eGFP- cells expressing CD45 and/or H LA-DR.
  • I Flow cytometry quantification of number of CD45+ and/or HLA-DR+ reprogrammed cells 3, 6 and 9 days after transfection with the 3 RNA platforms.
  • RNA construct refers to an arrangement or sequence of one or more ribonucleic acid molecule(s).
  • the term RNA construct includes for example RNA sequences designed or engineered for applications such as gene expression and/or therapeutic purposes, and can for example form part of cell delivery systems, such as, but not limited to vectors or plasmids, or non-viral cell delivery systems, such as, but not limited to, lipid-based delivery systems, polymer-based delivery systems or electroporation-mediated delivery systems.
  • RNA or “mi-RNA”, “miR”, “mi-R”, microRNA and micro-RNA are used interchangeably herein.
  • pri-miRNA or “primary miRNA” as used herein refers single strand RNA which transcribed from DNA and has a hairpin loop structure containing miRNA and its complementary strand.
  • pre-miRNA is produced from pri-miRNA partially by cleavage by an intranuclear enzyme called Drosha.
  • miRNA, or pri-miRNA thereof as used herein therefore relates to both mature the microRNA and its precursor form, encompassing the mature miRNA as well as the primary transcript.
  • isomiRs refer to heterogeneous variants of individual miRNAs that can differ in length or sequence from the archetype miRNA, also known as canonical miRNA. isomiRs include 5' isomiRs (displaying sequence changes at 5' end of the archetype form), 3' isomiRs (displaying changes at the 3' end), polymorphic isomiRs (displaying changes within the sequence), and mixed type isomiRs (wherein at least two of the above listed changes occur), as defined in Wagner et al. 2024. “miRNA mimic” as used herein refer to a class of molecules that can be used to imitate the gene silencing ability of one or more miRNAs.
  • miRNA mimic refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics can be designed as mature molecules (e.g. single stranded) or mimic precursors (e.g., pri- or pre-miRNAs). “miRNA mimic” as used herein cover both mimics of canonical miRNA sequences, and isomiRs mimics.
  • encapsulation refers to the process of enclosing or packaging RNA molecules within a protective carrier or delivery system. Encapsulation may be performed to enhance stability, delivery efficiency, and targeted delivery of RNA molecules and RNA-based therapeutics to the cells. Encapsulation methods include, but not limited to, liposomes, lipid nanoparticles (LNPs), polymers and polymer nanoparticles, cationic polymers, and exosomes.
  • LNPs lipid nanoparticles
  • polymers and polymer nanoparticles cationic polymers, and exosomes.
  • contacting a cell with a composition is defined as bringing said cell and said composition in physical contact with one another, for example under conditions appropriate for internalization.
  • the term includes for example introduction of compositions of nucleic acids intracellularly, such as, but not limited to, transfection (non-viral delivery), transduction (viral vector delivery), electroporation, lipofection, sonoporation, and microinjection.
  • the term also includes allowing effectors, such as drugs or small molecules, to perform their effects on said cells.
  • MSCs Mesenchymal stem cells
  • chondrocytes chondrocytes
  • adipocytes adipocytes
  • PSC pluripotent stem cell
  • multipotent refers to cells that can give rise to several other cell types, but those cell types are limited in number.
  • An example of multipotent cells is hematopoietic cells - blood stem cells that can develop into several types of blood cells but cannot develop into brain cells for example.
  • somatic cell refers to any cell other than a germ cell, such as an egg or a sperm, which does not directly transfer its DNA to the next generation. Typically, somatic cells have limited or no pluripotency.
  • Reprogramming refers herein to the process of converting of differentiating cells from one cell type into another. “Reprogramming” thus refers to reprogramming cells to a different type or lineage. In particular, in some embodiments of the present invention, reprogramming refers to converting or transdifferentiating any type of cell into a type 1 conventional dendritic cell or an antigen-presenting cell.
  • replicon refers to an RNA molecule capable of autonomous replication within a host cell.
  • ml- ⁇ P-modified mRNA refers to a RNA modified to contain the synthetic pyrimidine nucleoside N1 -Methylpseudouridine (abbreviated ml- ⁇ P).
  • ml- ⁇ P synthetic pyrimidine nucleoside N1 -Methylpseudouridine
  • miR-142 and “miR-142a” can be used interchangeably.
  • EGFP EGFP
  • eGFP green fluorescent protein
  • the present invention provides RNA-based cell reprogramming strategies, in particular RNA-based reprogramming of any cell type into a dendritic cell or and antigen presenting cell.
  • the present invention relates to a composition
  • a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of: Pll.1 , IRF8, and BATF3, wherein the RNA construct is selected from the group consisting of: self-amplifying RNA (saRNA), circular RNA (circRNA), non-modified RNA, modified RNA (modRNA) and transamplifying RNA (taRNA).
  • saRNA self-amplifying RNA
  • circRNA circular RNA
  • modRNA modified RNA
  • taRNA transamplifying RNA
  • RNA types suitable for encoding and expressing the transcription factors, or combinations thereof in cells may be used in the compositions or methods and uses as described herein.
  • the combination of at least two transcription factors encoded by the composition comprising one or more RNA construct(s) is: a. PU.1 and IRF8; b. PU.1 and BATF3; or c. IRF8 and BATF3.
  • the combination of at least two transcription factors encoded by the composition comprising one or more RNA construct(s) is PU.1 and IRF8, or PU.1 and BATF3.
  • the combination of at least two transcription factors is PU.1 , IRF8, and BATF3.
  • the combination of at least two transcription factors in the 5’ to 3’ order is: a. PU.1 , IRF8, BATF3; b. PU.1 , BATF3, IRF8; c. IRF8, BATF3, PU.1 ; d. IRF8, PU.1 , BATF3; e. BATF3, IRF8, PU.1 ; or f. BATF3, PU.1 , IRF8.
  • the composition comprises 2, such as 3 RNA constructs encoding the combination of at least two transcription factors
  • each of the at least two transcription factors is encoded by a single RNA construct, optionally further encoding other genes such as reporter genes, such as fluorescent reporter genes, for example GFP, BFP or RFP.
  • the composition comprises 3 RNA constructs, wherein the combination of at least two transcription factors is Pll.1 , IRF8, and BATF3, and wherein each of the RNA constructs encodes one of Pll.1 , IRF8, and BATF3.
  • the RNA construct(s) are identical or different.
  • the RNA constructs may be all modified RNAs.
  • PU.1 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:13 or SEQ ID NO:14.
  • IRF8 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO: 11 or 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% sequence identity to SEQ ID NO: SEQ ID NO:11 or SEQ ID NO:12.
  • BATF3 is encoded by a polynucleotide sequence with at least 90% sequence identity to SEQ ID NO:15 or 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:15 or SEQ ID NO:16.
  • PU.1 is of a polypeptide sequence with at least 90% sequence identity to SEQ ID NO:19 or SEQ ID NQ: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:19 or SEQ ID NQ:20.
  • IRF8 is of a polypeptide sequence with at least 90% sequence identity to SEQ ID NO:17 or 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: 17 or SEQ ID NO: 18.
  • BATF3 is of a polypeptide sequence with at least 90% sequence identity to SEQ ID NO:21 or 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:21 or SEQ ID NO:22.
  • Direct reprogramming of cells by expression of the combinations of transcription factors as described herein can be significantly improved by expression of miRNAs or miRNA mimics.
  • the present invention provides compositions of isomiRs for use in cell reprogramming.
  • RNA-based co-expression of the transcription factors as described herein allows direct reprogramming of cells and reprogramming efficiency can be significantly improved by co-transfection with miRNA mimics.
  • One aspect of the present invention relates to a composition comprising one or more miRNA mimics, wherein said one or more miRNA mimics are isomiRs, for use in cell reprogramming.
  • the one or more miRNA mimics are isomiRs of miRNA 124a-1, miRNA-124-1, miRNA-126a, miRNA-126, miRNA-142, and/or miRNA- 150.
  • Another aspect of the invention relates to a composition
  • a composition comprising one or more miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics, selected from the group consisting of: miR-124, miR-124a-1 , miR-124-1, miR-124-3p, miR-124- 5p, miR-142a, miR-142, miR-126, miR-126a, miR-150, miR-142-3p-1 , miR-142-3p-3, and miR-142-5p-1.
  • said miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics a. downregulate the gene expression of SP1, KLF5, and/or RELA, preferably wherein said miRNAs, or primary miRNAs (pri- miRNAs) thereof, and/or miRNA mimics are of the miR-124 family, even more preferably wherein said miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics are miR- 124-3p or isomiRs thereof; and/or b. downregulate the gene expression of KLF10, PATZ1 , and/or ELK3 preferably wherein said miRNAs, or primary miRNAs (pri- miRNAs) thereof, and/or miRNA mimics are of the miR-142 family.
  • said miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics a. downregulate the gene expression of SP1, KLF5, and/or RELA, preferably wherein said miRNAs, or primary miRNAs (pri- miRNAs) thereof, and/or miRNA mimics are of the miR-124 family, even more preferably wherein said miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics are miR- 124-3p or isomiRs thereof; and/or b. downregulate the gene expression of KLF10, PATZ1 , and/or ELK3 preferably wherein said miRNAs, or primary miRNAs (pri- miRNAs) thereof, and/or miRNA mimics are of the miR-142 family.
  • miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics are of the miR-124 family, preferably miR-124-3p or miR-124-5p
  • said miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics downregulate the protein expression of STAT3, MAPK14, and/or RELA, preferably wherein STAT3, MAPK14, and RELA represent the nodes with the highest connectivity to differentially expressed proteins in the miR-124 network, as identified through protein interaction analysis using STRING as described in Example 8 (Szklarczyk et al., 2023).
  • miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics are of the miR-142 family
  • said miRNAs, or primary miRNAs (pri- miRNAs) thereof, and/or miRNA mimics downregulate the protein expression of CCND1, TGFBR1 , and/or RAB2, preferably wherein CCND1 , TGFBR1 , and/or RAB2 represent the nodes with the highest connectivity to differentially expressed proteins in the miR-142 network, as identified through protein interaction analysis using STRING as described in Example 8 (Szklarczyk et al., 2023).
  • miR-142-3p-1 downregulates the protein expression of CCND1 , preferably wherein CCND1 represents the node with the highest connectivity to differentially expressed proteins in the miR-142 network, as identified through protein interaction analysis using STRING as described in Example 8 (Szklarczyk et al., 2023).
  • miR-142-3p-1 and miR-142-3p downregulate the protein expression of TGFBR1 , and/or RAB2, preferably wherein TGFBR1 , and/or RAB2 represent the node with the highest connectivity to differentially expressed proteins in the miR-142 network, as identified through protein interaction analysis using STRING as described in Example 8 (Szklarczyk et al., 2023).
  • miR-142-5p-1 downregulates the protein expression of TGFBR1 , preferably wherein TGFBR1 represents the node with the highest connectivity to differentially expressed proteins in the miR-142 network, as identified through protein interaction analysis using STRING as described in Example 8 (Szklarczyk et al., 2023).
  • the composition comprises more than one miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics. This may be the case for example in order to improve reprogramming efficiency and/or fidelity in the methods described herein, such as improving cDC1 cell reprogramming.
  • the composition comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8 miRNAs, or primary miRNAs (pri-miRNAs) thereof, and/or miRNA mimics, selected from the group consisting of: miR-124, miR-124a-1 , miR-124-1 , miR-124-3p, miR-124-5p, miR-142a, miR-142, miR-126, miR-126a, miR-150, miR-142-3p-12, miR- 142-3p-3, and miR-142-5p-1.
  • miRNA mimics selected from the group consisting of: miR-124, miR-124a-1 , miR-124-1 , miR-124-3p, miR-124-5p, miR-142a, miR-142, miR-126, miR-126a, miR-150, miR-142-3p-12, miR- 142-3p-3, and miR-142-5
  • the one or more miRNAs are the two miRNAs miR-124 and miR-142.
  • the one or more miRNAs are the two miRNAs miR-124 and miR-142a.
  • the composition comprises at least one or more miRNA mimic(s).
  • miRNA mimics include canonical miRNA mimics and isomiRs mimics.
  • the composition comprises at least one, such as at least two, such as at least three, such as at least four, such as at least five miRNA mimic(s).
  • said at least one, such as at least two, such as at least three, such as at least four, such as at least five miRNA mimic(s) are isomiRs.
  • the isomiRs are 5' isomiRs, 3' isomiRs, polymorphic isomiRs, and/or mixed type isomiRs. In preferred embodiments, the isomiRs are 5' isomiRs.
  • the miRNA mimic(s) are the isomiRs miR-142-3p-1 , miR- 142-3p-3 and/or miR-142-5p-1.
  • the composition comprises or consists of miR-142-3p, miR-142- 3p-1 and miR-142-5p-1.
  • the composition comprises or consists of miR-124-3p and miR- 142-5p-1.
  • the at least one or more miRNAs is a primary miRNA (pri- miRNA).
