WO2023237754A2 - Cyclosporine h et ses utilisations - Google Patents

Cyclosporine h et ses utilisations Download PDF

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WO2023237754A2
WO2023237754A2 PCT/EP2023/065527 EP2023065527W WO2023237754A2 WO 2023237754 A2 WO2023237754 A2 WO 2023237754A2 EP 2023065527 W EP2023065527 W EP 2023065527W WO 2023237754 A2 WO2023237754 A2 WO 2023237754A2
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cell
cells
csh
hspc
vector
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WO2023237754A3 (fr
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Anna Christina KAJASTE-RUDNITSKI
Ilaria CASTIGLIONI
Giulia UNALI
Francesco PIRAS
Nadia COLTELLA
Federico ROSSARI
Luigi Maria NALDINI
Georgia FOUSTERI
Adriana STUCCHI
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Fondazione Telethon
Ospedale San Raffaele SRL
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Ospedale San Raffaele SRL
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    • C12N2501/20—Cytokines; Chemokines
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Definitions

  • the present invention relates to the use of cyclosporin H (CsH) in cell and gene therapy, in particular for reducing or preventing T cell exhaustion and/or loss of T cell effector functions; increasing T cell engraftment and/or persistence; and increasing survival of and/or engraftment by haematopoietic stem and/or progenitor cells (HSPCs).
  • CsH cyclosporin H
  • haematopoietic system is a complex hierarchy of cells of different mature cell lineages. These include cells of the immune system that offer protection from pathogens, cells that carry oxygen through the body and cells involved in wound healing. All these mature cells are derived from a pool of haematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation into any blood cell lineage. HSCs have the ability to replenish the entire haematopoietic system.
  • HSCs haematopoietic stem cells
  • HCT Haematopoietic cell transplantation
  • GvHD graft- versus-host disease
  • Gene therapy approaches based on the transplantation of genetically modified autologous HSCs offer potentially improved safety and efficacy over allogeneic HCT. They are particularly relevant for patients lacking a matched donor.
  • stem cell gene therapy is based on the genetic modification of a relatively small number of stem cells. These persist long-term in the body by undergoing self-renewal, and generate large numbers of genetically “corrected” progeny. This ensures a continuous supply of corrected cells for the rest of the patient’s lifetime.
  • HSCs are particularly attractive targets for gene therapy since their genetic modification will be passed to all the blood cell lineages as they differentiate. Furthermore, HSCs can be easily and safely obtained, for example from bone marrow, mobilised peripheral blood and umbilical cord blood.
  • HSCs and their progeny require a technology which permits stable integration of the corrective DNA into the genome, without affecting HSC function. Accordingly, the use of integrating recombinant viral systems such as y-retroviruses, lentiviruses and spumaviruses has dominated this field (Chang, A.H. et al. (2007) Mol. Ther. 15: 445-456). Therapeutic benefits have already been achieved in y-retrovirus-based clinical trials for Adenosine Deaminase Severe Combined Immunodeficiency (ADA-SCID; Aiuti, A. et al. (2009) N. Engl. J. Med.
  • ADA-SCID Adenosine Deaminase Severe Combined Immunodeficiency
  • lentiviruses have been employed as delivery vehicles in the treatment of X-linked adrenoleukodystrophy (ALD; Cartier, N. et al. (2009) Science 326: 818-823), and very recently for metachromatic leukodystrophy (MLD; Biffi, A. et al. (2013) Science 341 : 1233158) and WAS (Aiuti, A. et al. (2013) Science 341 : 1233151).
  • ALD X-linked adrenoleukodystrophy
  • MLD metachromatic leukodystrophy
  • lentiviruses are among the best available platforms for cell transduction, difficulties remain with the methods employed for the genetic modification of cells, in particular haematopoietic stem and progenitor cells. For example, for gene therapy to be efficacious, effective gene transfer into target cells must be reached without inducing detrimental effects on their biological properties.
  • CAR T cell therapy has emerged as a potential effective strategy for the treatment of both solid tumor and hematological malignancies (Marofi et al, 2021 ; Zhang et al, 2020).
  • TAE tumor microenvironment
  • ROS reactive oxygen species
  • CAR T cell exhaustion is considered one of the major limitations to their efficacy, and it is associated with poor responses in cancer patients receiving immunotherapy (Delgoffe et al, 2021). Moreover, the resting T stem memory compartment that would guarantee long-lived efficacy of the treatment remains difficult to target during CAR T cell generation. Therefore, the development of strategies aimed at mitigating exhaustion, thus maintaining CAR T effector functions and persistence, as well as improving lentiviral transduction efficacy across T cell subsets is important for improving the efficacy and clinical outcomes of CAR T cell therapies.
  • CsH provides advantages by improving the quality of cell products due to its capacity to transiently dampen cellular metabolism.
  • HSC exposed to CsH had upregulation of pathways that are mainly involved in metabolic processes such as Kreb-cycle and fatty acid oxidation.
  • genes related to lipid metabolism and T cell receptor signaling pathway were downregulated in cells exposed to CsH as compared to controls. Accordingly, mass spectrometry metabolomics revealed a significant CsH-induced increase in acylcarnitines and free fatty acids at steady state in HSC, thus confirming a potential effect of CsH in inducing relevant metabolic alterations.
  • Oxidative stress is proposed to play a major role in impairing HSPC function in specific disease contexts such as Fanconi Anemia (FA).
  • FA Fanconi Anemia
  • the inventors have also found that, several pathways related to oxidative stress and metabolic pathways were significantly enriched in HSPC from FA patients. Interestingly and in line with its metabolic effects, exposure to CsH mitigated upregulation of these pathways. Thus, CsH may be used to preserve FA HSPC during their ex vivo culture in the context of corrective gene therapy applications.
  • T cells transduced in the presence of CsH resulted in lower percentages of exhausted cells in culture as compared to controls. This effect was confirmed also in the context of murine T cells. Growing evidence indicates that exhausted T cells undergo metabolic alterations, leading to poor responsiveness to immune-checkpoint-blockade and lower efficacy of CAR T cell therapies.
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for: (a) reducing or preventing T cell exhaustion and/or loss of T cell effector functions; and/or (b) increasing T cell engraftment and/or persistence.
  • CsH cyclosporin H
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for reducing or preventing T cell exhaustion.
  • CsH cyclosporin H
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for reducing or preventing loss of T cell effector functions. In another aspect, the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing T cell engraftment.
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing T cell persistence.
  • CsH cyclosporin H
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing regulatory T (Treg) cell transduction efficiency.
  • CsH cyclosporin H
  • Teg regulatory T
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for improving the immunomodulatory profile of a regulatory T (Treg) cell, for example a Treg cell transduced or transfected with a vector.
  • Improved immunomodulatory profile may, for example, comprise increased immunoregulatory cytokine (e.g. IFNy and/or IL-10, preferably IFNy and IL-10) production by the Treg cell, and/or decreased proinflam matory cytokine (e.g. TNF) production by the Treg cell.
  • the invention provides a T cell for use in a method of therapy, wherein the method comprises contacting the T cell with cyclosporin H (CsH) or a derivative thereof.
  • CsH cyclosporin H
  • T cell exhaustion and/or loss of T cell effector functions is reduced or prevented.
  • T cell engraftment and/or persistence is increased.
  • the therapy is treatment or prevention of cancer. In one embodiment, the therapy is treatment or prevention of infection.
  • the T cell is transduced or transfected with a vector
  • the T cell is transduced with a viral vector.
  • the vector is a retroviral vector or a lentiviral vector.
  • the vector comprises one or more nucleotide sequence encoding a chimeric antigen receptor (CAR)
  • the vector comprises one or more nucleotide sequence encoding a T cell receptor (TCR).
  • TCR T cell receptor
  • the T cell is a chimeric antigen receptor (CAR) T cell.
  • CAR chimeric antigen receptor
  • the T cell comprises an exogenous T cell receptor (TCR).
  • TCR exogenous T cell receptor
  • the T cell is a CD4+ T cell.
  • the T cell is a CD8+ T cell.
  • the T cell is a regulatory T (Treg) cell. In one embodiment, the T cell is a CD4+CD25+ Treg cell. In one embodiment, the T cell is a CD4+CD25+CD127lo Treg cell. In one embodiment, the T cell is a CD4+CD25+FOXP3+ Treg cell.
  • Treg regulatory T
  • the T cell is a stimulated T cell.
  • the T cell is a primary T cell.
  • the T cell is a T stem memory cell.
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for:
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for reducing or preventing NK cell exhaustion. In another aspect, the invention provides use of cyclosporin H (CsH) or a derivative thereof for reducing or preventing loss of NK cell effector functions. In another aspect, the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing NK cell engraftment. In another aspect, the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing NK cell persistence.
  • the invention provides a NK cell for use in a method of therapy, wherein the method comprises contacting the NK cell with cyclosporin H (CsH) or a derivative thereof.
  • CsH cyclosporin H
  • NK cell exhaustion and/or loss of NK cell effector functions is reduced or prevented.
  • NK cell engraftment and/or persistence is increased.
  • the therapy is treatment or prevention of cancer. In one embodiment, the therapy is treatment or prevention of infection.
  • the NK cell is transduced or transfected with a vector
  • the NK cell is transduced with a viral vector.
  • the vector is a retroviral vector or a lentiviral vector.
  • the vector comprises one or more nucleotide sequence encoding a chimeric antigen receptor (CAR)
  • the NK cell is a chimeric antigen receptor (CAR) NK cell.
  • CAR chimeric antigen receptor
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing survival of and/or engraftment by haematopoietic stem and/or progenitor cells (HSPCs).