  • At least one of the miRNA(s), or primary miRNA thereof, and/or miRNA mimics is encoded by a sequence selected from the group consisting of: a. miRNA 124a-1 of SEQ ID NO: 1; b. miRNA-124-1 of SEQ ID NO: 2; c. miRNA-126a of SEQ ID NO: 3; d. miRNA-126 of SEQ ID NO: 4 e. miRNA-142 of SEQ ID NO: 5 or SEQ ID NO: 6 f. miRNA-150 of SEQ ID NO: 7 or SEQ ID NO: 8 g. miRNA-124-3p mimic of SEQ ID NO: 9; h. miRNA-124-5p mimic of SEQ ID NO: 10; i.
  • At least one of the one or more RNA construct(s), or at least one of the one or more miRNA(s), or pri-miRNAs thereof, and/or miRNA mimics, is encapsulated.
  • the encapsulation is selected from the group consisting of: nanoparticle encapsulation, polymeric encapsulation, viral encapsulation, exosome encapsulation, and lipid nanoparticle encapsulation.
  • the one or more RNA construct(s) are naked RNA or RNA not encapsulated or incorporated into a viral particle, for example a lentiviral particle, such as naked non-modified or modified RNA, or non-modified or modified RNA not encapsulated or incorporated into a viral particle, for example a lentiviral particle.
  • compositions of RNA construct(s) encoding the reprogramming transcription factors as described herein and the compositions of miRNAs, or pri-miRNAs thereof, and/or miRNA mimics as described herein are provided as a single composition
  • one aspect of the present invention relates to a composition
  • a composition comprising: a. the composition comprising one or more RNA construct(s) as described herein; and b. the composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein.
  • compositions of RNA construct(s) encoding the reprogramming transcription factors as described herein and/or the compositions of miRNAs, or pri-miRNAs thereof, and/or miRNA mimics as described herein further comprise compounds inhibiting Polycomb Repressive Complex 2 (PRC2), such as SUZ12 or EZH2 inhibitors, preferably GSK-126.
  • PRC2 Polycomb Repressive Complex 2
  • the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics as described herein may be beneficially encoded by one or more construct(s) or vector(s), for example, but not limited to, construct(s) or vector(s) facilitating their entry and/or expression in cells.
  • compositions comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics as described herein.
  • At least one of the one or more miRNA(s) is encoded as a pri-miRNA.
  • the one or more vector(s) may be a viral, or a non-viral vector.
  • the viral vector(s) may be selected from the group consisting of: adenovirus, lentivirus, retrovirus, alphaviruses, flavivirus, rhabdovirus, Sendai virus, pox virus, adeno- associated virus (AAV), and herpes simplex virus (HSV).
  • the non-viral vector(s) may be selected from the group consisting of: liposomes, polymeric nanoparticles, dendrimers, cationic polymers, nanogels, inorganic nanoparticles, carbon nanotubes, hydrogels, electroporation, and ultrasound-mediated vectors
  • the at least two transcription factors such as the at least two transcription factors selected from the group consisting of: Pll.1, IRF8, and BATF3, are encoded as polycistronic constructs, optionally separated by 2A peptides.
  • the at least two transcription factors such as the at least two transcription factors selected from the group consisting of: Pll.1, IRF8, and BATF3, are encoded as monocistronic constructs.
  • compositions of the present invention further comprise one or more inhibitor(s) of interferons (IFN inhibitors), preferably wherein the IFN inhibitor(s) are selected from the group consisting of: kinase inhibitors, JAK/STAT inhibitors and virus proteins neutralizing type I interferons, even more preferably wherein the IFN inhibitor(s) are selected from the group consisting of B18R and ruxolitinib, yet even more preferably wherein the IFN inhibitor is ruxolitinib.
  • IFN inhibitors interferons
  • IFN inhibitors include, but not limited to, Tofacitinib, Baricitinib, Ritlecitinib, Abrocitinib, Upadacitinib and Delgocitinib, and viral proteins know as innate inhibiting proteins (I IPs), including but not restricted to HSV- 2 Us1, HSV-1 Us1, HSV-1Us11, Orf OV20.0L, BVDV Npro, PIV-5 V, MERS-CoV M, MERS-CoV ORF4a, Langat NS5, Influenza NS1 , vaccinia virus immune evasion protein E3, vaccinia virus immune evasion protein K3 and vaccinia virus immune evasion protein B18.
  • I IPs innate inhibiting proteins
  • the composition is a pharmaceutical composition.
  • the present invention provides methods for reprogramming cells, said methods making use of miRNA mimics, in particular isomiRs.
  • the invention relates to a method for reprogramming or inducing a cell, said method comprising the steps of: a. Contacting a cell with a composition comprising one or more constructs or vectors encoding a combination of at least two transcription factors; b. Expressing the at least two transcription factors; c. Contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics; whereby the cell is reprogrammed or induced.
  • said reprogramming is direct cell reprogramming, or direct cell fate reprogramming.
  • the one or more miRNA mimic(s) are isomiRs.
  • the one or more isomiRs are 5’ isomiRs.
  • the isomiRs of the composition or methods of the present invention may preferably be isomiRs of the miRNAs of the compositions and methods of the present invention.
  • the one or more miRNA mimics are isomiRs of miRNA 124a-1, miRNA-124-1, miRNA-126a, miRNA-126, miRNA-142, and/or miRNA-150.
  • the one or more isomiRs are isomiRs of miR-142.
  • the one or more isomiRs are the isomiRs miR-142-3p-1, miR-142-3p-3 and/or miR-142-5p-1.
  • One aspect of the present invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of: a. Contacting a cell with a composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3; b. Expressing the at least two transcription factors; c.
  • composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics; whereby a dendritic cell or antigen-presenting cell is obtained.
  • said reprogramming is direct cell reprogramming, or direct cell fate reprogramming.
  • the one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of Pll.1 , IRF8 and BATF3 comprise both RNA constructs as described herein, wherein the RNA constructs encode one or more of Pll.1, IRF8 and BATF3, and one or more viral vectors such as lentiviruses, adenoviruses or adeno associated viruses encoding one or more of PU .1 , I RF8 and BATF3.
  • Another aspect of the present invention relates to a method for reprogramming or inducing a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of: a. Contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1, IRF8, and BATF3; b. Expressing the at least two transcription factors; whereby a dendritic cell or antigen-presenting cell is obtained.
  • the method comprises the steps of: a. Contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1, IRF8, and BATF3; b. Expressing the at least two transcription factors; c. Contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics; whereby a dendritic cell or antigen-presenting cell is obtained.
  • said reprogramming is direct cell reprogramming, or direct cell fate reprogramming.
  • RNA types exhibit different payload expression levels and kinetics, as in Example 11.
  • the present invention shows that repeating step a. of contacting and b. of expressing of the methods herein, also called “redosing”, has the advantage of rescuing the fading of the transgene expression levels over time and prolonging transgene expression of linear (ml- ⁇ P-modified mRNA), circular and self-amplifying RNA-encoded transgenes.
  • the steps a. of contacting a cell with a composition comprising one or more RNA construct(s) and step b. of expressing are repeated.
  • the step a. of contacting a cell with a composition comprising one or more RNA construct(s) and step b. of expressing are repeated twice, such as repeated three times, such as repeated 4 times, such as repeated 5 times, such as repeated 6 times, such as 7 times, for example at least 8 times.
  • the step a. of contacting a cell with a composition comprising one or more RNA construct(s) and step b. of expressing are performed on day 0, day 3 and day 6, wherein day 0 is defined as the first-time step a. of contacting a cell with a composition comprising one or more RNA construct(s) is performed.
  • the step a. of contacting a cell with a composition comprising one or more RNA construct(s) and step b. of expressing are performed on day 0, day 3 and day 7, wherein day 0 is defined as the first-time step a. of contacting a cell with a composition comprising one or more RNA construct(s) is performed.
  • the methods comprise the step a. of contacting the cells once with one or more saRNA construct(s) .
  • the methods comprise performing the step a. of contacting the cells and step b. of expressing twice at days 0 and 3 with one or more circular RNA construct(s) wherein day 0 is defined as the first-time step a. of contacting a cell with a composition comprising one or more circular RNA construct(s) is performed.
  • the methods comprise performing the step a. of contacting the cells and step b. of expressing three times at days 0, 3 and 7 with one or more linear RNA construct(s), wherein day 0 is defined as the first-time step a. of contacting a cell with a composition comprising one or more linear RNA construct(s) is performed.
  • the step c. of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is not performed prior to the step a.
  • RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3 and/or the step b. of expressing the at least two transcription factors.
  • the step c. of contacting the cells with a composition comprising miRNA mimics comprises miR-142 mimics, such as miR-142-3p, miR-142-3p-1 , miR- 142-3p-3 and/or miR-142-5p-1 , and is performed on day 0, day 3 and day 6 following the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3.
  • miR-142 mimics such as miR-142-3p, miR-142-3p-1 , miR- 142-3p-3 and/or miR-142-5p-1 , and is performed on day 0, day 3 and day 6 following the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3.
  • step c. comprises contacting the cells with a composition comprising one or more construct s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, and the method further comprises a step of expressing the one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics.
  • composition comprising one or more construct(s) or vector(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1, IRF8, and BATF3 is as described herein in the section Compositions.
  • the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is as described herein in the section Compositions.
  • the step of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed prior to, simultaneously, and/or after, the step of contacting a cell with a composition comprising one or more constructs or vectors, or with a composition comprising one or more RNA construct(s).
  • the step of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed simultaneously to the step of contacting a cell with a composition comprising one or more constructs or vectors, or with a composition comprising one or more RNA construct(s).
  • the step of expressing the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed prior to, simultaneously, and/or after, the step of expressing the at least two transcription factors. In even more preferred embodiments, the step of expressing the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, is performed simultaneously to the step of expressing the at least two transcription factors.
  • the step c. of the methods comprises contacting the cells with miRNA-124 and/or miRNA-142 simultaneously to the step of expressing the at least two transcription factors.
  • the step c. of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is repeated.
  • the step c. of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is repeated after the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1 , IRF8, and BATF3 and/or the step b. of expressing the at least two transcription factors, for example repeated twice, such as repeated three times, such as repeated 4 times, such as repeated 5 times, such as repeated 6 times, such as 7 times, for example at least 8 times.
  • the step c. of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed on day 0, day 3 and day 6 following the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of Pll.1 , IRF8, and BATF3 and/or the step b. of expressing the at least two transcription factors.
  • the step c. of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is not performed prior to the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1 , IRF8, and BATF3 and/or the step b. of expressing the at least two transcription factors.
  • the step c. of contacting the cells with a composition comprising miRNA mimics comprises miR-142 mimics, such as miR-142-3p, miR-142-3p-1 , miR- 142-3p-3 and/or miR-142-5p-1 , and is performed on day 0, day 3 and day 6 following the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1 , IRF8, and BATF3.
  • miR-142 mimics such as miR-142-3p, miR-142-3p-1 , miR- 142-3p-3 and/or miR-142-5p-1 , and is performed on day 0, day 3 and day 6 following the step a. of contacting a cell with a composition comprising one or more RNA construct(s) encoding the combination of at least two transcription factors selected from the group consisting of PU.1 , IRF8, and BATF3.
  • RNA construct(s) of the present invention comprise of consist of self-amplifying RNA (saRNA).
  • saRNA typically include, but not limited to, replication elements of positive-strand RNA viruses, to be able to replicate and increase the production of the antigens they encode, such as for example the transcription factors as described herein.
  • saRNAs can comprise, polyadenylation signals at the 3’ end to enhance their stability and promote translation, as well as modified cap structures on their 5’ end.
  • the one or more RNA construct(s) comprise or consist of self-amplifying RNA.
  • the self-amplifying RNA is selected from the group consisting of: Venezuelan equine encephalitis (VEE) virus, Semliki forest virus, Sindbis virus, classical swine fever virus, tick-borne encephalitis virus, alphavirus chimera based on the Venezuelan equine encephalitis virus, and Sindbis virus replicons.
  • VEE Venezuelan equine encephalitis
  • RNA constructs of the present invention comprise or consist of circular RNA, forming a closed-loop structure created by a back-splicing event.
  • circular RNAs have been engineered for example for optimal protein expression, including N6-methyladenosine (m6A) incorporation, vector topology, number of stop codons, 5' and 3' untranslated regions (UTRs), IRES sequences and synthetic aptamers.
  • m6A N6-methyladenosine
  • UTRs 5' and 3' untranslated regions
  • IRES sequences synthetic aptamers.
  • circular RNA are typically more stable than linear RNAs due to, for example, their resistance to exonucleases.
  • the one or more RNA construct(s) comprise or consist of circular RNA (circRNA).
  • the one or more RNA construct(s) comprise or consist of linear RNA.
  • the linear RNA molecule further comprises a 3' poly(A) tail, a Kozak sequence, a open reading frame, a 3' untranslated region, a 5' untranslated region, a 5' cap or any combination thereof.