  • CsH cyclosporin H
  • HSPCs haematopoietic stem and/or progenitor cells
  • the invention provides a haematopoietic stem and/or progenitor cell (HSPC) for use in a method of treating Fanconi Anemia, wherein the method comprises contacting the HSPC with cyclosporin H (CsH) or a derivative thereof.
  • HSPC haematopoietic stem and/or progenitor cell
  • the HSPC is transduced or transfected with a vector.
  • the HSPC is transduced with a viral vector.
  • the vector is a retroviral vector or a lentiviral vector.
  • the CsH or derivative thereof is at a concentration of about 1-50 pM. In another embodiment, the CsH or derivative thereof is at a concentration of about 5-50 pM.
  • the CsH or derivative thereof is at a concentration of about 1-40 or 5- 40 pM. In another embodiment, the CsH or derivative thereof is at a concentration of about 1- 30 or 5-30 pM. In another embodiment, the CsH or derivative thereof is at a concentration of about 1-20 or 5-20 pM. In another embodiment, the CsH or derivative thereof is at a concentration of about 1-15 or 5-15 pM.
  • the CsH or derivative thereof is at a concentration of about 1-15, 2- 14, 3-13, 4-12, 5-11 , 6-10 or 7-9 pM.
  • the concentration of CsH may be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 pM.
  • the concentration of CsH or a derivative thereof is about 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 pM.
  • the concentration of CsH or a derivative thereof is about 8 pM.
  • the T cell or HSPC is contacted with the CsH or derivative thereof at the beginning of the cell culture. In one embodiment, the T cell or HSPC is contacted with the CsH or derivative thereof before transduction or transfection with a vector.
  • the T cell or HSPC is contacted twice with the CsH or derivative thereof.
  • the T cell or HSPC is: (a) contacted with the CsH or derivative thereof at the same time as transduction or transfection with a vector; and/or
  • the T cell or HSPC is cultured for 16 days or less before administration to a subject. In one embodiment, the T cell or HSPC is cultured for 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6 or 5 days or less before administration to a subject.
  • the invention provides a method of cell therapy comprising the steps of:
  • the method further comprises the step of transducing or transfecting the cell with a vector.
  • the invention provides a method of gene therapy comprising the steps of:
  • the cell is a T cell. In one embodiment, the cell is a haematopoietic stem and/or progenitor cell (HSPC).
  • HSPC haematopoietic stem and/or progenitor cell
  • the T cell is a chimeric antigen receptor (CAR) T cell.
  • the T cell comprises an exogenous T cell receptor (TCR).
  • the T cell is a CD4+ T cell. In one embodiment, the T cell is a CD8+ T cell.
  • the T cell is a regulatory T (Treg) cell. In one embodiment, the T cell is a CD4+CD25+ Treg cell. In one embodiment, the T cell is a CD4+CD25+CD127lo Treg cell. In one embodiment, the T cell is a CD4+CD25+FOXP3+ Treg cell.
  • Treg regulatory T
  • the cell is a T cell and wherein: (a) T cell exhaustion and/or loss of T cell effector functions is reduced or prevented; and/or
  • T cell engraftment and/or persistence is increased.
  • the cell is a HSPC and wherein HSPC survival and/or engraftment is increased.
  • the invention provides a method of transducing a population of T cells, preferably regulatory T (Treg) cells, comprising the steps of:
  • the efficiency of transduction may be increased, for example compared to a method of transduction lacking the contacting of the population of T cells with CsH or a derivative thereof.
  • FIG. 1 Impact of CsH on HSPC global proteome.
  • A. Global proteomic profile of cord blood- derived CD34+ cells from three different healthy donors, upon CsH exposure.
  • FIG. 2 CsH-induced increase in acylcarnitines and free fatty acids at steady state in HSC.
  • A General scheme of the transport of free fatty acids from the cytosol to the mitochondrial matrix. Carnitine palmityl transferase (CPT1), Carnitineacylcarnitine translocase (CACT)
  • CPT1 Carnitine palmityl transferase
  • CACT Carnitineacylcarnitine translocase
  • B Box plots of the single acylcarnitines and free fatty acids, expressed as normalized levels (normalized to the DMSO groups and autoscaled in MetaboAnalyst v5.0) shown as floating bars (min to max with line at mean).
  • FIG. 3 Metabolic alterations upon CsH exposure in primary HSPC.
  • C LIMAP plots of Single-cell RNASeq analysis comparing CD34+ HSPC from healthy donor (HD) and patients affected by Fanconi Anemia (FA).
  • D Heat map of pathways significantly enriched in FA vs HD HSPC.
  • Figure 4 - CsH does not alter HSPC composition or proliferation ex vivo.
  • MFI mean fluorescence intensity
  • FIG. 5 Metabolic alterations upon CsH exposure in primary T cells.
  • C Gene expression analysis of the most significant metabolic genes downregulated upon CsH exposure, expressed in fold change vs control (dmso), normalized to 18S ribosomal RNA.
  • FIG. 6 CAR-T cell manufacturing in the presence of CsH partially prevents exhaustion.
  • Exhausted T cells are identified as LAG3+PD1+CTLA4+ or LAG3+PD1+TIM3+ cells in human (A) and murine (B) primary T cells, acquired from flow cytometry analysis, 4 days after transduction, with or w/o CsH.
  • C Transduction efficiency, expressed as percentage of NGFR+ cells, in T cell stem memory (TSCM) cells, within CD3+ CD4+ and CD8+ populations, transduced with or w/o CsH.
  • TSCM cells are identified as CD62L+CD45RA+ cells.
  • D. Proliferation analysis are expressed as mean fluorescence intensity (MFI) of eFluor 670 cell labelling dye, at the indicated time points.
  • MFI mean fluorescence intensity
  • FIG. 7 - CsH enhances Treg transduction efficiency. Transduction efficiency of CD19+ CAR Treg cells from 4 independent experiments and 4 HD assessed with FC at day 15 post transduction (LV MOI:10).
  • (a) Representative FC plots show CAR expression (%NGFR+) after gating on CD25+CD127lo cells,
  • Figure 8 - CsH enhances PD-1 expression levels of CD19 CAR Tregs.
  • (a) Graphs shows the expansion curve from day 0 to day 14 of both untransduced cells and CAR-T regs transduced with or without CsH at different MOI (2-5-10).
  • (b) Data display % CD25+ CD127lo cells gating on CD4+; the % and gMFI of FoxP3+, the % of CTLA-4+ and % with gMFI of PD- 1 cells gating on CD25+ CD127lo Treg cells. Results in (b) show data pooled from 4 or 2 HD after transduction with CD19 CAR LV at MOI 10.
  • Figure 9 - CsH increases both IL-10 and IFNy production while reducing TNF expression in CAR Tregs.
  • FIG 10 The effect of CsH on Tregs’ suppressive function.
  • CsH-treated vs DMSO- treated CD 19 CAR Tregs were co-cultured with heterologous PBMC, previously stained with CSFE, in the presence of anti-CD3/CD28-coated beads at various PBMC:Treg ratios.
  • Teff gated on CD8+ proliferation was assessed by FC.
  • FIG 11 Effect of CsH on counteracting the generation of exhausted CAR T cells in vitro.
  • Cyclosporin H (CsH, CAS No. 83602-39-5) is a cyclic undecapeptide having the following structure: CsH is known to selectively antagonise the formyl peptide receptor, however unlike cyclosporin A (CsA), CsH does not bind cyclophilin to evoke immunosuppression. CsA mediates immunosuppression as a complex with the host peptidyl-prolyl isomerase cyclophilin A (CypA). This inhibits the Ca 2+ -dependent phosphatase calcineurin and consequent activation of pro-inflammatory cytokines such as IL-2 (Sokolskaja, E. et al. (2006) Curr. Opin. Microbiol. 9: 404-8).
  • Solutions of CsH for use in the present invention may be prepared using routine methods known in the art.
  • the concentration at which CsH or a derivative thereof is applied to a population of cells may be adjusted for different vector systems to optimise the effect disclosed herein.
  • a skilled person may therefore select a suitable concentration of CsH or a derivative thereof to maximise the effect disclosed herein while minimising any toxicity.
  • the present invention encompasses the use of CsH and derivatives of CsH.
  • the CsH derivatives of the present invention are those which reduce or prevent T cell exhaustion, and/or increase T cell engraftment and/or persistence; or increase survival of and/or engraftment by haematopoietic stem and/or progenitor cells (HSPCs).
  • HSPCs haematopoietic stem and/or progenitor cells
  • CsH derivatives of the present invention may have been developed for increased solubility, increased stability and/or reduced toxicity.
  • CsH derivatives of the invention are preferably of low toxicity for mammals, in particular humans.
  • CsH derivatives of the invention are of low toxicity for haematopoietic stem and/or progenitor cells; and/or T cells.
  • ACT adoptive T cell therapy
  • TCR tumor-specific T-cell Receptor
  • CAR chimeric antigen receptor
  • exhaustion a dysfunctional state characterized by a loss of effector functions.
  • a hallmark of exhaustion is the upregulation of a panel of inhibitory receptors (I Rs) including PD-1 , CTLA-4, LAG-3, Tim-3 and 2B4.
  • I Rs inhibitory receptors
  • exhaustion may refer to increased expressed of one or more of PD-1 , CTLA-4, LAG-3, Tim-3 and 2B4 (for example LAG-3, PD-1 and CTLA-4; or LAG-3, PD-1 and Tim-3).
  • the uses and methods of the invention reduce the number of LAG-3+ PD-1+CTLA-4+ cells in a population of T cells. In some embodiments, the uses and methods of the invention reduce the number of LAG-3+PD-1+Tim-3+ cells in a population of T cells. The reduction may be, for example, a reduction of at least 1 %, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.