  • the modified RNA can comprise at least one modified nucleoside selected from the group consisting of, but not restricted to, 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O- dimethyluridine (m4U), 2-thiouridine (s2U), 2' fluorouridine, pseudouridine, 2'-O- methyluridine (Um), 2'deoxy uridine (2' dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2'-O-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O- trimethyladenosine (m62Am), 2'-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2'- O-methylguanosine (mCm), 7-methylguanosine (m7G), 2'- O-methylguanosine (m7G
  • the one or more RNA construct(s) comprise or consist of non-modified RNA. In other embodiments, the one or more RNA construct(s) comprise or consist of modified RNA. In further embodiments, the one or more RNA construct(s) comprise or consist of linear modified RNA, such as linear ml- ⁇ P-modified mRNA.
  • Trans-amplifying RNAs are split-vector derivatives of self-amplifying RNAs (saRNAs), as they combine a non-replicating mRNA encoding an alphaviral replicase and a transreplicon RNA coding for the transgene.
  • saRNAs self-amplifying RNAs
  • the one or more RNA construct(s) comprise or consist of trans-amplifying RNA (taRNA).
  • taRNA trans-amplifying RNA
  • the taRNA can be engineered for faster replication, greater potency and improved immunogenicity.
  • RNA molecules may be modified to integrate, but not limited to, synthetic nucleosides, such as N1 -Methylpseudouridine (ml- ⁇ P) in order to enhance translation of the proteins encoded by said RNAs.
  • synthetic nucleosides such as N1 -Methylpseudouridine (ml- ⁇ P)
  • the one or more RNA construct(s) comprise or consist of modified RNA.
  • the modified RNA is a ml- ⁇ P-modified mRNA.
  • the modified RNA is linear modified RNA, such as linear ml- ⁇ P-modified mRNA.
  • the methods further comprise a step of inhibiting the IFN pathway of the cell.
  • the step of inhibiting the IFN pathway of the cell is performed prior to, during, and/or after the step of contacting a cell with a composition comprising one or more constructs or vectors, or with a composition comprising one or more RNA construct(s).
  • the step of inhibiting the IFN pathway of the cell is performed prior to, during, and/or after the step of expressing the at least two transcription factors.
  • the step of inhibiting the IFN pathway is performed using small molecules, viral proteins including, but not limited to, innate inhibiting proteins, delivery of shRNAs, siRNAs, miRNAs and miRNA mimetics targeting downstream mediators and/or effectors of IFN signaling.
  • viral proteins including, but not limited to, innate inhibiting proteins, delivery of shRNAs, siRNAs, miRNAs and miRNA mimetics targeting downstream mediators and/or effectors of IFN signaling.
  • the step of inhibiting the IFN pathway is performed by contacting the cells with one or more IFN inhibitors.
  • the one or more IFN inhibitors are selected from the group consisting of: kinase inhibitors, JAK/STAT inhibitors and virus proteins neutralizing type I interferons.
  • the one or more IFN inhibitors are selected from the group consisting of: B18R and ruxolitinib.
  • the one or more IFN inhibitor is ruxolitinib. This may be the case in particular for methods described herein making use of linear RNA, circular RNA or saRNA, for example methods of cDC1 reprogramming using linear RNA, circular RNA or saRNA, preferably wherein reprogramming efficiency is measured as induction of CD45 and MHC-II surface marker expression.
  • B18R is a Vaccinia virus protein that neutralizes type I interferons (IFNa, I FNb, IFNe,k,t,d,z,w,v) by inhibiting activation of interferon-mediated signal transduction.
  • Ruxolitinib belongs to the Janus kinase (JAK) inhibitors family, with selectivity for JAK1 and JAK2 subtypes.
  • IFN inhibitors include Tofacitinib, Baricitinib, Ritlecitinib, Abrocitinib, Upadacitinib and Delgocitinib, and viral proteins know as innate inhibiting proteins (I IPs), including but not restricted to HSV-2 Us1 , HSV-1 Us1, HSV-1Us11, Orf OV20.0L, BVDV Npro, PIV-5 V, MERS-CoV M, MERS- CoV ORF4a, Langat NS5, Influenza NS1 , vaccinia virus immune evasion protein E3, vaccinia virus immune evasion protein K3 and vaccinia virus immune evasion protein B18.
  • I IPs innate inhibiting proteins
  • the IFN inhibitors are Upadacitinib, Filgotinib, or Baricitinib.
  • the inventors have showed herein that said inhibitors enhance cell reprogramming efficiency in some applications. This may be the case for example in methods of reprogramming cells to cDC1 cells, as measured by CD45 and/or HLA-DR positivity of reprogrammed cells, such as reprogrammed cDC1 cells from human dermal fibroblasts (HDFs).
  • HDFs human dermal fibroblasts
  • the one or more IFN inhibitor is present at a concentration between 0 and 100pM, preferably between 10nM and 50pM, even more preferably between 100nM and 25pM, yet even more preferably between 100nM and 10pM.
  • the cell is a human or non-human cell. In preferred embodiments, the cell is a human cell.
  • the cell is a human fibroblast, such as a human dermal fibroblast (HDF), or a human embryonic fibroblast (HEF).
  • a human fibroblast such as a human dermal fibroblast (HDF), or a human embryonic fibroblast (HEF).
  • the cell is a cancer cell, such as a cancer cell line, for example a primary cancer cell line.
  • the cell is a melanoma cancer cell, such as the A2058 cancer cell line, or a sarcoma cancer cell, for example Leiomyosarcoma, such as the SK-LMS-1 cancer cell line.
  • the cell is a mouse cell. In further embodiments, the cell is a mouse embryonic fibroblast (MEF). In some embodiments of the methods of the present invention, the reprogrammed or induced cell is an induced dendritic cell (iDC). In preferred embodiments, the reprogrammed or induced cell is a human or non-human iDC, such as a mouse iDC.
  • iDC induced dendritic cell
  • the reprogrammed or induced cell is a conventional type 1 dendritic cell (cDC1 cell).
  • the reprogrammed or induced cell expresses CD45, XCR1 and/or MHC-II. This is for example the case when the cell is a mouse cell, for example a mouse dendritic cell, such as a mouse cDC1 cell.
  • the reprogrammed or induced cell expresses CD45, XCR1 and MHC-II.
  • the reprogrammed or induced cell expresses CD45, CD226 and/or HLA-DR. This is for example the case when the cell is a human cell, for example a human dendritic cell, such as a human cDC1 cell.
  • the reprogrammed or induced cell expresses CD45, CD226 and HLA-DR. In other embodiments, the reprogrammed or induced cell expresses CD226. In yet other embodiments, the reprogrammed cell further expresses CD141.
  • the reprogramming or induction is in vivo, in vitro, or ex vivo.
  • the method further comprises a step of culturing the cell in a cell media, wherein the culturing step is conducted before, during, and/or after expressing the at least two transcription factors.
  • the cell is cultured during at least 2 days, such as at least 4 days, such as at least 5 days, such as at least 6 days, such as at least 8 days, such as at least 9 days, such as at least 10 days, such as at least 12 days.
  • the cell culture media comprises one or more IFN inhibitor(s) selected from the group consisting of: kinase inhibitors, JAK/STAT inhibitors, and virus proteins neutralizing type I interferons.
  • the one or more IFN inhibitor(s) are selected form the group consisting of B18R and ruxolitinib. In even more preferred embodiments, the one or more IFN inhibitor is ruxolitinib.
  • the method further comprises culturing the cell in a cell media comprising one or more cytokines selected form the group consisting of: IFN(3, IFNy, TNFa, IFNa, IL-1 p, IL-6, CD40I, Flt3l, GM- CSF, IFN-A1 , IFN-co, IL-2, IL-4, IL-15, prostaglandin 2, SCF and oncostatin M (OM).
  • cytokines selected form the group consisting of: IFN(3, IFNy, TNFa, IFNa, IL-1 p, IL-6, CD40I, Flt3l, GM- CSF, IFN-A1 , IFN-co, IL-2, IL-4, IL-15, prostaglandin 2, SCF and oncostatin M (OM).
  • the method further comprises culturing the cell in a cell media comprising one or more epigenetic modifiers, such as histone deacetylase inhibitors.
  • the histone deacetylase inhibitor is valproic acid.
  • the step of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed prior to, simultaneously, and/or after the step of expressing the at least two transcription factors.
  • the step of contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics is performed no more than 6h, such as no more than 12h, such as no more than 24 day, such as no more than 36h, such as no more than 48h, such as no more than 72h, such as no more than 96h, such as no more than 120h , such as no more than 144h after the step of expressing the at least two transcription factors.
  • the methods further comprise the steps of: a. assessing the expression of CD45, XCR1 , CD141 , CD226, HLA- DR, HLA-ABC, CD40, and/or MHC-II in the cell, after the step of expressing the at least two transcription factors; b.
  • a higher level of expression of CD45, XCR1 , CD141, CD226, HLA-DR, HLA- ABC, CD40, and/or MHC-II preferably a higher level of expression of XCR1, even more preferably a higher level of expression of CD226 after contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, indicates increased cell reprogramming fidelity.
  • the methods further comprising the steps of: a. assessing the expression of CD45, XCR1 , CD141 , CD226, HLA- DR, HLA-ABC, CD40, and/or MHC-II in the cell, after the step of the step of expressing the at least two transcription factors; b.
  • a higher level of expression of CD45, XCR1 , CD141, CD226, HLA-DR, HAL- ABC, CD40, and/or MHC-II preferably a higher level of expression of CD45 and MHC- II, even more preferably a higher level of expression of CD45 and HLA-DR after contacting the cells with a composition comprising one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, or with a composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, indicates increased cell reprogramming efficiency.
  • the dendritic cell or antigen-presenting cell displays increased cell reprogramming efficiency and/or fidelity.
  • the increased cell reprogramming efficiency and/or fidelity may be assessed as described herein.
  • the reprogramming efficiency and/or fidelity increases with increasing concentration of the miRNA(s), or pri-miRNAs thereof, and/or miRNA mimics, and/or with increasing concentration of the one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics.
  • the assessment of the levels of expression of CD45, XCR1, CD141 , CD226, HLA-DR, HLA-ABC, CD40, and/or MHC-II in the cell is performed using flow cytometry and/or single-cell RNA sequencing (scRNAseq).
  • the step c. of comparing the levels of expression of CD45, XCR1 , CD141, CD226, HLA-DR, HLA-ABC, CD40. and/or MHC-II is performed by comparing the percentage of cells expressing CD45, XCR1, CD 141 , CD226, HLA-DR, HLA-ABC, CD40, and/or MHC-II of step a. of the method, with the percentage of cells expressing CD45, XCR1 , CD141 , CD226, HLA- DR, HLA-ABC, CD40, and/or MHC-II of step b of the method.
  • Another aspect of the present invention relates to a method of improving cell reprogramming fidelity of a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of the methods as described herein.
  • a further aspect of the present invention relates to a method of improving cell reprogramming efficiency of a cell into a dendritic cell or antigen-presenting cell, said method comprising the steps of the methods as described herein.
  • the cell reprogramming efficiency is improved by at least 25%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 500%.
  • the cell reprogramming efficiency is improved by at least 0.25- fold, such as at least 0.5-fold, such as at least 1-fold, such as at least 1.5-fold, such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold, such as at least 1.9- fold, such as at least 2-fold, such as at least 2.1 -fold, such as at least 2.2-fold, such as at least 2.3-fold, such as at least 2.4-fold, such as at least 2.5-fold, such as at least 2.6- fold, such as at least 2.7-fold, such as at least 2.8-fold, such as at least 2.9-fold, such as at least 3-fold, such as at least 5-fold, such as at least 10-fold.
  • at least 0.5-fold such as at least 1-fold
  • at least 1.5-fold such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold
  • at least 1.9- fold such as at least 2-fold
  • the cell reprogramming efficiency is improved by at least 1.5-fold, such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold, such as at least 1.9-fold, such as at least 2-fold, such as at least 2.1 -fold, such as at least 2.2-fold, such as at least 2.3-fold, such as at least 2.4-fold, such as at least 2.5- fold, such as at least 2.6-fold, such as at least 2.7-fold, such as at least 2.8-fold, such as at least 2.9-fold, such as at least 3-fold, such as at least 5-fold, such as at least 10- fold.
  • at least 1.5-fold such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold, such as at least 1.9-fold, such as at least 2-fold, such as at least 2.1 -fold, such as at least 2.2-fold, such as at least 2.3-fold, such as at least 2.4-fold, such as at least 2.5-
  • the cell reprogramming fidelity is improved by at least 25%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 200%, such as at least 300%, such as at least 500%.
  • the cell reprogramming fidelity is improved by at least 0.25-fold, such as at least 0.5-fold, such as at least 1-fold, such as at least 1.5-fold, such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold, such as at least 1.9- fold, such as at least 2-fold, such as at least 2.1 -fold, such as at least 2.2-fold, such as at least 2.3-fold, such as at least 2.4-fold, such as at least 2.5-fold, such as at least 2.6- fold, such as at least 2.7-fold, such as at least 2.8-fold, such as at least 2.9-fold, such as at least 3-fold, such as at least 5-fold, such as at least 10-fold.
  • the cell reprogramming fidelity is improved by at least 1.5-fold, such as at least 1.6-fold, such as at least 1.7-fold, such as at least 1.8-fold, such as at least 1.9-fold, such as at least 2-fold, such as at least 2.1 -fold, such as at least 2.2-fold, such as at least 2.3-fold, such as at least 2.4-fold, such as at least 2.5-fold, such as at least 2.6-fold, such as at least 2.7-fold, such as at least 2.8-fold, such as at least 2.9- fold, such as at least 3-fold, such as at least 5-fold, such as at least 10-fold.
  • Another aspect of the present invention relates to a cell comprising the one or more RNA construct(s), or the one or more construct(s) or vector(s) as described herein.
  • the cell is a mammalian cell, such as a human or murine cell.
  • the cell is selected from the group consisting of: a stem cell, a differentiated cell, and a cancer cell, wherein: a. 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 or a hematopoietic stem cell; b. the differentiated cell is any somatic cell, such as a fibroblast, for example a dermal fibroblast or an embryonic fibroblast, or a hematopoietic cell, such as a monocyte.
  • a fibroblast for example a dermal fibroblast or an embryonic fibroblast
  • a hematopoietic cell such as a monocyte.
  • the cell is a cancer cell, such as a cancer cell line, for example a primary cancer cell line. In other embodiments, the cell is a melanoma cancer cell, or a sarcoma cancer cell.
  • the cell is a reprogrammed human dendritic cell or human antigen-presenting cell, such as a human type 1 conventional dendritic cell.
  • the cell expresses one or more surface marker(s) selected from the group consisting of: CLEC9A, CD45, CD141 , XCR1, MHC-II, HLA- DR, HLA-ABC. CD40, and CD226.
  • a further aspect of the present invention relates to a reprogrammed or induced cell obtained by the method as described herein.
  • the reprogrammed or induced cell is an induced dendritic cell (iDC), preferably wherein the reprogrammed or induced cell is a human or non-human iDC, such as a mouse iDC.
  • iDC induced dendritic cell
  • the reprogrammed or induced cell is a human or non-human iDC, such as a mouse iDC.
  • the reprogrammed or induced cell is a conventional type 1 dendritic cell (cDC1 cell).
  • the reprogrammed or induced cell expresses CLEC9A, CD45, CD141 , XCR1 , CD226, HLA-DR, HLA-ABC, CD40, and/or MHC-II.
  • the reprogrammed or induced cell expresses CD45, XCR1 and/or MHC-II.
  • the reprogrammed or induced cell expresses HLA-ABC, and CD40.
  • the reprogrammed or induce cell expresses CLEC9A, CD141, HLA-DR and/or CD226.
  • kits-of-parts comprising: a.
  • the composition comprising one or more RNA construct(s) as described herein.
  • the composition comprising one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein.
  • a further aspect of the present invention relates to a kit-of-parts comprising: a.
  • the composition comprising one or more RNA construct(s) as described herein.
  • One or more IFN inhibitors selected form the group consisting of: kinase inhibitors, JAK/STAT inhibitors such as JAK1 and/or JAK2 inhibitors, and virus proteins neutralizing type I interferons, preferably wherein the one or more INF inhibitors are selected form the group consisting of: B18R, Ruxolitinib, Fedratinib, Upadacitinib, Filgotinib, and Baricitinib, more preferably selected from the group consisting of: B18R and ruxolitinib, even more preferably wherein the one or more IFN inhibitor is ruxolitinib.
  • kits-of-parts comprising: a.
  • the composition comprising one or more RNA construct(s) as described herein.
  • the composition comprising one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics, as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics as described herein.
  • One or more IFN inhibitors selected form the group consisting of: kinase inhibitors, JAK/STAT inhibitors, such as JAK1 and/or JAK2 inhibitors, and virus proteins neutralizing type I interferons, preferably wherein the one or more IFN inhibitors are selected form the group consisting of: B18R, Ruxolitinib, Fedratinib, Upadacitinib, Filgotinib, and Baricitinib, more preferably selected from the group consisting of:B18R and ruxolitinib, even more preferably wherein the one or more IFN inhibitor is ruxolitinib.
  • kits-of-parts comprising: a. A composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors. b. The composition comprising one or more constructs or vectors encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein.
  • a further aspect of the present invention relates to a kit-of-parts comprising: a.
  • a composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of PU.1, IRF8, and BATF3 b.
  • the composition comprising one or more constructs or vectors encoding the one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics, as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein.
  • kits-of-parts comprising: a. A composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors b.
  • the composition comprising one or more constructs or vectors encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri- miRNAs thereof, and/or miRNA mimics, as described herein c.
  • One or more IFN inhibitors selected form the group consisting of: kinase inhibitors, JAK/STAT inhibitors, and virus proteins neutralizing type I interferons, preferably wherein the one or more IFN inhibitors are selected form the group consisting of B18R and ruxolitinib, even more preferably wherein the one or more IFN inhibitor is ruxolitinib.
  • a further aspect of the present invention relates to a kit-of-parts comprising: a.
  • a composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3 b.
  • the composition comprising one or more constructs or vectors encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein, or the composition comprising one or more construct(s) or vector(s) encoding the one or more miRNAs, or pri-miRNAs thereof, and/or miRNA mimics, as described herein.
  • c A composition comprising one or more constructs or vectors encoding the combination of at least two transcription factors selected from the group consisting of Pll.1, IRF8, and BATF3 b.
  • the composition comprising one or more constructs or vectors encoding the one or more miRNAs, or pri-miRNAs thereof, and/
  • One or more IFN inhibitors selected form the group consisting of: kinase inhibitors, JAK/STAT inhibitors, and virus proteins neutralizing type I interferons, preferably wherein the one or more IFN inhibitors are selected form the group consisting of B18R and ruxolitinib, even more preferably wherein the one or more IFN inhibitor is ruxolitinib.
  • Medical uses and method of treatment preferably use the group consisting of: kinase inhibitors, JAK/STAT inhibitors, and virus proteins neutralizing type I interferons, preferably wherein the one or more IFN inhibitors are selected form the group consisting of B18R and ruxolitinib, even more preferably wherein the one or more IFN inhibitor is ruxolitinib.
  • compositions for use in medicine.
  • cell the reprogrammed or induced cell, or the kit-of-parts as described herein, for use in medicine.
  • the cancer is selected from the group consisting of: colorectal cancer, head and neck cancer, basal cell carcinoma, cervical dysplasia, sarcoma, germ cell tumor, retinoblastoma, glioblastoma, lymphoma, Hodgkin's lymphoma, nonHodgkin’s lymphoma, blood cancer, prostate cancer, ovarian cancer, cervix cancer, oesophageal cancer, uterus cancer, vaginal cancer, breast cancer, gastric cancer, oral cavity cancer, naso-pharynx cancer, trachea cancer, larynx cancer, bronchi cancer, bronchioles cancer, lung cancer, pleural cancer, bladder and urothelial cancer, hollow organs cancer, esophagus cancer, stomach cancer, bile duct cancer, intestine cancer, colon cancer, rectum cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gall bladder cancer, spleen cancer, brain cancer,
  • the cancer is selected from the group consisting of: head and neck cancer, colorectal cancer, melanoma, breast cancer, lung cancer, liver cancer, lymphoma, bladder and urothelial cancer, pancreatic cancer and glioblastoma. In even more preferred embodiments, the cancer is selected from the group consisting of: melanoma and sarcoma.
  • a method of treating cancer or infectious diseases comprising administering to an individual in need thereof the composition, the cell, the reprogrammed or induced cell, or the kit-of-parts as described herein.
  • compositions, the cell, the reprogrammed or induced cell, or the kit-of-parts as described herein for the manufacture of a medicament for the treatment of cancer or infectious diseases.
  • Selected mouse and human pri-miRNA sequences together with approximately 300 bp upstream and downstream genomic sequence of the hairpin or hairpin cluster, were amplified from mouse or human genomic DNA using PCR. Resulting amplicons were size-selected with gel electrophoresis, purified and cloned into a constitutive lentiviral vector under control of the SFFV promoter using the Takara In-Fusion kit, according to the manufacturer’s protocol. After cloning, plasmids were purified from bacterial cultures with the GeneJET Plasmid Miniprep Kit. Large scale plasmid production for lentivirus generation was performed using GenElute HP Plasmid DNA Maxiprep Kit. Insert regions of all plasmids used in this study were validated using Sanger sequencing.
  • mice All mouse experiments were performed by following the Swedish guidelines and with approval form a local ethics committee.
  • Mouse embryonic fibroblasts were isolated from E13.5 Clec9a-tdTomato reporter mice as previously described (Rosa et al. 2018). MEFs were expanded until passage 2-4 before reprogramming. Human dermal fibroblasts were reprogrammed at passage 7-12. All cells were cultured in complete DMEM - Dulbecco’s modified eagle medium with high glucose, 2 mM GlutaMAX Supplement, 1 mM sodium pyruvate, 10 pg/ml Penicillin/Streptomycin and 10 % fetal bovine serum (FBS) - at 5 % CO2 and 37 °C. Fibroblasts were cultured in completed DMEM, on cell culture dishes pre-coated in 0.1 % gelatin for 15-30 minutes at 37 °C.
  • FBS fetal bovine serum
  • 293Ts were grown in 15 cm tissue culture plates to 70-90 % confluency and transfected in serum-free DMEM with a mixture of 7.5 pg of psPAX2.G plasmid, 2.5 pg of pMD2 plasmid and 10 pg of the transfer plasmid coding for PIB TFs or pri-miRNA, together with 60 pL of PEI (1 mg/ml) in 2 mL of OptiMEM Reduced Serum Medium (Thermo Fisher Scientific). After 5 hours, transfection medium was replaced with complete DMEM. Next day it was changed again to 12 mL complete DMEM for collection.
  • Virus-containing medium was collected every 8-16 hours to a total of three times, after which the virus was filtered using a 0.45 pm low protein binding filter, concentrated 50-100X using Lenti-X Concentrator (Takara) and stored at -80 °C.
  • fibroblasts were plated on 12- or 6-well plates, or 10 cm dishes at 250.000 cells per plate/dish.
  • cells were transduced with lentivirus containing PIB TFs and pri-miR sequences with polybrene at a final concentration of 8 pg/mL overnight.
  • medium was changed back to complete DMEM.
  • Medium was changed every 3 days.
  • PIB or miRNA lentivirus titers were varied. When miRNA-titers were varied, PIB virus volume was kept constant at 10 pL.
  • miR-124 and miR-142 were cotransduced together, half the volume of each was used, for an equivalent total titer of the mixture to the individual miRNA vectors.
  • cytokine expression assay For fluorescence-activated cell sorting, cells were raised and stained as described above for flow cytometry. MEFs for the cytokine expression assay were sorted on BD FACSMelody and HDFs for RNA-seq and ATAC-seq were sorted on BD FACSymphony S6.
  • Clec9a-tdT mouse embryonic fibroblast
  • Clec9a-tdT MEFs expression of tdTomato marks the acquisition of a DC fate during DC reprogramming (Rosa et al. 2018).
  • XCR1 cDC1 -restricted marker marking high cDC1 identity of reprogrammed cells.
  • miRNA candidates increased Clec9a-tdT reporter activation compared PIB alone (Fig. 1B)
  • miR-124 increased the percentage of CD45 + MHC-II + cells to 44.1 ⁇ 6.6 % compared to 25.3 ⁇ 4.7 % with PIB alone (Fig. 1 C) , suggesting that miR-124 increases reprogramming efficiency by promoting successful cDC1 reprogramming.
  • miRNA candidates decreased reprogramming efficiency (%CD45+ MHC-II+ cells) when combined with PIB, confirming that pri-miRNA overexpression by itself does not increase reprogramming efficiency.
  • XCR1 expression including miR-124, miR-126a, miR-142a and miR-150, suggesting that these miRNAs allow the generation of reprogrammed DCs with statistically higher cDC1 fidelity when compared to PIB overexpression alone.
  • miR-142a induced the highest increase (3.5-fold) in XCR1 + cells (23.7 ⁇ 1.1 %) compared to PIB alone (6.8 ⁇ 1.0 %) (Fig. 1 D, E).
  • miR-124 and miR-142a increase cDC1 reprogramming efficiency and fidelity of reprogrammed DCs when co-expressed with PIB.
  • Example 3 miR-124 and miR-142 show additive effect in DC reprogramming.
  • miR- 124 and miR-142a promoted a dose dependent increase in the proportion of successfully reprogrammed cells based on the expression of CD45, MHCII and XCR1.
  • Clec9a-tdT reporter activation was not affected by miR-124, miR- 142 or the combination at day 2, 4, 6 or 9 of reprogramming (Fig. 2E, left)
  • the percentage of CD45+ MHC-II+ XCR1+ reprogrammed DCs increased with the miRNAs on days 6 and 9 (Fig. 2E, right), suggesting that miRNAs increase the velocity and fidelity of cDC1 reprogramming.
  • reprogrammed cells generated with PIB together with miR-124 and/or miR-142 were functional and responded to inflammatory stimulation by secreting proinfl am matory cytokines including IL-12, CXCL10 and TFNa at reprogramming day 9 (Fig. 2F).