  • I Rs Inhibitory receptors
  • T cells are a variety of molecules expressed by T cells, involved in the regulation of motility, cytokine production and effector functions, thus orchestrating peripheral self-tolerance maintenance and acting as breaks for T cell responses.
  • Their ligands are widely upregulated on a broad spectrum of tumor cells and other cells of the tumor microenvironment, thus the interaction between ligands and inhibitory receptors dampens anti-tumor immunity and results in T cell exhaustion and tumor escape.
  • I Rs such as PD-1 , LAG-3 or Tim-3, in the absence of activation markers, on antigen-specific T cells represents a hallmark of T cell exhaustion.
  • CTLA-4 knockout mice develop lymphoproliferative disorders, that cause extensive tissue damage and death in the first month of age.
  • PD-1 deficient mice develop autoimmune diseases.
  • Phenotypic characterization of human T cells from healthy subjects point to a dynamic and variegate expression of the IR profile, that varies according to T cell subset, differentiation and activation.
  • I Rs can be also expressed by early differentiated TSCM, as shown in subjects vaccinated against yellow fever.
  • the term “survival” as used herein refers to the ability of the cells, for example haematopoietic stem and/or progenitor cells to remain alive (e.g. not die or become apoptotic) during in vitro or ex vivo culture.
  • Cells for example haematopoietic stem and/or progenitor cells may undergo, for example, increased apoptosis following transduction with a vector during cell culture; thus, the surviving cells may have avoided apoptosis and/or cell death. Cell survival may be readily analysed by the skilled person.
  • the numbers of live, dead and/or apoptotic cells in a cell culture may be quantified at the beginning of culture and/or following culture for a period of time (e.g. about 6 or 12 hours, or 1 , 2, 3, 4, 5, 6, 7 or more days; preferably, the period of time begins with the transduction of the cells with a vector).
  • the effect of an agent on cell survival may be assessed by comparing the numbers and/or percentages of live, dead and/or apoptotic cells at the beginning and/or end of the culture period between experiments carried out in the presence and absence of the agent, but under otherwise substantially identical conditions.
  • Cell numbers and/or percentages in certain states may be quantified using any of a number of methods known in the art, including use of haemocytometers, automated cell counters, flow cytometers and fluorescence activated cell sorting machines. These techniques may enable distinguishing between live, dead and/or apoptotic cells.
  • apoptotic cells may be detected using readily available apoptosis assays (e.g.
  • phosphatidylserine PS
  • Annexin V which binds to exposed PS
  • apoptotic cells may be quantified through use of fluorescently-labelled Annexin V), which may be used to complement other techniques.
  • engraftment refers to the ability of the cells to populate and/or survive in a subject following their transplantation, i.e. in the short and/or long term after transplantation.
  • engraftment may refer to the number and/or percentages of haematopoietic cells descended from the transplanted haematopoietic stem and/or progenitor cells (e.g. graft-derived cells) that are detected about 1 day to 24 weeks, 1 day to 10 weeks, or 1-30 days or 10-30 days after transplantation.
  • engraftment may be evaluated in the peripheral blood as the percentage of cells deriving from the human xenograft (e.g. positive for the CD45 surface marker), for example.
  • engraftment is assessed at about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 days after transplantation.
  • engraftment is assessed at about 4, 5, 6, 7, 8, 9, 10 ,11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 weeks after transplantation.
  • engraftment is assessed at about 16-24 weeks, preferably 20 weeks, after transplantation.
  • Engraftment may be readily analysed by the skilled person.
  • the transplanted cells may be engineered to comprise a marker (e.g. a reporter protein, such as a fluorescent protein), which can be used to quantify the graft-derived cells.
  • a marker e.g. a reporter protein, such as a fluorescent protein
  • Samples for analysis may be extracted from relevant tissues and analysed ex vivo (e.g. using flow cytometry). Transduction efficiency
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for increasing regulatory T (Treg) cell transduction efficiency.
  • CsH cyclosporin H
  • Teg regulatory T
  • Increasing the efficiency of transduction refers to an increase in the transduction of the cells (e.g. Treg cells) contacted with an agent (e.g. CsH or a derivative thereof), in comparison to the transduction achieved in the absence of the agent but under otherwise substantially identical conditions.
  • An increased efficiency of transduction may therefore allow the multiplicity of infection (MOI) and/or the transduction time required to achieve effective transduction to be reduced.
  • MOI multiplicity of infection
  • the percentage of cells transduced by the vector is increased (e.g. by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%).
  • the vector copy number per cell is increased (e.g. by at least 1-fold, 2-fold, 3- fold, 4-fold, 5-fold or 10-fold). Preferably both are achieved at the same time.
  • Methods for determining the percentage of cells transduced by a vector are known in the art. Suitable methods include flow cytometry, fluorescence-activated cell sorting (FACS) and fluorescence microscopy. The technique employed is preferably one which is amenable to automation and/or high throughput screening.
  • a population of cells may be transduced with a vector which harbours a reporter gene.
  • the vector may be constructed such that the reporter gene is expressed when the vector transduces a cell.
  • Suitable reporter genes include genes encoding fluorescent proteins, for example green, yellow, cherry, cyan or orange fluorescent proteins.
  • qPCR quantitative PCR
  • Methods for determining vector copy number are also known in the art.
  • the technique employed is preferably one which is amenable to automation and/or high throughput screening. Suitable techniques include quantitative PCR (qPCR) and Southern blot-based approaches. Immunomodulatory profile
  • the invention provides use of cyclosporin H (CsH) or a derivative thereof for improving the immunomodulatory profile of a regulatory T (Treg) cell, for example a Treg cell transduced or transfected with a vector.
  • CsH cyclosporin H
  • Treg regulatory T
  • Improving the immunomodulatory profile may refer to an improvement in the immunomodulatory profile of the cells contacted with an agent (e.g. CsH or a derivative thereof) in comparison to cells not contacted with the agent but under otherwise substantially identical conditions.
  • an agent e.g. CsH or a derivative thereof
  • Improved immunomodulatory profile may, for example, comprise increased immunoregulatory cytokine (e.g. IFNy and/or IL-10, preferably IFNy and IL-10) production by the Treg cell.
  • the immunoregulatory cytokine production may be increased by, for example, at least 1-fold, 2- fold, 3-fold, 4-fold, 5-fold or 10-fold.
  • Improved immunomodulatory profile may, for example, comprise decreased proinflammatory cytokine (e.g. TNF) production by the Treg cell.
  • the proinflammatory cytokine production may be decreased by, for example, at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold or 10-fold.
  • Improved immunomodulatory profile may, for example, comprise both increased immunoregulatory cytokine production and decreased proinflammatory cytokine production by the Treg cell.
  • cell is a T cell.
  • T cells are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TOR) on the cell surface.
  • B cells B cells and natural killer cells (NK cells)
  • TOR T-cell receptor
  • the T cells are resting T cells. Resting CD4+ T cells are quiescent. In one embodiment, the T cells are unstimulated T cells. In one embodiment, the T cells are stimulated T cells. Once stimulated, resting T cells proliferate and generate a large clone of antigen-specific cells. In one embodiment, the T cells are CD4+ T cells. In one embodiment, the T cells are CD8+ T cells. In one embodiment, the T cells are CD3+ T cells. In one embodiment, the T cells are Stem memory T cells; Central Memory T cells; Effector Memory T cells; and/or terminally differentiated effector memory T cells.
  • the T cell is a regulatory T (Treg) cell.
  • Treg cells are a subpopulation of T cells that modulate the immune system and maintain tolerance to self-antigens.
  • Treg cells are immunosuppressive and may suppress induction and proliferation of effector T cells.
  • the T cell is a CD4+CD25+ Treg cell. In one embodiment, the T cell is a CD4+CD25+CD127lo Treg cell. In one embodiment, the T cell is a CD4+CD25+FOXP3+ Treg cell.
  • a stem cell is able to differentiate into many cell types.
  • a cell that is able to differentiate into all cell types is known as totipotent. In mammals, only the zygote and early embryonic cells are totipotent. Stem cells are found in most, if not all, multicellular organisms. They are characterised by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialised cell types.
  • the two broad types of mammalian stem cells are embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialised embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialised cells, but also maintaining the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.
  • HSCs Haematopoietic stem cells
  • HSCs are multipotent stem cells that may be found, for example, in peripheral blood, bone marrow and umbilical cord blood. HSCs are capable of self-renewal and differentiation into any blood cell lineage. They are capable of recolonising the entire immune system, and the erythroid and myeloid lineages in all the haematopoietic tissues (such as bone marrow, spleen and thymus). They provide for life-long production of all lineages of haematopoietic cells.
  • Haematopoietic progenitor cells have the capacity to differentiate into a specific type of cell. In contrast to stem cells however, they are already far more specific: they are pushed to differentiate into their “target” cell. A difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times. Haematopoietic progenitor cells can be rigorously distinguished from HSCs only by functional in vivo assay (i.e. transplantation and demonstration of whether they can give rise to all blood lineages over prolonged time periods).
  • the haematopoietic stem and progenitor cells of the invention comprise the CD34 cell surface marker (denoted as CD34+).
  • HSPC Haematopoietic stem and progenitor cell
  • a population of haematopoietic stem and/or progenitor cells may be obtained from a tissue sample.
  • a population of haematopoietic stem and/or progenitor cells may be obtained from peripheral blood (e.g. adult and foetal peripheral blood), umbilical cord blood, bone marrow, liver or spleen.
  • peripheral blood e.g. adult and foetal peripheral blood
  • umbilical cord blood e.g. umbilical cord blood
  • bone marrow e.g., hematomatopoietic stem and/or progenitor cells
  • liver or spleen e.g., liver or spleen.
  • these cells are obtained from peripheral blood or bone marrow. They may be obtained after mobilisation of the cells in vivo by means of growth factor treatment.
  • Mobilisation may be carried out using, for example, G-CSF, plerixaphor or combinations thereof.
  • Other agents such as NSAIDs and dipeptidyl peptidase inhibitors, may also be useful as mobilising agents.
  • stem cell growth factors GM-CSF and G-CSF are now performed using stem cells collected from the peripheral blood, rather than from the bone marrow. Collecting peripheral blood stem cells provides a bigger graft, does not require that the donor be subjected to general anaesthesia to collect the graft, results in a shorter time to engraftment and may provide for a lower longterm relapse rate.
  • Bone marrow may be collected by standard aspiration methods (either steady-state or after mobilisation), or by using next-generation harvesting tools (e.g. Marrow Miner).
  • haematopoietic stem and progenitor cells may also be derived from induced pluripotent stem cells.
  • HSCs are typically of low forward scatter and side scatter profile by flow cytometric procedures. Some are metabolically quiescent, as demonstrated by Rhodamine labelling which allows determination of mitochondrial activity. HSCs may comprise certain cell surface markers such as CD34, CD45, CD133, CD90 and CD49f. They may also be defined as cells lacking the expression of the CD38 and CD45RA cell surface markers. However, expression of some of these markers is dependent upon the developmental stage and tissue-specific context of the HSC. Some HSCs called “side population cells” exclude the Hoechst 33342 dye as detected by flow cytometry. Thus, HSCs have descriptive characteristics that allow for their identification and isolation.
  • CD38 is the most established and useful single negative marker for human HSCs.
  • Human HSCs may also be negative for lineage markers such as CD2, CD3, CD14, CD16, CD19, CD20, CD24, CD36, CD56, CD66b, CD271 and CD45RA. However, these markers may need to be used in combination for HSC enrichment.
  • CD34 and CD133 are the most useful positive markers for HSCs.
  • HSCs are also positive for lineage markers such as CD90, CD49f and CD93. However, these markers may need to be used in combination for HSC enrichment.
  • the haematopoietic stem and progenitor cells are CD34+CD38- cells.
  • a differentiated cell is a cell which has become more specialised in comparison to a stem cell or progenitor cell. Differentiation occurs during the development of a multicellular organism as the organism changes from a single zygote to a complex system of tissues and cell types. Differentiation is also a common process in adults: adult stem cells divide and create fully- differentiated daughter cells during tissue repair and normal cell turnover. Differentiation dramatically changes a cell’s size, shape, membrane potential, metabolic activity and responsiveness to signals. These changes are largely due to highly-controlled modifications in gene expression. In other words, a differentiated cell is a cell which has specific structures and performs certain functions due to a developmental process which involves the activation and deactivation of specific genes.
  • a differentiated cell includes differentiated cells of the haematopoietic lineage such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells, T cells, B-cells and NK- cells.
  • differentiated cells of the haematopoietic lineage can be distinguished from stem cells and progenitor cells by detection of cell surface molecules which are not expressed or are expressed to a lesser degree on undifferentiated cells.
  • Suitable human lineage markers include CD33, CD13, CD14, CD15 (myeloid), CD19, CD20, CD22, CD79a (B), CD36, CD71 , CD235a (erythroid), CD2, CD3, CD4, CD8 (T) and CD56 (NK).
  • isolated population of cells may refer to the population of cells having been previously removed from the body.
  • An isolated population of cells may be cultured and manipulated ex vivo or in vitro using standard techniques known in the art.
  • An isolated population of cells may later be reintroduced into a subject. Said subject may be the same subject from which the cells were originally isolated or a different subject.
  • a population of cells may be purified selectively for cells that exhibit a specific phenotype or characteristic, and from other cells which do not exhibit that phenotype or characteristic, or exhibit it to a lesser degree.
  • a population of cells that expresses a specific marker such as CD34
  • a population of cells that does not express another marker such as CD38
  • Purification or enrichment may result in the population of cells being substantially pure of other types of cell.
  • Purifying or enriching for a population of cells expressing a specific marker may be achieved by using an agent that binds to that marker, preferably substantially specifically to that marker.
  • An agent that binds to a cellular marker may be an antibody, for example an anti-CD34 or anti- CD38 antibody.
  • antibody refers to complete antibodies or antibody fragments capable of binding to a selected target, and including Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques.
  • alternatives to classical antibodies may also be used in the invention, for example “avibodies”, “avimers”, “anticalins”, “nanobodies” and “DARPins”.
  • the agents that bind to specific markers may be labelled so as to be identifiable using any of a number of techniques known in the art.
  • the agent may be inherently labelled, or may be modified by conjugating a label thereto.
  • conjugating it is to be understood that the agent and label are operably linked. This means that the agent and label are linked together in a manner which enables both to carry out their function (e.g. binding to a marker, allowing fluorescent identification or allowing separation when placed in a magnetic field) substantially unhindered. Suitable methods of conjugation are well known in the art and would be readily identifiable by the skilled person.
  • a label may allow, for example, the labelled agent and any cell to which it is bound to be purified from its environment (e.g. the agent may be labelled with a magnetic bead or an affinity tag, such as avidin), detected or both.
  • Detectable markers suitable for use as a label include fluorophores (e.g. green, cherry, cyan and orange fluorescent proteins) and peptide tags (e.g. His tags, Myc tags, FLAG tags and HA tags).
  • a number of techniques for separating a population of cells expressing a specific marker are known in the art. These include magnetic bead-based separation technologies (e.g. closed- circuit magnetic bead-based separation), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g. using affinity columns or beads, such biotin columns to separate avidin-labelled agents) and microscopy-based techniques.
  • magnetic bead-based separation technologies e.g. closed- circuit magnetic bead-based separation
  • flow cytometry e.g. flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g. using affinity columns or beads, such biotin columns to separate avidin-labelled agents) and microscopy-based techniques.
  • FACS fluorescence-activated cell sorting
  • affinity tag purification e.g. using affinity columns or beads, such biotin columns to separate avidin-labelled agents
  • microscopy-based techniques e.g. using magnetic bead
  • Clinical grade separation may be performed, for example, using the CliniMACS® system (Miltenyi). This is an example of a closed-circuit magnetic bead-based separation technology.
  • dye exclusion properties e.g. side population or rhodamine labelling
  • enzymatic activity e.g. ALDH activity
  • a vector is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell.
  • the vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid, or facilitating the expression of the protein encoded by a segment of nucleic acid.
  • Vectors may be non-viral or viral.
  • vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes and viruses.
  • the vector may be single stranded or double stranded. It may be linear and optionally the vector comprises one or more homology arms.
  • the vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest.
  • the vectors used in the invention may be, for example, plasmid or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.
  • Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transformation, transfection and transduction.
  • techniques are known in the art, for example transduction with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors, Sleeping Beauty vectors; direct injection of nucleic acids and biolistic transformation.
  • Non-viral delivery systems include but are not limited to DNA transfection methods.
  • transfection includes a process using a non-viral vector to deliver a gene to a target cell.
  • Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nature Biotechnology (1996) 14: 556) and combinations thereof.
  • CFAs cationic facial amphiphiles
  • vector includes an expression vector, i.e. a construct capable of in vivo or in vitro/ex vivo expression. Expression may be controlled by a vector sequence, or, for example in the case of insertion at a target site, expression may be controlled by a target sequence.
  • a vector may be integrated or tethered to the cell’s DNA.
  • Viral delivery systems include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors and baculoviral vectors.
  • AAV adeno-associated viral
  • Nucleotide of interest The vector may comprise a nucleotide of interest (NOI).
  • NOI nucleotide of interest
  • the nucleotide of interest gives rise to a therapeutic effect.
  • the vector comprises one or more nucleotide sequence encoding a chimeric antigen receptor (CAR). In one embodiment, the vector comprises one or more nucleotide sequence encoding a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the CAR may be, for example, a CD19 CAR or a GD2 CAR.
  • An example nucleotide sequence encoding a CD19 CAR is:
  • An example nucleotide sequence encoding a GD2 CAR is: ATGGAGTTCGGTCTGAGTTGGCTCTTCCTGGTGGCCATCTTGAAGGGCGTGCAGTGTTCGCGGGACAT
  • the nucleotide sequence encoding a CAR comprises or consists of a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13 or 14.
  • the nucleotide sequence encoding a CAR comprises or consists of the nucleotide sequence of SEQ ID NO: 13 or 14.
  • Suitable NOIs include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, anti-oxidant molecules, engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, immune co-stimulatory molecules, immunomodulatory molecules, anti-sense RNA, microRNA, shRNA, siRNA, ribozymes, miRNA target sequences, a transdomain negative mutant of a target protein, toxins, conditional toxins, antigens, tumour suppressor proteins, growth factors, transcription factors, membrane proteins, surface receptors, anti-cancer molecules, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives thereof (such as derivatives with an associated reporter group).
  • the NOIs may also encode pro-drug activating enzymes.
  • NOI is the beta-globin chain which may be used for gene therapy of thalassemia/sickle cell disease.
  • NOIs also include those useful for the treatment of other diseases requiring non- urgent/elective gene correction in the myeloid lineage such as: chronic granulomatous disease (CGD, e.g. the gp91 phox transgene), leukocyte adhesion defects, other phagocyte disorders in patients without ongoing severe infections and inherited bone marrow failure syndromes (e.g. Fanconi anaemia), as well as primary immunodeficiencies (SCIDs).