  • miR-124 and miR-142a have an additive effect in DC reprogramming mediated by overexpression of PIB by enhancing the velocity of reprogramming and the fidelity of reprogrammed DCs.
  • reprogrammed cells generated with PIB together with miR-124 and/or miR-142a are functional cDC1-like cells.
  • miR-124 and miR-142 increase efficiency of cDC1 reprogramming mediated by PU.1, IRF8 and BATF3 in human cells.
  • the inventors also measured the expression of CD226 as a surrogate marker for high cDC1 fidelity of reprogrammed DCs.
  • the inventors observed that miR-142 increased the fidelity of reprogramming by increasing CD226 expression 2.3-fold (29.1 ⁇ 9.6 % compared to 12.9 ⁇ 6.5 %) compared to PIB alone (Fig. 3B, C).
  • miR-124 increases efficiency of the cell fate conversion by increasing the surface expression of the successful DC reprogramming markers CD45 and MHC-II/HLA-DR
  • miR-142 enhances cDC1 fidelity of reprogrammed DCs measured by surface expression of the cDC1 restricted markers XCR1 (in the mouse) and CD226 (in the human).
  • Example 5 miR-124 and miR-142 enhances cDC1 reprogramming efficiency by repressing the original cell fate and promoting cDC1 gene expression
  • HDFs or iDCs were sorted for reprogramming markers CD45 and HLA-DR.
  • Empty vector-transduced control HDFs were sorted on day 3 using the parent live cell gate.
  • Human DC controls were isolated from buffy coats with the Pan-DC Enrichment Kit (Miltenyi Biotec) and sorted for cDC1s (HLA- DR+ CD11c+ CD141+), cDC2s (HLA-DR+ CD11c+ CD1c+ CD141-) and pDCs (HLA- DR+ CD11c- CD123+). 1000-2000 cells were sorted and their RNA extracted using TRIzol (Thermo Fisher Scientific) extraction.
  • RNA-seq libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v3 (Takara) according to manufacturer’s instructions. Two technical replicate RNA-seq library pools of 79-samples each were sequenced in a single run with the S2 reagent kit, split with a lane divider, on a NovaSeq 6000 (Illumina) sequencer.
  • Resulting gene counts were further processed with R package DESeq2 (Love, Huber, and Anders 2014) and normalized using RLE method.
  • DESeq2 package and used for performing differential expression analysis based on Wald test.
  • the inventors defined upregulated genes by a fold change (FC) > 0.5 and Benjamini Hochberg (BH) corrected p-value ⁇ 0.05 and downregulated genes by FC ⁇ - 0.5 and BH-corrected p-value ⁇ 0.05.
  • FC fold change
  • BH Benjamini Hochberg
  • CLEC9A and XCR1 expression and miRNA target downregulation were quantified with CPM normalization.
  • Downregulated gene threshold in miRNA target prediction analysis was a mean fold change > 2 across three biological replicates.
  • Cell type enrichment was performed against ARCHS4 using EnrichR.
  • RNA-seq total RNA sequencing
  • the inventors further validated this finding by detecting expression of the cDC1- specific genes CLEC9A, THBD (encoding CD141) and XCR1 (Fig. 4C).
  • miR-124 also showed an increase in cDC1 gene expression at day 3 compared to scrambled control (Fig. 4B) and allowed expression of XCR1, which was not detected at any point in reprogrammed DCs transduced with PIB and scrambled miRNA (Fig. 4C).
  • the inventors investigated the genes downregulated in reprogrammed DCs by miRNA overtime.
  • miR-124 was predicted to directly target 57 % (224/392) of downregulated genes compared to 38 % by miR-142 (113/300) (Fig. 4F), suggesting that miR-124 directly represses a higher percentage of genes downregulated at early stages of DC reprogramming.
  • miR-124 and miR-142 repress the original cell fate and promote cDC1 gene expression, with miR-124 acting primarily early in reprogramming and miR-142 driving increasing iDC fidelity over time.
  • miR-124 induces a permissive chromatin state in the context of cDC1 reprogramming
  • HDFs or iDCs were sorted for reprogramming markers CD45 and HLA-DR.
  • Empty vector-transduced control HDFs were sorted on day 3 using the parent live cell gate.
  • Human DC controls were isolated from buffy coats with the Pan-DC Enrichment Kit (Miltenyi Biotec) and sorted for cDC1s (HLA- DR+ CD11c+ CD141+), cDC2s (HLA-DR+ CD11c+ CD1c+ CD141-) and pDCs (HLA- DR+ CD11c- CD123+).
  • the inventors sorted 5000-10000 cells per sample and followed a previously a published protocol (Buenrostro et al.
  • the nf-core (Ewels et al. 2020) ATAC-seq pipeline version 1.2.2 was used with default parameters. Reads were mapped to the GRCh38 reference genome using BWA (H. Li and Durbin 2009). Peak calling was performed with MACS2 separately for each sample. A combined peak list for all samples was obtained by using BEDTools (Quinlan and Hall 2010). Finally, read counts on a combined peak list were calculated using featurecounts. The resulting read counts were processed with R package DESeq2 (Quinlan and Hall 2010) (REF) and normalized using the RLE method. For peak annotation ChlPseeker R library (Yu, Wang, and He 2015)) was used.
  • the inventors sought to characterize the effects of the miRNAs on PIB-driven chromatin remodeling. To do this, an assay for transposase-accessible chromatin with sequencing (ATAC-Seq) was performed at reprogramming days 3, 6 and 9 using HDFs co-transduced with PIB and miR-124, miR-142 or scrambled (non-targeting) pri-miRNA sequence (Fig. 4A). The inventors analyzed chromatin accessibility in regions containing cDC1s genes and observed that the majority was accessible from as early as day 3 (Fig. 5A), which goes in line with our previous findings in DC reprogramming of cancer cells (Zimmermannova et al. 2023).
  • miR-124 increased accessibility of cDC1 genes compared to miR-142 or the scrambled control (Fig. 5A). Accordingly, miR-124 induced a gain of 3503 accessible peaks and a loss of 1299 accessible peaks compared to scrambled control on day 3, indicating an early induction of permissive chromatin landscape (Fig. 5B).
  • miR-142 showed an opposite effect on chromatin accessibility, with 77.6 % less differentially accessible peaks when compared to cells treated with miR-124, with the majority (858 peaks; 79,7 %) being less accessible when compared to the scrambled control at day 3 Peaks with increased accessibility upon miR-124 overexpression were enriched in Polycomb Repressive Complex (PRC) members SLIZ12 and EZH2, as well as in transcriptional repressor REST (Fig. 5C), indicating a possible role in H3K27me3 mark inhibition in increasing Pll.1 binding site accessibility in early reprogramming. Interestingly, both miRNAs showed an enrichment for SPI1 (Pll.1) motifs in regions with higher accessibility (Fig.
  • CD45+ HLA-DR+ cells were sorted into 350 pL of RNeasy Micro (Qiagen) lysis buffer from which total RNA was later isolated according to the manufacturer’s protocol.
  • DNA libraries were prepared using the SuperScript IV Reverse Transcriptase (Invitrogen) with random hexamer primers.
  • XCR1 expression was quantified by qPCR using TaqMan probes (ThermoFisher Scientific), with PPIA used as the reference gene.
  • miRNA mimics are chemically synthesized double-stranded RNA molecules imitating mature miRNA duplexes.
  • the inventors started by generating miRNA mimics for miR-124 (Fig. 6A), and transfected miRNA mimics on days 0, 3 and 6 of reprogramming to simulate constitutive pri-miRNA expression. miR-124-3p replicated the increase in reprogramming efficiency in a dose-dependent manner (Fig. 6A).
  • miR-124-3p was added on days -2, 0, 3 or 6 (Fig. 6D). Surprisingly, pre-treatment or day 6 addition of miR-124-3p did not increase reprogramming efficiency (Fig. 6E), underscoring the importance of cooperation between the PIB transcription factors and miR-124 for enhanced cDC1 reprogramming efficiency.
  • XCR1 was primarily upregulated by the canonical 3’ strand of miR-142 by qPCR (Fig. 6H). miRNA mimics had comparable effect on HEF reprogramming, with miR-124-3p, miR- 142-3p-1 , miR-142-3p-1 and miR-142 combination increasing reprogramming efficiency compared to the scrambled control (Fig. 6I). The miR-142-3p, miR-142-3p,5p and miR-142-3p-1 increased XCR1 expression in HEFs (Fig. 6J).
  • the inventors also detected a notable additive effect of co-transfection with all 8 miR-142 mimics. Taken together, the inventors selected the canonical miR-142-3p and the isomiRs miR-142- 3p-1, miR-142-3p-3 and miR-142-5p-1 for follow-up analyses, alongside miR-124-3p. When added together, these mimics showed limited additive effects on reprogramming efficiency to CD45+ HLA-DR+ i DC, apart from the combination of miR-124-3p and miR- 142-5p-1 (Fig. 6K).
  • the inventors integrated transcriptomic and genomic accessibility data using GRaNPA (Kamal et al. 2023) to identify miRNA targets which could mediate their effect in DC reprogramming (Fig. 7A). They identified SP1 and KLF5 as the most significant TFs targeted by miR-124-3p, and KLF10, PATZ1 and ELK3 by validated miR-142 strands (Fig. 7B). Considering the role of miRNAs in target downregulation, the inventors investigated whether GRaNPA target predictions were downregulated upon hairpin overexpression and confirmed that SP1 , KLF5 and RELA were significantly downregulated on co-transduction with miR-124 (Fig. 7C).
  • Linear ml- ⁇ P-modified monocistrionic and bicistronic mRNAs coding for human PU.1, PU.1-T2A-mTagBFP2 (PU.1-BFP), IRF8, IRF8-T2A-miRFP670nano (IRF8-RFP), BATF3, BATF3-T2A-mNeonGreen (BATF3-GFP) and GFP were generated and in vitro synthesized.
  • Monocistronic linear mRNAs were co-transfected together with miRNA mimics using Lipofectamine RNAiMAX with a total of 1 pg of mRNAs combined with a mass ratio and the miRNA mimics for a final concentration of 1 nM.
  • the three linear ml- ⁇ P-modified bicistronic mRNAs were co-transfected using Lipofectamine RNAiMAX with a total of 100ng of mRNAs in the presence or absence of ruxolitinib, Baricitinib, Fedratinib, Upadacitinib or Filgotinib, and reprogramming efficiency was profiled 60h after transfection.
  • RNA-mediated delivery of transcription factors allows cDC1 reprogramming in human cells
  • the inventors transfected HDFs with modified linear mRNAs encoding human Pll.1 , IRF8, BATF3 or GFP with or without miRNA mimics on days 0, 2, 4 and 6 and profiled cDC1 reprogramming efficiency at day 9 (Fig. 8A).
  • the inventors observed expression of the cDC1 reprogramming marker CD45 in transfected HDFs, suggestion that cDC1 reprogramming is possible using modRNA.
  • cDC1 reprogramming efficiency at day 3 induced lentiviral vector-transduced GFP+ cells (Gated in GFP+) and mRNA+ cells (gated in BFP+RFP+GFP+) (Fig. 8D).
  • miR- 124-3p increased reprogramming efficiency compared to either PIB mRNA alone or the scrambled control, and that PIB mRNA with no mimics, miR-142-3p-1, miR-142-3p-3 and the combination of all 5 miRNA mimics increased the yield of CD45+ iDCs when compared to reprogramming mediated by lentiviral vectors (Fig. 8E).
  • the inventors transduced human dermal fibroblasts with lentiviral vectors encoding Pll.1, IRF8 and BATF3 and profiled reprogramming yield in the presence of different IFN inhibitors, including Ruxolitinib (rux), Baricitinib (Bari, JAK1/JAK2 inhibitor), Fedratinib (Fedra, JAK2 inhibitor), Upadacitinib (Upa, JAK1 inhibitor) and Filgotinib (Filgo, JAK 1 inhibitor), at 3 different concentrations 9 days after transduction.
  • Ruxolitinib rux
  • Baricitinib Bari, JAK1/JAK2 inhibitor
  • Fedratinib Fredra, JAK2 inhibitor
  • Upadacitinib Upa, JAK1 inhibitor
  • Filgotinib Filgotinib
  • Example 10 saRNA encoding PU.1, IRF8 and BATF3 induces cDC1 reprogramming
  • Synthetic polycistronic self-amplifying RNAs were generated based on the Venezuelan equine encephalitis (VEE) genome (Yoshioka et al. 2013; Yoshioka and Dowdy 2017) encoding the human sequences of PU.1 , IRF8 and BATF3 separated by 2A selfcleaving peptides in a tricistronic cassette (saRNA-PIB).
  • saRNA-PIB and GFP- encoding saRNA saRNA-GFP
  • saRNA-GFP were synthesized in vitro and cells co-transfected with saRNA-PIB (0.5ug) and saRNA-GFP (0.5ug), using Lipofectamin Messenger Max. After transfection, the media was daily replaced by fresh culture media with or without B18R, ruxolitinib or both.