  • CCD chronic granulomatous disease
  • gp91 phox transgene e.g. the gp91 phox transgene
  • leukocyte adhesion defects e.g. the gp91 phox transgene
  • other phagocyte disorders in patients without ongoing severe infections and inherited bone marrow failure syndromes e.g. Fanconi anaemia
  • SCIDs primary immunodeficiencies
  • NOIs also include those useful in the treatment of lysosomal storage disorders and immunodeficiencies.
  • the cells of the invention may be formulated for administration to subjects with a pharmaceutically acceptable carrier, diluent or excipient.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline, and potentially contain human serum albumin.
  • Handling of cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.
  • the invention provides a population of cells of the invention, which may be used as disclosed herein.
  • the cell(s) may be for use in therapy, for example for use in gene therapy.
  • the use may be as part of an adoptive cell therapy, for example and adoptive T cell therapy.
  • the adoptive cell therapy may, for example, be adoptive Treg cell therapy.
  • the therapy e.g. using Treg cells
  • haematopoietic stem and/or progenitor cell transplantation is the transplantation of blood stem cells derived from the bone marrow (in this case known as bone marrow transplantation) or blood.
  • HSCT Haematopoietic stem cell transplantation
  • Stem cell transplantation is a medical procedure in the fields of haematology and oncology, most often performed for people with diseases of the blood or bone marrow, or certain types of cancer.
  • HSCTs Many recipients of HSCTs are multiple myeloma or leukaemia patients who would not benefit from prolonged treatment with, or are already resistant to, chemotherapy.
  • Candidates for HSCTs include paediatric cases where the patient has an inborn defect such as severe combined immunodeficiency or congenital neutropenia with defective stem cells, and also children or adults with aplastic anaemia who have lost their stem cells after birth.
  • Other conditions treated with stem cell transplants include sickle-cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing’s Sarcoma, Desmoplastic small round cell tumour and Hodgkin’s disease.
  • the cell(s) is administered as part of an autologous stem cell transplant procedure.
  • the cell(s) is administered as part of an allogeneic stem cell transplant procedure.
  • autologous stem cell transplant procedure it is to be understood that the starting cell(s) (which may then be genetically engineered) is obtained from the same subject as that to which the final cell(s) is administered. Autologous transplant procedures are advantageous as they avoid problems associated with immunological incompatibility and are available to subjects irrespective of the availability of a genetically matched donor.
  • allogeneic stem cell transplant procedure it is to be understood that the starting cell(s) (which may then be genetically engineered) is obtained from a different subject as that to which the final cell(s) is administered.
  • the donor will be genetically matched to the subject to which the cells are administered to minimise the risk of immunological incompatibility.
  • the subject is subjected to a mild myeloablative, reduced intensity or non- myeloablative conditioning regimen before administration of the HSPCs.
  • Suitable doses of cell(s) are such as to be therapeutically and/or prophylactically effective.
  • the dose to be administered may depend on the subject and condition to be treated, and may be readily determined by a skilled person.
  • Haematopoietic progenitor cells provide short term engraftment. Accordingly, gene therapy by administering haematopoietic progenitor cells would provide a non-permanent effect in the subject. For example, the effect may be limited to 1-6 months following administration of the haematopoietic progenitor cells. An advantage of this approach would be better safety and tolerability, due to the self-limited nature of the therapeutic intervention.
  • haematopoietic progenitor cell gene therapy may be suited to treatment of acquired disorders, for example cancer, where time-limited expression of a (potentially toxic) anticancer nucleotide of interest may be sufficient to eradicate the disease.
  • the invention may be, for example, useful in the treatment of a disease selected from the group consisting of mucopolysaccharidosis type I (MPS-1), chronic granulomatous disorder (CGD), Fanconi anaemia (FA), sickle cell disease, Pyruvate kinase deficiency (PKD), Leukocyte adhesion deficiency (LAD), metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD), GM2 gangliosidosis, thalassemia, cancer, a genetic disease and a blood disease.
  • MPS-1 mucopolysaccharidosis type I
  • CCD chronic granulomatous disorder
  • FA Fanconi anaemia
  • PDD Pyruvate kinase deficiency
  • LAD Leukocyte adhesion deficiency
  • MLD metachromatic leukodystrophy
  • GLD globoid cell leukodystrophy
  • the invention may also be, for example, useful in the treatment of mucopolysaccharidoses disorders and other lysosomal storage disorders.
  • agents for use in the invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • the skilled person can readily determine an appropriate dose of one of the agents of the invention to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific agent employed, the metabolic stability and length of action of that agent, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.
  • a “subject” refers to either a human or non-human animal.
  • non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats.
  • the non-human animal may be a companion animal.
  • the subject is a human.
  • HSPC Human CD34+ hematopoietic stem/progenitor cells
  • HSPCs were cultured at a concentration of 1 x 10 6 cells per milliliter, in serum-free StemSpan medium (StemCell Technologies) supplemented with penicillin (100 lll/ml), streptomycin (100 g/ml), 100 ng/ml recombinant human stem cell factor (rhSCF), 20 ng/ml recombinant human thrombopoietin (rhTPO), 100 ng/ml recombinant human Flt3 ligand (rhFItS), and 20 ng/ml recombinant human IL6 (rhlL6) (all from Peprotech) 24 hours (prestimulation). Cells were then exposed to 8uM Cyclosporyn H (CsH) or to DMSO (as control) for 16 hours, and then collected for proteomic or metabolomics analysis.
  • CsH Cyclosporyn H
  • DMSO as control
  • PBMCs Peripheral blood mononuclear cells
  • CD3+ T cells were isolated and stimulated using magnetic beads (ratio celkbead 1 :1) conjugated with anti-CD3/anti-CD28 antibodies (Dynabeads human T-activator CD3/CD28, Thermo Fisher).
  • CD4+ and CD8+ T cells were isolated by immune-magnetic separation using CD4 or CD8 T-cell isolation kits (Miltenyi Biotech) according the manufacturer’s instructions, and stimulated using Dynabeads.
  • Cells were maintained in RPMI medium supplemented with 10% FBS, 1% penicillin/streptomycin, 2% glutamine, 1X Non-Essential Amino Acids, 1mM Sodium Pyruvate, IL-7 (5 ng/ml; PreproTech) and IL-15 (5 ng/ml; PreproTech).
  • Dynabeads were removed after 3 days of culture and primary T cells were transduced with the given lentiviral vector, at the indicated multiplicity of infection (MOI), in the presence or absence of CsH 8uM, and diluted 1 :1 with fresh medium after 16 hours. Cells were collected 4 days after transduction for flow cytometry, molecular and functional analyses. All cells were cultured in a 5% CO2 humidified atmosphere at 37°C.
  • Bone marrow-derived CD34+ cells (BM-CD34 + ) from 2 healthy donors and 3 FANCA patients were cultured in CellGenix GMP SCGM serum-free medium, Xeno-free [20802-0500], supplemented with: 1 % P/S - 2% Glutamine - 100ng/ml hSCF - 100ng/ml hFlt3 - 100ng/ml hTPO - 20ng/ml IL-3 - 10ug/ml infliximab [anti TNFalpha] - 1mM N-Acetylcistein. After 10 hours of stimulation, cells were exposed to 8uM CsH for 12 hours and then collected for scRNA-seq experiments.
  • Viral vectors Viral vectors
  • Lentiviral vectors were produced by transient transfection in 293T cells and were all VSV-g pseudotyped and concentrated by ultracentrifugation as already described (Montini et al, 2006).
  • the previously reported Anti-CD19 CAR sequence J.N. Brudno et al., Nat Med 2020
  • the NGFR is expressed from the minimal CMV promoter in the opposite orientation.
  • Cytofluorimetric analyses were performed on FACS Canto II (BD Pharmingen) equipped with DIVA Software, and analyzed either with the FSC express software. Fluorescence Minus One (FMO) stained cells were used as controls. Single-color controls CompBeads (BD Pharmingen) were used as compensation beads to set gating parameters and optimize voltages.
  • FMO Fluorescence Minus One
  • antibodies are: Anti human CD3 Percp5.5 (BioLegend - 300328), Anti human CD4 PB (BD Pharmingen - 558116), Anti human CD8 APC-H7 (BD Pharmingen - 641400), Anti human PD1 APC (BD Pharmingen - 329908), Anti human CD366 (Tim-3) PEcy7 (BD Pharmingen - 345014), Anti human CD223 (Lag-3) PE (BD Pharmingen - 369306), Anti human CD152 (CTLA-4) PEcy7 (BioLegend - 349913), Anti human CD271 (LNGFR) APC (Miltenyi Biotec 130-113-418), Anti human CD34 PEcy7 (BD Pharmingen - 348811) , Anti human CD133/2 (293C3) PE (Miltenyi Biotec 130-113-186), Anti human CD90 APC (BD Pharmingen - 559869), Anti human CD62L PE
  • the proliferation assay was performed with Cell Proliferation Dye eFluor® 670 (Affimetrix, eBioscience), according to the manufacturer's instructions. During cell division, this dye will be distributed equally between daughter cells, and can be measured as successive halving of the fluorescence intensity. Cells were stained and analyzed at flow cytometry at different time points. Rainbow Calibration Particles (BD Pharmingen) were analyzed before every acquisition as an internal control for calibration.
  • the cell cycle analysis was performed by Ki67 (BD Pharmingen) and Hoechst (Invitrogen) staining, according to the manufacturer's instructions. Cells were stained and analyzed at flow cytometry, 24 hours after CsH exposure.
  • RNA extraction, RT-qPCR and gene expression analysis RNA extraction from cells was performed using the RNeasy Plus micro Kit (QIAGEN), according to manufacturer’s instructions.
  • the extracted mRNAs were reverse transcribed (RT) using the SuperScript Vilo kit (Invitrogen).
  • qPCR were performed using Fast Sybr green per mastermix (Thermofisher) and run using the Viia7 Real-Time PCR system (Thermofisher).
  • Relative quantification values were calculated as the fold-change expression of the gene of interest over its expression in the reference sample, by the formula 2-AACt.
  • the expression was normalized using the housekeeping gene 18S ribosomal RNA, whose expression is not affected upon metabolic variation.
  • Oxygen consumption rate was measured on the SeahorseXFe96 Analyzer (Agilent Technologies, Santa Clara, CA, USA) using SeaHorse XF Cell Mito Stress Test, following the manufacturer’s instructions. Briefly, on the day of the assay, cells were counted and attached to 96-well Seahorse cell culture microplates, precoated with CorningTMCell-Tak (Sacco, Cadorago, Italy) according to the manufacturer’s instructions, at a density of 250000 cells per well.
  • Results were normalized by cell number, measured at the end of the experiment using the CyQUANT Cell Proliferation Assays (Thermo FisherScientific). Data are expressed as pmol of oxygen per minute per arbitrary units (pmol/min/a.u.).
  • mPB-CD34+ cells from three different healthy donors (100,000 minimum per sample), exposed or not to CsH as described above, were pelleted and flash-frozen in liquid nitrogen for shipment to the Biological Mass Spectrometry Core Facility at University of Colorado Denver. For analysis, cells were lysed and analyzed as previously described (Haudek-Prinz et al, 2012). Levels of acylcarnitines and free fatty acids have been normalized to the DMSO groups and autoscaled in MetaboAnalyst v 5.0. Statistical analysis was performed using 2way ANOVA with Holm-Sidak’s multiple comparisons test (* p ⁇ 0.05, ** p ⁇ 0.01). Graphs and statistical analysis prepared using GraphPad Prism v 9.1.1.
  • Single cell RNA-sequencing and analysis scRNA-seq libraries were generated using a microfluidics-based approach on Chromium Controller (10x Genomics) using the Chromium Single Cell 3' Reagent Kit v3.1 according to the manufacturer’s instructions.
  • concentration of the scRNA-seq libraries was determined using Qubit v3.0, and size distribution was assessed using an Agilent 4200 TapeStation system. Libraries were sequenced on an Illumina NovaSeq instrument (paired-end, 150-bp read length). Raw data from scRNA-seq was analyzed and processed as previously described (Giordano et al, 2022). Functional enrichment analysis was performed on lists of differentially expressed genes. Heatmaps were generated using the R package pheatmap (v1.0.12).
  • Hematopoietic stem and progenitor cells represent the ideal candidates for gene therapy applications thanks to their self-renewal potential, their capability to propagate the entire hematopoietic lineage, and the tolerogenic effect on host immunity (Naldini, 2015). Nevertheless, HSPCs display a low gene manipulation efficiency making it necessary to further enhance ex vivo gene transfer efficacy.
  • Cyclosporine H CsH
  • LV lentiviral vector
  • CsH increases steady state levels of Acylcarnitines and many free fatty acids in mPB-CD34+ cells
  • CsH treated cells confirms the potential impact on HSPC metabolism, and more specifically could be in line with an alteration in fatty acids oxidation.
  • CsH induces metabolic alteration on HSPC from both healthy donors and Fanconi anemia patients
  • T cell exhaustion is a state characterized by sequential phenotypic and functional changes, occurring during many chronic infections and cancer (Feldman et al, 2015; Krebs et al, 2013). Exhausted T cells display distinctive patterns of cytokine receptors, transcription factors and effector molecules, as well as sustained expression of inhibitory receptors, finally culminated in the loss of T-cell functions (Blackburn et al, 2009; Crawford & Wherry, 2009). Growing evidence indicates that exhausted T cells undergo metabolic alterations, associated with distinct signaling cascades and epigenetic landscapes, which leads to poor responsiveness to immune-checkpoint-blockade and lower T cell functionality (Franco et al, 2020).
  • CsH is able to increase LV vector transduction in HSPC and T cells (Petrillo et al., 2018) and have investigated its impact on the biological properties of these clinically relevant target cells in the context of ex vivo cell and gene therapy applications.
  • Fanconi anemia commonly develop progressive bone marrow failure and have a high risk of cancer.
  • the prominent role of the FA protein family involves DNA damage response and/or repair and ssignificant evidence supports excessive apoptosis of HSPC, induced by oxidative and other sources of stress, as a critical factor in the pathogenesis of bone marrow failure and leukemia progression in FA (Du et al, 2008).
  • LV-mediated HSC gene therapy may constitute a new safe and efficient approach for the treatment/prevention of the bone marrow failure (BMF) characteristic of FA patients. Nevertheless, the isolation and ex-vivo manipulation of FA HSCs remains challenging due to low recovery and vulnerability of these cells to environmental triggers (Tolar et al., 2012).
  • CsH has the capacity to mitigate activation of several pathways potentially harmful for HSC biological integrity such as oxidative phosphorylation DNA damage and repair pathways as well as Myc-driven transcirptional programms that have recently been shown to Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia (Rodriguez et al, 2021). Moreover, CsH treated HSPC showed a significant upregulation in free fatty acid content. High fatty acid levels mimicking the bone marrow microenvironment have been shown to enable maintenance of engraftable quiescent HSCs ex vivo (Kobayashi et al, 2019) and sustained HSC functions (Dong et al, 2021).
  • fatty acids have also been show to modulated CD8+ T cell responses and improve cacner immnuotherapies (Luu et al, 2021).
  • Chimeric antigen receptor (CAR)-T cells show great promise in treating cancers and viral infections.
  • most protocols developed to expand T cells require relatively long periods of time in culture, potentially leading to progression toward populations of terminally differentiated effector memory cells.
  • adoptively transferred T cells should express a less differentiated phenotype because those cell subsets circulate to lymphoid organs and are capable of robust expansion (Redeker & Arens, 2016).
  • T cells defined as cells expressing the lymphoid homing molecules CD62L and CCR7
  • a failure of infused T cells to persist has been correlated with the absence of CD4 + T cells in the CD8 + T cell product (Patel et al, 2016).
  • the use of PBMCs in the production of the CAR-T cells in this protocol allows the final cultures to contain both antigen-specific CD8 + T cells and CD4 + T cells.
  • PBMCs are activated with anti-CD3 and anti-CD28 along with interleukin 2 (IL- 2) at a relatively high density.
  • IL-2 interleukin 2
  • CD4 + T cells are more refractory to lentiviral transduction as compared to CD8+ T cells (Kerkar et al, 2011).
  • the use of CsH during lentiviral transduction could offer the possibility to generate CAR-T cells achieving gene marking also in the relevant T stem memory compartment (Gattinoni et al, 2011) while better preserving their antitumoral capacities.
  • T cell exhaustion plays a major role in limiting CAR T efficacy, and it is associated with poor responses in cancer patients receiving immunotherapy (Delgoffe et al., 2021). This status can be promoted by the immunosuppressive conditions within the tumor microenvironment, as the increase of reactive oxygen species (ROS) levels, or by excessive CAR signaling itself, as a result of high antigen burden or tonic signaling induced by clustering of CAR molecules (Long et al, 2015; Lynn et al, 2019). For these reasons, the inhibition of CAR signaling (Rest), as well as T cell metabolic reprogramming, have been proposed to reverse CAR-T exhaustion (Weber et al., 2021).
  • ROS reactive oxygen species
  • T cells undergo a sort of transient metabolic delay, which could be exploited to reduce ROS production, but also to coax the cells into a resting state, thus inhibiting tonic signaling and potentially preventing exhaustion.
  • Tregs were purified from buffy coats using anti-CD4 and anti-CD25 magnetic beads (Treg isolation kit, Miltenyi). Cells were activated with anti- CD3/CD28-coated beads and kept in culture with 1000U/ml of IL-2 and rapamycin (100nM) to foster Treg enrichment. After 30 h of stimulation, Tregs were transduced with the LV vector encoding for CD19 CAR (MOI 10) +/- CsH (8pM). Transduction efficiency was evaluated by FC at 8- and 15-days post transduction by assessing the percentage of NGFR+ cells (Fig. 7). Our results show that CsH exposure enhances human Treg transduction efficiency measured both as %NGFR, gMFI on CD4+ (Fig. 7a-b) and gMFI on FOXP3+ cells (Fig. 7c).
  • CsH does not alter the survival and growth potential of human CD19 CAR Tregs but increases their PD-1 surface levels.
  • CsH improves the immunomodulatory profile of human CD19 CAR Tregs.
  • CsH does not alter the in vitro suppressive function of CD19 CAR Tregs.
  • functional assays to evaluate the in vitro functionality of CsH-treated CD19 CAR Tregs.
  • CsH vs DM SO-treated CAR Tregs were added at different PBMC:Treg ratios, and cells were activated with anti-CD3/CD28-coated beads. Tconv proliferation was evaluated after 5 days of culture.
  • CsH-treated CAR-Tregs showed a potent suppressive capacity (expressed as suppression %) similar to DMSO-treated Tregs (Fig. 10). This result indicated that LV transduction in the presence of CsH increases CAR expression without impairing Tregs’ suppressive properties in vitro.
  • Tregs were transduced at 10 MOI with the lentiviral vector encoding the CD19 CAR, with or without 8pM of CsH in DMSO.
  • Cell culture was maintained for 15 days by refreshing the culture medium every 2 days with cRPMI, r-IL2 and rapamycin (100nM) (Sigma).