  • Clec9a-tdT MEFs were co-transfected at day 0 with saRNA-PIB and saRNA-GFP and cDC1 reprogramming efficiency was evaluated be flow cytometry quantification of tdTomato+ reprogrammed cDC1-like cells.
  • saRNA replication was previously shown to elicit strong interferon (IFN) responses that can affect protein expression and reprogramming efficiency
  • IFN interferon
  • the inventors also tested whether treatment of transfected cells with the IFN inhibitors B18R (Vaccinia virus protein that neutralizes type I interferons) and ruxolitinib (JAK1/JAK2 inhibitor) could allow higher reprogramming efficiency.
  • the inventors observed that saRNA-PIB allowed tdTomato reporter activation and expression of CD45 and MHC-II, suggesting that saRNA can induce cDC1 reprogramming (Fig 9. A-C).
  • Example 11 Linear, circular and self-amplifying RNA platforms exhibit different payload expression levels and kinetics.
  • Human leiomyosarcoma cell line SK-LMS-1 was cultured in complete DMEM - Dulbecco’s modified eagle medium with high glucose, 2 mM GlutaMAX Supplement, 1 mM sodium pyruvate, 10 pg/ml Penicillin/Streptomycin and 10 % fetal bovine serum (FBS) - at 5 % CO2 and 37 °C.
  • FBS fetal bovine serum
  • Linear ml- ⁇ P-modified RNA, circular RNA, and self-amplifying RNA, coding for EGFP were in vitro synthesized and purified by Genescript.
  • SK- LMS-1 cells were plated in 48-well plates.
  • cells reached 70-90 % confluency, and medium was changed to complete DM EM with ruxolitinib at a final concentration of 1 pM.
  • cells were transfected with linear, circular, or selfamplifying RNA coding for EGFP, together with 0.2 pL of Lipofectamine RNAiMAX in 20 pL of OptiMEM Reduced Serum Medium.
  • RNA varied.
  • cells were raised with TrypLE Express and resuspended in complete DM EM with ruxolitinib at a final concentration of 1 pM.
  • a fraction of the cells from each well were transferred to new 48-well plates to avoid over-confluency due to high growth rate of SK-LMS-1 cells.
  • redosing experiment cells were transfected on day 0, 3, and 6, respectively.
  • eGFP-encoding modified linear, circular and saRNAs Fig. 10A-F
  • MFI mean fluorescence intensity
  • eGFP+ cells After day 2 post transfection, frequency of eGFP+ cells decreased overtime for the 3 platforms, with saRNA and circular RNA showing fastest and slowest decline of frequency of eGFP+ cells overtime, respectively. Moreover, the inventors detected the presence of a small population of eGFP+ cells expressing high levels of eGFP from day 1 to day 8 in cultures specifically in saRNA-treated cultures, suggesting that saRNA can amplify within transfected cells allowing high eGFP expression even 8 days after transfection.
  • RNA allowed the highest frequency of eGFP+ cells at day 8 post transfection (between 20-40% eGFP+ cells) when compared to linear RNA ( ⁇ 20% eGFP+ cells) and saRNA ( ⁇ 5% eGFP+ cells) (Fig. B-E).
  • Fig. 10G the inventors asked whether redosing allows prolonged transgene expression mediated by the 3 RNA platforms (Fig. 10G). They demonstrated that redosing allows higher frequency of eGFP+ cells overtime for the 3 platforms, associated with increased frequency of eGFP+ positive cells (Fig. 10H) and enhanced eGFP MFI (Fig. 101).
  • RNA allows highest transgene expression at day 2 post transfection (early timepoint), followed by circular RNA and saRNA, and that saRNA shows highest transgene expression at day 8 (late timepoint).
  • the different RNA platforms show distinct kinetics of frequency of transfected cells, with circular RNA allowing higher frequency of eGFP+ transfected cells at day 8 post transfection when compared to linear and saRNA.
  • these data also suggest that repeated transfections can enhance the frequency of transfected cells overtime.
  • Example 12 Linear, circular and self-amplifying RNA platforms allow cDC1 reprogramming in cancer cells.
  • SK-LMS-1 cells were plated in 48-well plates. On day 0 of reprogramming, cells reached 70-90 % confluency, and medium was changed to complete DMEM containing 1 pM ruxolitinib. Cells were then transfected with linear, circular, or self-amplifying RNA coding for PIB TFs, together with 0.2 pL of Lipofectamine RNAiMAX in 20 pL of OptiMEM Reduced Serum Medium. For linear and circular RNAs encoding monocistronic PIB TFs, three PIB TFs were mixed at 1:1 :1 molar ratio.
  • RNAs were varied.
  • cells were raised with TrypLE Express and resuspended in complete DM EM with ruxolitinib at a final concentration of 1 pM.
  • a fraction of the cells from each well were transferred to a new well in 48-well plates to avoid crowding due to the high growth rate of SK-LMS-1 cells.
  • RNA platforms could induce cDC1 reprogramming measured by the frequency of cancer cells expressing CD45 and/or HLA-DR in live (for linear and circular RNA) or eGFP+ (for saRNA) cells 3 days after transfection (Fig. 11B).
  • eGFP+ for saRNA
  • RNA constructs as those in example 12 were used, following the same transfection protocol.
  • cells were treated with 1 dose (day 0), two doses (day 0 and 3), and three doses (day 0, 3, and 7) of linear, circular, or selfamplifying RNA coding for PIB TFs.
  • day 0 1 dose
  • day 0 and 3 two doses
  • day 0 3, and 7 three doses
  • day 0, 3, and 7 of linear, circular, or selfamplifying RNA coding for PIB TFs.
  • day 3 and 6 of reprogramming cells were subcultured to avoid crowding.
  • RNA platforms are functional antigen-presenting cells ( Figure 12F, G).
  • Reprogramming efficiency decreased over time to frequencies of CD45+ and/or HLA- DR+ cells below 5% independently of the reprogramming platform.
  • circular RNA allowed higher reprogramming efficiency at day 6 with one single dose at day 0 when compared to linear and saRNA.
  • a small population of SK-LMS-1 cells coexpressing CD45 and HLA-DR was detected specifically in cultures transfected with PIB-encoding saRNA.
  • two doses of circular RNA at day 0 and 3 allowed highest reprogramming efficiency at day 9 (approx. 40% CD45+ and/or HLA-DR+ cells) when compared to 2 doses of linear or saRNA (approx. 10% CD45+ and/or HLA-DR+ cells).
  • saRNA allowed highest number of reprogrammed cells at day 9.
  • circular RNA allowed the highest number of reprogrammed cells at day 9.
  • linear RNA allowed the highest number of reprogrammed cells.
  • cDC1 type 1 conventional dendritic cells
  • This reprogramming remodels the tumor microenvironment, induces tumor regression, and establishes long-term systemic anti-tumor immunity in mouse syngeneic models (Ascic et al. 20242024). It also exhibited strong synergistic effects when combined with immune checkpoint blockade (ICB) therapy.
  • IRB immune checkpoint blockade
  • RNA modalities described herein provide an alternative non-viral system for direct in vivo tumor cell reprogramming.
  • RNA-based reprogramming will further enhance T-cell infiltration and overall antitumor efficacy.
  • Nanocarriers include for example exosomes, lipossomes, lipid-based, sylicon-based, polymer-based and peptide-based nanoparticles. The feasibility of these delivery methods for RNA has been widely established in the literature.
  • the inventors have also previously demonstrated that adoptive transfer of in vitro reprogrammed or in vitro transduced cancer cells (generated with viral vectors encoding DC reprogramming factors) induces antitumor efficacy as monotherapy and in combination with ICB (Zimmermannova et al. 2023, Ascic et al. 2024).
  • RNA modalities described herein also provide an alternative non-viral system for tumor cell transfection and reprogramming in vitro.
  • RNAs encoding Pll.1 , IRF8 and BATF3 have been shown herein to allow efficient DC reprogramming of cancer cells.
  • such reprogrammed cancer cells will be administered in vivo for cancer immunotherapy or will be used in vitro for other applications including neoantigen identification or expansion of lymphocytes from peripheral blood and tumors.
  • SEQ. ID NO. 1 Mus musculus microRNA 124a- 1 (Mir124a-1, NR_029813.1) AGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCCATACAATTAAG GCACGCGGTGAATGCCAAGAATGGGGCTG
  • SEQ. ID NO. 2 Homo sapiens microRNA 124-1 (Mir124-1 , NR_029668.1), DNA sequence AGGCCTCTCTCTCCGTGTTCACAGCGGACCTTGATTTAAATGTCCATACAATTAAG GCACGCGGTGAATGCCAAGAATGGGGCTG SEQ. ID NO. 3: Mus musculus microRNA 126a (Mir126a, NR_029541.1), DNA sequence
  • SEQ. ID NO. 4 Homo sapiens microRNA 126 (Mir126, NR_029695.1), DNA sequence CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTCAAACTCGT ACCGTGAGTAATAATGCGCCGTCCACGGCA
  • SEQ. ID NO. 5 Mus musculus microRNA 142 (Mir142, NR_029555.1), DNA sequence ACCCATAAAGTAGAAAGCACTACTAACAGCACTGGAGGGTGTAGTGTTTCCTACTT TATGGATG
  • SEQ. ID NO. 6 Homo sapiens microRNA 142 (Mir142, NR_029683.1), DNA sequence GACAGTGCAGTCACCCATAAAGTAGAAAGCACTACTAACAGCACTGGAGGGTGTA GTGTTTCCTACTTTATGGATGAGTGTACTGTG
  • SEQ. ID NO. 7 Mus musculus microRNA 150 (Mir150, NR_029560.1), DNA sequence CCCTGTCTCCCAACCCTTGTACCAGTGCTGTGCCTCAGACCCTGGTACAGGCCTG GGGGATAGGG
  • SEQ. ID NO. 8 Homo sapiens microRNA 150 (Mir150, NR_029703.1), DNA sequence CTCCCCATGGCCCTGTCTCCCAACCCTTGTACCAGTGCTGGGCTCAGACCCTGGT ACAGGCCTGGGGGACAGGGACCTGGGGAC
  • SEQ. ID NO. 11 Mus musculus interferon regulatory factor 8 (IRF8), transcript variant 1 (NM_001301811.1), DNA sequence ATGTGTGACCGGAACGGCGGGCGGCGGCTGCGGCAGTGGCTGATCGAACAGAT
  • SEQ. ID NO. 12 Homo sapiens interferon regulatory factor 8 (IRF8), transcript variant
  • SEQ. ID NO. 13 Mus musculus spleen focus forming virus (SFFV) proviral integration oncogene (Spi 1 ), transcript variant 2 (NM_011355.2), DNA sequence
  • SEQ. ID NO. 14 Homo sapiens spleen focus forming virus (SFFV) proviral integration oncogene (Spi1), transcript variant 2 (NM_003120.3), DNA sequence
  • SEQ. ID NO. 15 Mus musculus basic leucine zipper ATF-like transcription factor 3 (BATF3) (NM_030060.2), DNA sequence
  • SEQ. ID NO. 16 Homo sapiens basic leucine zipper ATF-like transcription factor 3 (BATF3) (NM_018664.3), DNA sequence ATGTCGCAAGGGCTCCCGGCCGCCGGCAGCGTCCTGCAGAGGAGCGTCGCGGC
  • SEQ. ID NO. 17 Mus musculus interferon regulatory factor 8 (IRF8)
  • SEQ. ID NO. 18 Homo sapiens interferon regulatory factor 8 (IRF8), isoform 1
  • SEQ. ID NO. 19 Mus musculus spleen focus forming virus (SFFV) proviral integration oncogene (Spi1), isoform 2 (NP_035485.1), polypeptide sequence
  • SEQ. ID NO. 20 Homo sapiens spleen focus forming virus (SFFV) proviral integration oncogene (Spi1), isoform 2 (NP_003111.2), polypeptide sequence
  • SEQ. ID NO. 21 Mus musculus basic leucine zipper transcriptional factor ATF-like 3
  • SEQ. ID NO. 22 Homo sapiens basic leucine zipper transcriptional factor ATF-like 3
  • SEQ ID NO: 23 isomiR miR-142-3p-1
  • SEQ ID NO: 24 isomiR miR-142-3p-3
  • SEQ ID NO: 25 isomiR miR-142-5p-1
  • GRaNIE and GRaNPA inference and evaluation of enhancer-mediated gene regulatory networks. Molecular Systems Biology 19, e11627.

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Abstract

La présente invention concerne des compositions comprenant des ARN, leurs utilisations, et des méthodes d'expression génique à base d'ARN pour reprogrammer des cellules vers des cellules dendritiques classiques de type 1 ou des cellules présentatrices d'antigène. L'invention concerne en outre des cellules, des cellules reprogrammées et leurs utilisations.