  • Intranuclear staining was performed using fixation/permeabilization buffer solution (eBioscience), according to the manufacturer’s instructions. Stained cells were analyzed on a FACSCanto II (BD Biosciences), and data were analyzed with Diva software (BD Biosciences) and FlowJo software.
  • Cytokine production For intracellular cytokine staining, CAR Treg cells were stimulated with 1 :100 LAC (Leukocyte Activation Cocktail with BD GolgiPlugTM) (BD Biosciences) for 3 hours at 37°C with 5% of CO2. Then, intracellular cytokine production was stained and analyzed by flow cytometry, as previously described (Milardi et al. (2022) Eur J Immunol 52: 1171-1189).
  • LAC Leukocyte Activation Cocktail with BD GolgiPlugTM
  • Treg cells were co-cultured with PBMCs cells (labeled with 5pM of carboxy fluorescein succinimidyl ester fluorescent dye (CFSE) (Invitrogen) in PBS at 37°C for 8 minutes) at a ratio of 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16 (PBMCs: Treg cells) for 5 days.
  • CFSE carboxy fluorescein succinimidyl ester fluorescent dye
  • PBMCs peripheral blood mononuclear cells
  • CD4+ and CD8+ cells were isolated by immune-magnetic separation using CD4 or CD8 T-cell isolation kit (Miltenyi Biotech) according to the manufacturer’s instructions, and stimulated using magnetic beads (ratio celkbead 1 :1) conjugated with Dynabeads human T-activator CD3/CD28 (Day 0).
  • a second hit of CsH treatment was performed after two days, Staphylococcal enterotoxin B (SEB) was also added overnight, to boost T cell activation.
  • SEB Staphylococcal enterotoxin B
  • Cytofluorimetric analyses were performed at day 7 on FACS Canto II (BDPharmingen), and analyzed with the FSC express software. Exhausted cells were identified as the triple positive population for LAG3, TIM3 and PD1 surface markers.
  • Piras F Kajaste-Rudnitski A (2020) Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering. Gene Ther
  • cyclosporin H or a derivative thereof for: (a) reducing or preventing T cell exhaustion and/or loss of T cell effector functions; and/or (b) increasing T cell engraftment and/or persistence.
  • a T cell for use in a method of therapy comprising contacting the T cell with cyclosporin H (CsH) or a derivative thereof.
  • CsH cyclosporin H
  • T cell exhaustion and/or loss of T cell effector functions is reduced or prevented;
  • T cell engraftment and/or persistence is increased. 4.
  • T cell for use according to any preceding paragraph, wherein the T cell is transduced or transfected with a vector, optionally a viral vector.
  • T cell for use according to paragraph 5, wherein the vector is a retroviral vector or a lentiviral vector.
  • T cell for use according to paragraph 5 or 6, wherein the vector comprises one or more nucleotide sequence encoding a chimeric antigen receptor (CAR) and/or a T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • T cell for use according to any preceding paragraph, wherein the T cell is a chimeric antigen receptor (CAR) T cell and/or comprises an exogenous T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR exogenous T cell receptor
  • T cell for use according to any preceding paragraph, wherein the T cell is a CD4+ and/or CD8+ T cell.
  • CsH cyclosporin H
  • HSPCs haematopoietic stem and/or progenitor cells
  • a haematopoietic stem and/or progenitor cell for use in a method of treating Fanconi Anemia, wherein the method comprises contacting the HSPC with cyclosporin H (CsH) or a derivative thereof.
  • CsH cyclosporin H
  • T cell or HSPC for use according to any preceding paragraph, wherein the T cell or HSPC is contacted twice with the CsH or derivative thereof.
  • T cell or HSPC for use according to any preceding paragraph, wherein the T cell or HSPC is: (a) contacted with the CsH or derivative thereof at the same time as transduction or transfection with a vector; and/or
  • T cell or HSPC for use according to any preceding paragraph, wherein the T cell or HSPC is cultured for 16 days or less before administration to a subject.
  • a method of cell therapy comprising the steps of:
  • a method of gene therapy comprising the steps of:
  • T cell is a chimeric antigen receptor (CAR) T cell and/or comprises an exogenous T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR exogenous T cell receptor
  • T cell exhaustion and/or loss of T cell effector functions is reduced or prevented;
  • T cell engraftment and/or persistence is increased.

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Abstract

Utilisation de cyclosporine H (CsH) ou d'un dérivé de celle-ci pour : (a) la réduction ou la prévention de l'épuisement des cellules T et/ou de la perte des fonctions effectrices des cellules T ; et/ou (b) l'augmentation de la prise de greffe et/ou de la persistance des cellules T.
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Non-Patent Citations (63)

* Cited by examiner, † Cited by third party
Title
"Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS
AIUTI, A. ET AL., N. ENGL. J. MED., vol. 360, 2009, pages 447 - 458
AIUTI, A. ET AL., SCIENCE, vol. 341, 2013, pages 1233151
AUSUBEL, F.M. ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
BARQUINERO JSEGOVIA JCRAMIREZ MLIMON AGUENECHEA GPUIG TBRIONES JGARCIA JBUEREN JA: "Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice", BLOOD, vol. 95, 2000, pages 3085 - 3093
BLACKBURN SDSHIN HHAINING WNZOU TWORKMAN CJPOLLEY ABETTS MRFREEMAN GJVIGNALI DAWHERRY EJ: "Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection", NAT IMMUNOL, vol. 10, 2009, pages 29 - 37, XP055047746, DOI: 10.1038/ni.1679
CARTIER, N. ET AL., SCIENCE, vol. 326, 2009, pages 818 - 823
CAS , no. 83602-39-5
CHANG, A.H. ET AL., MOL. THER., 2007
CRAWFORD AWHERRY EJ: "The diversity of costimulatory and inhibitory receptor pathways and the regulation of antiviral T cell responses", CURR OPIN IMMUNOL, vol. 21, 2009, pages 179 - 186, XP026058394, DOI: 10.1016/j.coi.2009.01.010
D'ANDREA ADGROMPE M: "Molecular biology of Fanconi anemia: implications for diagnosis and therapy", BLOOD, vol. 90, 1997, pages 1725 - 1736, XP002962300
DELGOFFE GM, XU C, MACKALL CL, GREEN MR, GOTTSCHALK S, SPEISER DE, ZEHN D, BEAVIS PA: "The role of exhaustion in CAR T cell therapy", CANCER CELL, vol. 39, 2021, pages 885 - 888, XP086695075, DOI: 10.1016/j.ccell.2021.06.012
DONG SWANG QKAO YRDIAZ ATASSET IKAUSHIK STHIRUTHUVANATHAN VZINTIRIDOU ANIEVES EDZIECIATKOWSKA M ET AL.: "Chaperone-mediated autophagy sustains haematopoietic stem-cell function", NATURE, vol. 591, 2021, pages 117 - 123, XP037386556, DOI: 10.1038/s41586-020-03129-z
DU WADAM ZRANI RZHANG XPANG Q: "Oxidative stress in Fanconi anemia hematopoiesis and disease progression", ANTIOXID REDOX SIGNAL, vol. 10, 2008, pages 1909 - 1921
FELDMAN SAASSADIPOUR YKRILEY IGOFF SLROSENBERG SA: "Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors", SEMIN ONCOL, vol. 42, 2015, pages 626 - 639, XP009500295, DOI: 10.1053/j.seminoncol.2015.05.005
FERRARI GTHRASHER AJAIUTI A: "Gene therapy using haematopoietic stem and progenitor cells", NAT REV GENET, vol. 22, 2021, pages 216 - 234, XP037391962, DOI: 10.1038/s41576-020-00298-5
FRANCO FJACCARD AROMERO PYU YRHO PC: "Metabolic and epigenetic regulation of T-cell exhaustion", NAT METAB, vol. 2, 2020, pages 1001 - 1012, XP055895705, DOI: 10.1038/s42255-020-00280-9
GATTINONI LLUGLI EJI YPOS ZPAULOS CMQUIGLEY MFALMEIDA JRGOSTICK EYU ZCARPENITO C ET AL.: "A human memory T cell subset with stem cell-like properties", NAT MED, vol. 17, 2011, pages 1290 - 1297, XP055551014, DOI: 10.1038/nm.2446
GIORDANO AMSLUCIANI MGATTO FABOU ALEZZ MBEGHE CDELLA VOLPE LMIGLIARA AVALSONI SGENUA MDZIECIATKOWSKA M ET AL.: "DNA damage contributes to neurotoxic inflammation in Aicardi-Goutieres syndrome astrocytes", J EXP MED, vol. 219, 2022
GLIMM HOH IHEAVES CJ: "Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0", BLOOD, vol. 96, 2000, pages 4185 - 4193, XP086507540, DOI: 10.1182/blood.V96.13.4185
HACEIN-BEY-ABINA, S. ET AL., N. ENGL. J. MED., vol. 363, 2010, pages 1918 - 1927
HAUDEK-PRINZ VJKLEPEISZ PSLANY AGRISS JMESHCHERYAKOVA APAULITSCHKE VMITULOVIC GSTOCKL JGERNER C: "Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells", J PROTEOMICS, vol. 76, no. 150-162, 2012
HEFFNER GCBONNER MCHRISTIANSEN LPIERCIEY FJCAMPBELL DSMURNYY YZHANG WHAMEL ASHAW SLEWIS G ET AL.: "Prostaglandin E2 Increases Lentiviral Vector Transduction Efficiency of Adult Human Hematopoietic Stem and Progenitor Cells", MOL THER, vol. 26, 2018, pages 320 - 328, XP055561352, DOI: 10.1016/j.ymthe.2017.09.025
J.N. BRUDNO ET AL., NAT MED, 2020
KERKAR SPSANCHEZ-PEREZ LYANG SBORMAN ZAMURANSKI PJI YCHINNASAMY DKAISER ADHINRICHS CSKLEBANOFF CA ET AL.: "Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies", J IMMUNOTHER, vol. 34, 2011, pages 343 - 352
KLEBANOFF CAGATTINONI LRESTIFO NP: "Sorting through subsets: which T-cell populations mediate highly effective adoptive immunotherapy?", J IMMUNOTHER, vol. 35, 2012, pages 651 - 660
KOBAYASHI HMORIKAWA TOKINAGA AHAMANO FHASHIDATE-YOSHIDA TWATANUKI SHISHIKAWA DSHINDOU HARAI FKABE Y ET AL.: "Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo", CELL REP, vol. 28, 2019, pages 145 - 158
KREBS KBOTTINGER NHUANG LRCHMIELEWSKI MARZBERGER SGASTEIGER GJAGER CSCHMITT EBOHNE FAICHLER M ET AL.: "T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice", GASTROENTEROLOGY, vol. 145, 2013, pages 456 - 465, XP055236846, DOI: 10.1053/j.gastro.2013.04.047
LE BOURGEOIS TSTRAUSS LAKSOYLAR HIDANESHMANDI SSETH PPATSOUKIS NBOUSSIOTIS VA: "Targeting T Cell Metabolism for Improvement of Cancer Immunotherapy", FRONT ONCOL, vol. 8, 2018, pages 237
LILLEY, D.M.DAHLBERG, J.E.: "Methods in Enzymology", 1992, ACADEMIC PRESS, article "DNA Structures Part A: Synthesis and Physical Analysis of DNA"
LONG AHHASO WMSHERN JFWANHAINEN KMMURGAI MINGARAMO MSMITH JPWALKER AJKOHLER MEVENKATESHWARA VR ET AL.: "4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors", NAT MED, vol. 21, 2015, pages 581 - 590, XP055278553, DOI: 10.1038/nm.3838
LONG ET AL., NAT, 2015
LUU MRIESTER ZBALDRICH AREICHARDT NYUILLE SBUSETTI AKLEIN MWEMPE ALEISTER HRAIFER H ET AL.: "Microbial short-chain fatty acids modulate CD8(+) T cell responses and improve adoptive immunotherapy for cancer", NAT COMMUN, vol. 12, 2021, pages 4077, XP055905612, DOI: 10.1038/s41467-021-24331-1
LYNN RCWEBER EWSOTILLO EGENNERT DXU PGOOD ZANBUNATHAN HLATTIN JJONES RTIEU V ET AL.: "c-Jun overexpression in CAR T cells induces exhaustion resistance", NATURE, vol. 576, 2019, pages 293 - 300, XP036977360, DOI: 10.1038/s41586-019-1805-z
MAROFI FMOTAVALLI RSAFONOV VATHANGAVELU LYUMASHEV AVALEXANDER MSHOMALI NCHARTRAND MSPATHAK YJARAHIAN M ET AL.: "CAR T cells in solid tumors: challenges and opportunities", STEM CELL RES THER, vol. 12, 2021, pages 81, XP055971962, DOI: 10.1186/s13287-020-02128-1
MATAFORA VCORNO ACILIBERTO ABACHI A: "Missing Value Monitoring Enhances the Robustness in Proteomics Quantitation", J PROTEOME RES, vol. 16, 2017, pages 1719 - 1727
MILARDI ET AL., EUR J IMMUNOL, vol. 52, 2022, pages 1171 - 1189
MILLINGTON M, ARNDT A, BOYD M, APPLEGATE T, SHEN S: "Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells", PLOS ONE, vol. 4, pages e6461, XP055946049, DOI: 10.1371/journal.pone.0006461
NALDINI L: "Gene therapy returns to centre stage", NATURE, vol. 526, 2015, pages 351 - 360, XP055456380, DOI: 10.1038/nature15818
NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 556
OZOG STIMBERLAKE NDHERMANN KGARIJO OHAWORTH KGSHI GGLINKERMAN CMSCHEFTER LED'SOUZA SSIMPSON E ET AL.: "Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes", BLOOD, vol. 134, 2019, pages 1298 - 1311, XP086506808, DOI: 10.1182/blood.2019000040
PATEL SJONES RBNIXON DFBOLLARD CM: "T-cell therapies for HIV: Preclinical successes and current clinical strategies", CYTOTHERAPY, vol. 18, 2016, pages 931 - 942, XP055820575, DOI: 10.1016/j.jcyt.2016.04.007
PEARCE EL: "Metabolism in T cell activation and differentiation", CURR OPIN IMMUNOL, vol. 22, 2010, pages 314 - 320, XP027072472, DOI: 10.1016/j.coi.2010.01.018
PETRILLO CCESANA DPIRAS FBARTOLACCINI SNALDINI LMONTINI EKAJASTE-RUDNITSKI A: "Cyclosporin a and rapamycin relieve distinct lentiviral restriction blocks in hematopoietic stem and progenitor cells", MOL THER, vol. 23, 2015, pages 352 - 362, XP055206275, DOI: 10.1038/mt.2014.193
PETRILLO CTHORNE LGUNALI GSCHIROLI GGIORDANO AMSPIRAS FCUCCOVILLO IPETIT SJAHSAN FNOURSADEGHI M ET AL.: "Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells", CELL STEM CELL, vol. 23, 2018, pages 820 - 832
PIRAS FKAJASTE-RUDNITSKI A: "Antiviral immunity and nucleic acid sensing in haematopoietic stem cell gene engineering", GENE THER, 2020
PIRAS FRIBA MPETRILLO CLAZAREVIC DCUCCOVILLO IBARTOLACCINI SSTUPKA EGENTNER BCITTARO DNALDINI L ET AL.: "Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells", EMBO MOL MED, vol. 9, 2017, pages 1198 - 1211
POLAK, J.M.MCGEE, J.O'D.: "In Situ Hybridization: Principles and Practice", 1990, OXFORD UNIVERSITY PRESS
RAPPSILBER JISHIHAMA YMANN M: "Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics", ANAL CHEM, vol. 75, 2003, pages 663 - 670, XP055439282, DOI: 10.1021/ac026117i
REDEKER AARENS R: "Improving Adoptive T Cell Therapy: The Particular Role of T Cell Costimulation, Cytokines, and Post-Transfer Vaccination", FRONT IMMUNOL, vol. 7, 2016, pages 345, XP055459807, DOI: 10.3389/fimmu.2016.00345
RIO PNAVARRO SBUEREN JA: "Advances in Gene Therapy for Fanconi Anemia", HUM GENE THER, vol. 29, 2018, pages 1114 - 1123
RODRIGUEZ AZHANG KFARKKILA AFILIATRAULT JYANG CVELAZQUEZ MFURUTANI EGOLDMAN DCGARCIA DE TERESA BGARZA-MAYEN G ET AL.: "MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia", CELL STEM CELL, vol. 28, 2021, pages 33 - 47
RODRIGUEZ-GARCIA APALAZON ANOGUERA-ORTEGA EPOWELL DJGUEDAN S: "CAR-T Cells Hit the Tumor Microenvironment: Strategies to Overcome Tumor Escape", FRONT IMMUNOL, vol. 11, 2020, pages 1109, XP055846455, DOI: 10.3389/fimmu.2020.01109
ROE, B.CRABTREE, J.KAHN, A.: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
SAMBROOK, J., FRITSCH, E.F., MANIATIS, T.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SHYER JAFLAVELL RABAILIS W: "Metabolic signaling in T cells", CELL RES, vol. 30, 2020, pages 649 - 659, XP037261726, DOI: 10.1038/s41422-020-0379-5
SOKOLSKAJA, E. ET AL., CURR. OPIN. MICROBIOL., vol. 9, 2006, pages 404 - 8
TOLAR JBECKER PSCLAPP DWHANENBERG HDE HEREDIA CDKIEM HPNAVARRO SQASBA PRIO PSCHMIDT M ET AL.: "Gene therapy for Fanconi anemia: one step closer to the clinic", HUM GENE THER, vol. 23, 2012, pages 141 - 144
TYANOVA STEMU TSINITCYN PCARLSON AHEIN MYGEIGER TMANN MCOX J: "The Perseus computational platform for comprehensive analysis of (prote)omics data", NAT METHODS, vol. 13, 2016, pages 731 - 740, XP055904010, DOI: 10.1038/nmeth.3901
WANG CXSATHER BDWANG XADAIR JKHAN ISINGH SLANG SADAMS ACURINGA GKIEM HP ET AL.: "Rapamycin relieves lentiviral vector transduction resistance in human and mouse hematopoietic stem cells", BLOOD, vol. 124, 2014, pages 913 - 923, XP086511287, DOI: 10.1182/blood-2013-12-546218
WEBER EWPARKER KRSOTILLO ELYNN RCANBUNATHAN HLATTIN JGOOD ZBELK JADANIEL BKLYSZ D ET AL.: "Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling", SCIENCE, vol. 372, 2021, XP093032461, DOI: 10.1126/science.aba1786
ZHANG ZJIANG CLIU ZYANG MTANG XWANG YZHENG MHUANG JZHONG KZHAO S ET AL.: "B7-H3-Targeted CAR-T Cells Exhibit Potent Antitumor Effects on Hematologic and Solid Tumors", MOL THER ONCOLYTICS, vol. 17, 2020, pages 180 - 189
ZONARI EDESANTIS GPETRILLO CBOCCALATTE FELIDONNICI MRKAJASTE-RUDNITSKI AAIUTI AFERRARI GNALDINI LGENTNER B: "Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy", STEM CELL REPORTS, vol. 8, 2017, pages 977 - 990, XP002791722

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