PCT/EP2024/084945 2023-12-05 2024-12-05 Stratégies à base d'arn pour la reprogrammation de cellules dendritiques et leurs utilisations Pending WO2025120097A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3385373A1 (fr) * 2017-04-05 2018-10-10 Centro de Neurociências e Biologia Celular Compositions pour la reprogrammation de cellules en cellules dendritiques ou en cellules présentatrices d'antigènes, leurs procédés et utilisations
US20200017832A1 (en) * 2017-04-05 2020-01-16 Centro de Neurociencias e Biologia Cellular Compositions for reprogramming cells into dendritic cells or antigen presenting cells, methods and uses thereof
CA3218112A1 (fr) * 2021-05-19 2022-11-24 Fabio FIUZA ROSA Reprogrammation de cellules en cellules presentatrices d'antigenes ou cellules dendritiques conventionnelles de type 1
US20230332182A1 (en) * 2020-07-01 2023-10-19 Elevatebio Technologies, Inc. Compositions and methods for cellular reprogramming using circular rna

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3385373A1 (fr) * 2017-04-05 2018-10-10 Centro de Neurociências e Biologia Celular Compositions pour la reprogrammation de cellules en cellules dendritiques ou en cellules présentatrices d'antigènes, leurs procédés et utilisations
US20200017832A1 (en) * 2017-04-05 2020-01-16 Centro de Neurociencias e Biologia Cellular Compositions for reprogramming cells into dendritic cells or antigen presenting cells, methods and uses thereof
US20230332182A1 (en) * 2020-07-01 2023-10-19 Elevatebio Technologies, Inc. Compositions and methods for cellular reprogramming using circular rna
CA3218112A1 (fr) * 2021-05-19 2022-11-24 Fabio FIUZA ROSA Reprogrammation de cellules en cellules presentatrices d'antigenes ou cellules dendritiques conventionnelles de type 1

Non-Patent Citations (72)

* Cited by examiner, † Cited by third party
Title
"MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts", DEVELOPMENTAL CELL, vol. 46, no. 1, pages 73 - 84
ABERNATHY, DANIEL G.WOO KYUNG KIMMATTHEW J. MCCOYALLISON M. LAKEREBECCA OUWENGASEONG WON LEEXIAOYUN XING ET AL.: "MicroRNAs Induce a Permissive Chromatin Environment That Enables Neuronal Subtype-Specific Reprogramming of Adult Human Fibroblasts", CELL STEM CELL, vol. 21, no. 3, 2017, pages 332 - 348, XP085189930, DOI: 10.1016/j.stem.2017.08.002
ANOKYE-DANSOFREDERICKCHINMAY M. TRIVEDIDENISE JUHRMUDIT GUPTAZHENG CUIYING TIANYUZHEN ZHANG ET AL.: "Highly Efficient MiRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency", CELL STEM CELL, vol. 8, no. 4, 2011, pages 376 - 88, XP055547506, DOI: 10.1016/j.stem.2011.03.001
APARICIO-PUERTA, E.HIRSCH, P.SCHMARTZ, G.P.FEHLMANN, T.KELLER, V.ENGEL, A.KERN, F.HACKENBERG, M.KELLER, A.: "isomiRdb: microRNA expression at isoform resolution", NUCLEIC ACIDS RESEARCH, vol. 51, 2023, pages 0179 - 0185
ASCIC, E.AKERSTR6M, F.SREEKUMAR NAIR, M.ROSA, A.KUROCHKIN, I.ZIMMERMANNOVA, O.CATENA, X.ROTANKOVA, N.VESER, C.RUDNIK, M. ET AL.: "In vivo dendritic cell reprogramming for cancer immunotherapy", SCIENCE, vol. 386, 2024, pages 9083, XP093230336, DOI: 10.1126/science.adn9083
BALAN, SC ARNOLD-SCHRAUFA ABBASN COUESPELJ SAVORETF IMPERATOREA C VILLANIT P VU MANHN BHARDWAJM DALOD: "Large-Scale Human Dendritic Cell Differentiation Revealing Notch-Dependent Lineage Bifurcation and Heterogeneity", CELL REP, vol. 24, no. 7, 2018, pages 1902 - 1915, XP055705788, DOI: 10.1016/j.celrep.2018.07.033
BARRY, K CJ HSUM L BROZF J CUETOM BINNEWIESA J COMBESA E NELSON ET AL.: "A Natural Killer-Dendritic Cell Axis Defines Checkpoint Therapy-Responsive Tumor Microenvironments", NAT MED, vol. 24, no. 8, 2018, pages 1178 - 91, XP036928726, DOI: 10.1038/s41591-018-0085-8
BÖTTCHER, J PE BONAVITAP CHAKRAVARTYH BLEESM CABEZA-CABRERIZOS SAMMICHELIN C ROGERSE SAHAIS ZELENAYE SOUSA: "NK Cells Stimulate Recruitment of CDC1 into the Tumor Microenvironment Promoting Cancer Immune Control", CELL, vol. 172, no. 5, 2018, pages 1022 - 1037, XP085352323, DOI: 10.1016/j.cell.2018.01.004
BROZ, M LMIKHAIL BINNEWIESBIJAN BOLDAJIPOURAMANDA E NELSONJOSHUA L POLLACKDAVID J ERLEANDREA BARCZAK ET AL.: "Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity", CANCER CELL, vol. 26, no. 5, 2014, pages 638 - 52, XP029094865, DOI: 10.1016/j.ccell.2014.09.007
BUENROSTRO, JASON D.BEIJING WUHOWARD YCHANGWILLIAM J. GREENLEAF.: "ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide", CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, vol. 109, no. 1, 2015
CHENROBERTSEAN K. WANGJULIA A. BELKLAURA AMAYAZHIJIAN LIANGEL CARDENASBRIAN T. ABECHUN-KAN CHENPAUL A. WENDER: "Engineering Circular RNA for Enhanced Protein Production", NATURE BIOTECHNOLOGY, vol. 41, no. 2, 2023, pages 262 - 72
CONNORBRONWENERIN FIRMINAMY MCCAUGHEY-CHAPMANRUTH MONKKEVIN LEESOPHIE LIOTJOHANNES GEIGERCARSTEN RUDOLPHKATHRYN JONES: "Conversion of Adult Human Fibroblasts into Neural Precursor Cells Using Chemically Modified MRNA", HELIYON, vol. 4, no. 11, 2018, pages 00918
DOBINALEXANDERCARRIE A DAVISFELIX SCHLESINGERJORG DRENKOWCHRIS ZALESKISONALI JHAPHILIPPE BATUTMARK CHAISSONTHOMAS R GINGERAS: "STAR: Ultrafast Universal RNA-Seq Aligner", BIOINFORMATICS, vol. 29, no. 1, 2013, pages 15 - 21, XP055500895, DOI: 10.1093/bioinformatics/bts635
EWELSPHILIP A.ALEXANDER PELTZERSVEN FILLINGERHARSHIL PATELJOHANNES ALNEBERGANDREAS WILMMAXIME ULYSSE GARCIAPAOLO DI TOMMASOSVEN NA: "The Nf-Core Framework for Community-Curated Bioinformatics Pipelines", NATURE BIOTECHNOLOGY, vol. 38, no. 3, 2020, pages 276 - 78, XP037055456, DOI: 10.1038/s41587-020-0439-x
HAN SUN MURRAY ET AL: "TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets", vol. 16, no. 1, 1 January 2016 (2016-01-01), KP, pages 61, XP093151165, ISSN: 1598-2629, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770101/pdf/in-16-61.pdf> [retrieved on 20240416], DOI: 10.4110/in.2016.16.1.61 *
HEIDKAMP, G FJ SANDERC H K LEHMANNL HEGERN EISSINGA BARANSKAJ J LÜHR ET AL.: "Human Lymphoid Organ Dendritic Cell Identity Is Predominantly Dictated by Ontogeny, Not Tissue Microenvironment", SCI IMMUNOL, vol. 1, no. 6, 2016
HEINZSVENCHRISTOPHER BENNERNATHANAEL SPANNERIC BERTOLINOYIN C. LINPETER LASLOJASON X. CHENGCORNELIS MURREHARINDER SINGH: "Simple Combinations of Lineage-Determining Transcription Factors Prime Cis-Regulatory Elements Required for Macrophage and B Cell Identities", MOLECULAR CELL, vol. 38, no. 4, 2010, pages 576 - 89
HILDNER, KB T EDELSONW E PURTHAM DIAMONDH MATSUSHITAM KOHYAMAB CALDERON ET AL.: "Batf3 Deficiency Reveals a Critical Role for CD8alpha+ Dendritic Cells in Cytotoxic T Cell Immunity", SCIENCE, vol. 322, no. 5904, 2008, pages 1097 - 1100, XP055422537, DOI: 10.1126/science.1164206
HUANGHSI-YUANYANG-CHI-DUNG LINJING LIKAI-YAO HUANGSIRJANA SHRESTHAHSIAO-CHIN HONGYUN TANG ET AL.: "MiRTarBase 2020: Updates to the Experimentally Validated MicroRNA-Target Interaction Database", NUCLEIC ACIDS RESEARCH, 2019
JAYAWARDENATILANTHI M.BAKYTBEK EGEMNAZAROVELIZABETH A. FINCHLUNAN ZHANGJ. ALAN PAYNEKUMAR PANDYAZHIPING ZHANGPAUL ROSENBERGMARIA M: "MicroRNA-Mediated In Vitro and In Vivo Direct Reprogramming of Cardiac Fibroblasts to Cardiomyocytes", CIRCULATION RESEARCH, vol. 110, no. 11, 2012, pages 1465 - 73
KAMAL, A., ARNOLD, C., CLARINGBOULD, A., MOUSSA, R., SERVAAS, N.H., KHOLMATOV, M., DAGA, N., NOGINA, D., MUELLER-DOTT, S., REYES-P: "GRaNIE and GRaNPA : inference and evaluation of enhancer-mediated gene regulatory networks", MOLECULAR SYSTEMS BIOLOGY, vol. 19, 2023, pages 11627
KARLSEN, T.A.AAE, T.F.BRINCHMANN, J.E.: "Robust profiling of microRNAs and isomiRs in human plasma exosomes across 46 individuals", SCI REP, vol. 9, 2019, pages 19999
KIRKLING, M EU CYTLAKC M LAUK L LEWISA RESTEUA KHODADADI-JAMAYRANC W SIEBEL ET AL.: "Notch Signaling Facilitates In Vitro Generation of Cross-Presenting Classical Dendritic Cells", CELL REP, vol. 23, no. 12, 2018, pages 3658 - 3672
KOGUTIGORSANDRA M. MCCARTHYMARYNA PAVLOVADAVID P. ASTLINGXIAOMI CHENANA JAKIMENKOKENNETH L. JONES ET AL.: "High-Efficiency RNA-Based Reprogramming of Human Primary Fibroblasts", NATURE COMMUNICATIONS, vol. 9, no. 1, 2018, pages 745
KOZOMARAANAMARIA BIRGAOANUSAM GRIFFITHS-JONES: "MiRBase: From MicroRNA Sequences to Function", NUCLEIC ACIDS RESEARCH, vol. 47, no. D1, 2019, pages 155 - 62
KULESHOVMAXIM V.MATTHEW R. JONESANDREW D. ROUILLARDNICOLAS F. FERNANDEZQIAONAN DUANZICHEN WANGSIMON KOPLEV ET AL.: "Enrichr: A Comprehensive Gene Set Enrichment Analysis Web Server 2016 Update", NUCLEIC ACIDS RESEARCH, vol. 44, no. W1, 2016, pages 90 - 97
LAUTERBACH, HB BATHKES GILLESC TRAIDL-HOFFMANNC A LUBERG FEJERM A FREUDENBERG ET AL.: "Mouse CD8alpha+ DCs and Human BDCA3+ DCs Are Major Producers of IFN-Lambda in Response to Poly IC", J EXP MED, vol. 207, no. 12, 2010, pages 2703 - 17, XP008132858, DOI: 10.1084/jem.20092720
LIHENGRICHARD DURBIN: "Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform", BIOINFORMATICS, vol. 25, no. 14, 2009, pages 1754 - 60
LIJUN-HAOSHUN LIUHUI ZHOULIANG-HU QUJIAN-HUA YANG: "StarBase v2.0: Decoding MiRNA-CeRNA, MiRNA-NcRNA and Protein-RNA Interaction Networks from Large-Scale CLIP-Seq Data", NUCLEIC ACIDS RESEARCH, vol. 42, no. D1, 2014, pages 92 - 97
LOVEMICHAEL IWOLFGANG HUBERSIMON ANDERS: "Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2", GENOME BIOLOGY, vol. 15, no. 12, 2014, pages 550
LUNDSTROMKENNETH: "Trans-Amplifying RNA: Translational Application in Gene Therapy", MOLECULAR THERAPY, vol. 31, no. 6, 2023, pages 1507 - 8, XP093097368, DOI: 10.1016/j.ymthe.2023.03.015
MANDALPANKAJ KDERRICK J ROSSI: "Reprogramming Human Fibroblasts to Pluripotency Using Modified MRNA", NATURE PROTOCOLS, vol. 8, no. 3, 2013, pages 568 - 82
MANZANO, M.FORTE, E.RAJA, A.N.SCHIPMA, M.J.GOTTWEIN, E.: "Divergent target recognition by coexpressed 5'-isomiRs of miR-142-3p and selective viral mimicry", RNA, vol. 21, 2015, pages 1606 - 1620
MORIN, R.D.O'CONNOR, M.D.GRIFFITH, M.KUCHENBAUER, F.DELANEY, A.PRABHU, A.-L.ZHAO, Y.MCDONALD, H.ZENG, T.HIRST, M.: "Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells", GENOME RESEARCH, vol. 18, 2008, pages 610 - 621, XP055105830, DOI: 10.1101/gr.7179508
MUELLERPAULAMARKUS WOLFIENKATHARINA EKATCAJETAN IMMANUEL LANGDIRK KOCZANOLAF WOLKENHAUEROLGA HAHN ET AL.: "RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells", CELLS, vol. 9, no. 2, 2020, pages 504
MURTHYNIRENKUNWOO LEEPENGZHI YUNITHYA LINGAMPALLIHYUN JIN KIMRICHARD TANG.: "Peptide-Enhanced MRNA Transfection in Cultured Mouse Cardiac Fibroblasts and Direct Reprogramming towards Cardiomyocyte-like Cells", INTERNATIONAL JOURNAL OF NANOMEDICINE, March 1941 (1941-03-01)
O'BRIENJACOBHEYAM HAYDERYARA ZAYEDCHUN PENG: "Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation", FRONTIERS IN ENDOCRINOLOGY, vol. 9, 2018
PASCALEEMILIACARMEN CAIAZZAMARTINA PALADINOSILVIA PARISIFABIANA PASSAROMASSIMILIANO CAIAZZO: "MicroRNA Roles in Cell Reprogramming Mechanisms", CELLS, vol. 11, no. 6, 2022, pages 940
PATROROBGEET DUGGALMICHAEL I LOVERAFAEL A IRIZARRYCARL KINGSFORD.: "Salmon Provides Fast and Bias-Aware Quantification of Transcript Expression", NATURE METHODS, vol. 14, no. 4, 2017, pages 417 - 19
PEREIRA, C FB CHANGJ QIUX NIUD PAPATSENKOC E HENDRYN R CLARK ET AL.: "Induction of a Hemogenic Program in Mouse Fibroblasts", CELL STEM CELL, vol. 13, no. 2, 2013, pages 205 - 18, XP055329122, DOI: 10.1016/j.stem.2013.05.024
PEREIRA, C FI R LEMISCHKAK MOORE.: "Reprogramming Cell Fates: Insights from Combinatorial Approaches", ANN N YACAD SCI, vol. 1266, 2012, pages 7 - 17, XP071408578, DOI: 10.1111/j.1749-6632.2012.06508.x
PHAMPHUC VANNGOC BICH VUTHUY THI-THANH DAOHA THI-NGAN LELAN THI PHINGOC KIM PHAN: "Production of Endothelial Progenitor Cells from Skin Fibroblasts by Direct Reprogramming for Clinical Usages", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY - ANIMAL, vol. 53, no. 3, 2017, pages 207 - 16, XP036187524, DOI: 10.1007/s11626-016-0106-1
PIRESCRISTIANA FFÁBIO F ROSAILIA KUROCHKINCARLOS-FILIPE PEREIRA: "Understanding and Modulating Immunity With Cell Reprogramming", FRONTIERS IN IMMUNOLOGY, vol. 10, 2019, pages 2809, XP055776732, DOI: 10.3389/fimmu.2019.02809
POULIN, L FM SALIOE GRIESSINGERF ANJOS-AFONSOL CRACIUNJ L CHENA M KELLER ET AL.: "Characterization of Human DNGR-1+ BDCA3+ Leukocytes as Putative Equivalents of Mouse CD8alpha+ Dendritic Cells", J EXP MED, vol. 207, no. 6, 2010, pages 1261 - 71, XP055004992, DOI: 10.1084/jem.20092618
PRESKEYDAVIDTHOMAS F. ALLISONMARK JONESKAMEL MAMCHAOUICHRISTIAN UNGER.: "Synthetically Modified MRNA for Efficient and Fast Human IPS Cell Generation and Direct Transdifferentiation to Myoblasts", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 473, no. 3, 2016, pages 743 - 51, XP029528753, DOI: 10.1016/j.bbrc.2015.09.102
QUINLANAARON R.IRA M. HALL.: "BEDTools: A Flexible Suite of Utilities for Comparing Genomic Features", BIOINFORMATICS, vol. 26, no. 6, 2010, pages 841 - 42, XP055307411, DOI: 10.1093/bioinformatics/btq033
ROSA FÁBIO ET AL: "Direct Reprogramming of Mouse Embryonic Fibroblasts to Conventional Type 1 Dendritic Cells by Enforced Expression of Transcription Factors", vol. 10, no. 10, 1 January 2020 (2020-01-01), Sunnyvale, CA, USA, pages 1 - 19, XP055776737, ISSN: 2331-8325, Retrieved from the Internet <URL:http://dx.doi.org/10.21769/BioProtoc.3619> DOI: 10.21769/BioProtoc.3619 *
ROSA FÁBIO F. ET AL: "Direct reprogramming of fibroblasts into antigen-presenting dendritic cells", SCIENCE IMMUNOLOGY, vol. 3, no. 30, 14 December 2018 (2018-12-14), US, XP093110270, ISSN: 2470-9468, DOI: 10.1126/sciimmunol.aau4292 *
ROSAFÁBIO F.CRISTIANA F. PIRESILIA KUROCHKINEVELYN HALITZKITASNIM ZAHANNEJC ARHOLGA ZIMMERMANNOVA ET AL.: "Single-Cell Transcriptional Profiling Informs Efficient Reprogramming of Human Somatic Cells to Cross-Presenting Dendritic Cells", SCIENCE IMMUNOLOGY, vol. 7, no. 69, 2022, XP093230330, DOI: 10.1126/sciimmunol.abg5539
ROSAFÁBIO FCRISTIANA F PIRESILIA KUROCHKINANDREIA M GOMESALEXANDRA G FERREIRALUÍS G PALMAKRITIKA SHAIV ET AL.: "Direct Reprogramming of Fibroblasts into Antigen-Presenting Dendritic Cells", SCIENCE IMMUNOLOGY, vol. 3, no. 30, 2018
SALMONHELENEJULIANA IDOYAGAADEEB RAHMANMARYLÈNE LEBOEUFROMAIN REMARKSTEFAN JORDANMARIA CASANOVA-ACEBES ET AL.: "Expansion and Activation of CD103+ Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-L1 and BRAF Inhibition", IMMUNITY, vol. 44, no. 4, 2016, pages 924 - 38, XP029521260, DOI: 10.1016/j.immuni.2016.03.012
SCALAVINO VIVIANA ET AL: "Role of microRNAs in the Regulation of Dendritic Cell Generation and Function", vol. 21, no. 4, 15 February 2020 (2020-02-15), Basel, CH, pages 1319, XP093151163, ISSN: 1422-0067, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072926/pdf/ijms-21-01319.pdf> [retrieved on 20240416], DOI: 10.3390/ijms21041319 *
SCHLIMGENRYANJOHN HOWARDDAWN WOOLEYMAUREEN THOMPSONLINDSEY R. BADENOTTO O. YANGDAVID C. CHRISTIANI ET AL.: "Risks Associated With Lentiviral Vector Exposures and Prevention Strategies", JOURNAL OF OCCUPATIONAL & ENVIRONMENTAL MEDICINE, vol. 58, no. 12, 2016, pages 1159 - 66, Retrieved from the Internet <URL:https://doi.org/10.1097/JOM.0000000000000879>
SONTAG, SM FORSTERJ QINP WANEKS MITZKAH M SCHÜLERS KOSCHMIEDERS ROSE-JOHNK SERÉM ZENKE: "Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered IPS Cells", STEM CELLS, vol. 35, no. 4, 2017, pages 898 - 908, XP055876479, DOI: 10.1002/stem.2565
SPRANGERSTEFANIDAISY DAIBRENDAN HORTONTHOMAS F GAJEWSKI: "Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy", CANCER CELL, vol. 31, no. 5, 2017, pages 711 - 723, XP085004704, DOI: 10.1016/j.ccell.2017.04.003
SUN, Y.ORAVECZ-WILSON, K.MATHEWSON, N.WANG, Y.MCEACHIN, R.LIU, C.TOUBAI, T.WU, J.ROSSI, C.BRAUN, T.: "Mature T cell responses are controlled by microRNA-142", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 125, 2015, pages 2825 - 2840, XP055906116, DOI: 10.1172/JCI78753
SZKLARCZYK, D.KIRSCH, R.KOUTROULI, M.NASTOU, K.MEHRYARY, F.HACHILIF, R.GABLE, A.L.FANG, T.DONCHEVA, N.T.PYYSALO, S. ET AL.: "The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest", NUCLEIC ACIDS RESEARCH, vol. 51, 2023, pages 638 - 646
TAKAHASHIKAZUTOSHIKOJI TANABEMARI OHNUKIMEGUMI NARITATOMOKO ICHISAKAKIICHIRO TOMODASHINYA YAMANAKA: "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors", CELL, vol. 131, no. 5, 2007, pages 861 - 72
TAKAHASHIKAZUTOSHISHINYA YAMANAKA: "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors", CELL, vol. 126, no. 4, 2006, pages 663 - 76
WAGNER, V.MEESE, E.KELLER, A.: "The intricacies of isomiRs: from classification to clinical relevance", TRENDS IN GENETICS.
WANG, HY YANGJ LIUL QIAN.: "Direct Cell Reprogramming: Approaches, Mechanisms and Progress", NAT REV MOL CELL BIOL, no. 22, 2021, pages 410 - 24, XP037464714, DOI: 10.1038/s41580-021-00335-z
WARRENLUIGIPHILIP D. MANOSTIM AHFELDTYUIN-HAN LOHHU LIFRANK LAUWATARU EBINA ET AL.: "Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified MRNA", CELL STEM CELL, vol. 7, no. 5, 2010, pages 618 - 30
WCULEK, S KF J CUETOA M MUJALI MELEROM F KRUMMELD SANCHO.: "Dendritic Cells in Cancer Immunology and Immunotherapy", NAT REV IMMUNOL, vol. 20, 2019, pages 7 - 24, XP036976389, DOI: 10.1038/s41577-019-0210-z
XIEHUAFENGMIN YERU FENGTHOMAS GRAF.: "Stepwise Reprogramming of B Cells into Macrophages", CELL, vol. 117, no. 5, 2004, pages 663 - 76, XP055728800, DOI: 10.1016/S0092-8674(04)00419-2
YAPING SUN: "miR-142 controls metabolic reprogramming that regulates dendritic cell activation", THE JOURNAL OF CLINICAL INVESTIGATION, 8 April 2019 (2019-04-08), pages 1 - 14, XP093153294, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486330/pdf/jci-129-123839.pdf> DOI: 10.1172/JCI123839 *
YILUN WANG ET AL: "Elevated expression of miR-142-3p is related to the pro-inflammatory function of monocyte-derived dendritic cells in SLE", ARTHRITIS RESEARCH & THERAPY, vol. 18, no. 1, 16 November 2016 (2016-11-16), XP055672181, DOI: 10.1186/s13075-016-1158-z *
YOOANDREW S.ALFRED X. SUNLI LIALEKSANDR SHCHEGLOVITOVTHOMAS PORTMANNYULONG LICHRIS LEE-MESSERRICARDO E. DOLMETSCHRICHARD W. TSIEN: "MicroRNA-Mediated Conversion of Human Fibroblasts to Neurons", NATURE, vol. 476, no. 7359, 2011, pages 228 - 31, XP002702842, DOI: 10.1038/nature10323
YOSHIOKANAOHISAEDWIGE GROSHAI-RI LISHANTANU KUMARDEKKER C. DEACONCORNELIA MARONALYSSON R. MUOTRI ET AL.: "Efficient Generation of Human IPSCs by a Synthetic Self-Replicative RNA", CELL STEM CELL, vol. 13, no. 2, 2013, pages 246 - 54
YOSHIOKANAOHISASTEVEN F. DOWDY.: "Enhanced Generation of IPSCs from Older Adult Human Cells by a Synthetic Five-Factor Self-Replicative RNA", PLOS ONE, vol. 12, no. 7, 2017, pages 0182018
YUGUANGCHUANGLI-GEN WANGQING-YU HE: "ChIPseeker: An R/Bioconductor Package for ChIP Peak Annotation, Comparison and Visualization", BIOINFORMATICS, vol. 31, no. 14, 2015, pages 2382 - 83
ZHEN QIN ET AL: "miRNA-124 in Immune System and Immune Disorders", FRONTIERS IN IMMUNOLOGY, vol. 7, 4 October 2016 (2016-10-04), XP055566135, DOI: 10.3389/fimmu.2016.00406 *
ZIMMERMANNOVAOLGAALEXANDRA G. FERREIRAERVIN ASCICMARTA VELASCO SANTIAGOILIA KUROCHKINMORTEN HANSENOZCAN MET ET AL.: "Restoring Tumor Immunogenicity with Dendritic Cell Reprogramming", SCIENCE IMMUNOLOGY, vol. 8, no. 85, 2023

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