WO2013144409A2 - Vecteurs pour l'identification de la lignée hématopoïétique - Google Patents

Vecteurs pour l'identification de la lignée hématopoïétique Download PDF

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WO2013144409A2
WO2013144409A2 PCT/ES2013/070200 ES2013070200W WO2013144409A2 WO 2013144409 A2 WO2013144409 A2 WO 2013144409A2 ES 2013070200 W ES2013070200 W ES 2013070200W WO 2013144409 A2 WO2013144409 A2 WO 2013144409A2
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cells
hematopoietic
cell
nucleic acid
isolated
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PCT/ES2013/070200
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Spanish (es)
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WO2013144409A3 (fr
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Francisco MARTÍN MOLINA
Pilar MUÑOZ FERNÁNDEZ
Miguel GARCÍA TOSCANO
Karin BENABDELLAH
Marién COBO PULIDO
Per Anderson
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Fundación Pública Andaluza Progreso Y Salud
Instituto De Salud Carlos Iii
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention is encompassed within the field of biotechnology and more specifically within the field of cell biology and virology.
  • the present invention discloses the use of viral vectors, specifically, lentiviral vectors (LVs) for the identification and isolation of hematopoietic cells derived from pluripotent or multipotent cells.
  • viral vectors specifically, lentiviral vectors (LVs) for the identification and isolation of hematopoietic cells derived from pluripotent or multipotent cells.
  • Pluripotent cells such as iPSs (pluripotent induced cells) or ESCs (embryonic stem cells of the English “Embryonic Stem Cells”) are unique tools for the study of organogenesis, for the development of in vitro models of human genetic diseases (Cancer, neurodegenerative diseases, primary immunodeficiencies, etc.) as well as a potential source of cells for use in therapy.
  • the in vitro differentiation of said pluripotent cells towards hematopoietic lineage provides a unique tool for the study of human hematopoietic development in healthy individuals as well as in individuals presenting diseases related to the immune system.
  • these cells are a unique platform to carry out pharmacological tests (toxicological and / or therapeutic) in the different hematopoietic lines that can be derived from them.
  • a first aspect of the present invention relates to a method for the identification and / or isolation of hematopoietic progenitor cells comprising the following steps:
  • a pluripotent or multipotent stem cell or a group of pluripotent or multipotent stem cells with a nucleic acid molecule capable of integrating into the genome of said cell comprising a first nucleic acid sequence that is operably linked to a second sequence of nucleic acid, where the first nucleic acid sequence comprises the promoter sequence SEQ ID No 1 (proximal promoter) and where the second nucleic acid sequence comprises a marker gene;
  • Identify and / or isolate hematopoietic progenitor cells by analyzing the expression of the marker gene and optionally at least one hematopoietic marker present therein.
  • the first nucleic acid sequence comprises SEQ ID No. 2 (alternative promoter).
  • the nucleic acid molecule capable of integrating into the cell genome is SEQ ID No. 3 (WE vector) or SEQ ID No. 4 (AWE vector).
  • the marker gene encodes for at least one marker protein selected from the list consisting of GFP and eGFP (enhanced green fluorescence protein).
  • the hematopoietic progenitor cells identified and / or isolated are characterized as being negative for the following three phenotypic markers: CD45, CD31 and CD34.
  • the hematopoietic progenitor cells identified and / or isolated are characterized as being positive for CD34 and negative for CD45.
  • the hematopoietic progenitor cells identified and / or isolated are characterized as being positive for CD34 and CD45.
  • the stem cell is pluripotent and is selected from the list consisting of: induced pluripotent cells (iPSs), non-human embryonic stem cells, bone marrow stem cells, blood stem cells of umbilical cord, peripheral blood stem cells and body fat stem cells.
  • iPSs induced pluripotent cells
  • the pluripotent stem cell or group of pluripotent stem cells are induced pluripotent cells (iPSs) or non-human embryonic stem cells.
  • HPCs isolated hematopoietic progenitor cells
  • a third aspect of the invention relates to a population of isolated hematopoietic progenitor cells obtainable according to the method described in the first aspect of the invention or any of its preferred aspects.
  • a fourth aspect of the invention relates to a composition
  • a composition comprising an isolated hematopoietic progenitor cell or an isolated population of hematopoietic progenitor cells obtainable according to the method described in the first aspect of the invention or in any of its preferred aspects.
  • said composition is a pharmaceutical composition that optionally comprises a pharmaceutically acceptable carrier. More preferably, said composition further comprises a second active ingredient.
  • a fifth aspect of the invention refers to the composition of the fourth aspect of the invention for use as a medicament.
  • a hematopoietic nature preferably selected from primary immunodeficiencies and / or autoimmune diseases.
  • AWE and WE vectors mark hematopoietic lineage cells that derive from the differentiation of human pluripotent cells.
  • LTR Long Repeated Terminal Sequence, from English, Long Terminal Repeat.
  • eGFP Enhanced Green Fluorescent Protein.
  • CMV cytomegal virus promoter.
  • EF l-ot elongation factor 1-ot, from English, Elongation Factor 1-a.
  • the NT histogram corresponds to the control of the experiment, where embryonic cells AND-1 controls are shown without incubation with any type of vector. The vectors used in transduction are indicated at the top of the histograms.
  • D Flow cytometry histograms showing the absence of expression of the AWE vector in differentiated embryonic cells towards neural tissue (labeled with the A2B5 antibody in the 3 histograms on the left).
  • non-transduced cells (NT) and vectors expressing GFP were used under the constitutive promoter EF l-ot (pLVTHM).
  • the same histogram on the right shows the same cells transduced with the AWE vector but differentiated to the hematopoietic lineage (as indicated by CD45 marking; Y axis).
  • Figure 2 The expression kinetics of GFP directed by the AWE and WE vectors mimic the hematopoietic differentiation kinetics.
  • the graphs show the correlation between the percentages of GFP (A) and CD45 (B) expression in hESCs AND-1 cells transduced with the AWE, WE and pLVTHM lentiviral vectors at different days of differentiation (0, 10, 15 and 22) , as indicated in each graph.
  • FIG. 3 The transduction of pluripotent cells (hESCs) with the AWE and WE vectors allows visualizing progenitors that give rise to myeloid colonies.
  • Pluripotent cells (hESCs) transduced with the AWE and WE vectors were incubated in culture medium with H4434 methylcellulose (Stem Cell Technologies, USA) for 15 days.
  • the graph shows the efficiency of colony formation (CFU) of the transduced hESCs cells with the AWE and WE vectors by comparing them with the non-transduced hESCs pluripotent cells (NT).
  • CFU colony formation
  • the AWE and WE vectors express GFP in hemogenic progenitors and hematopoietic cells derived from pluripotent cells.
  • H9 pluripotent cells transduced with the AWE vector were incubated with hematopoietic half differentiation for 10 (upper histograms), 15 (central histograms) and 22 (lower histograms) days. Cells transduced on different days were isolated and the expression of markers CD45, CD31 and CD34 was analyzed. The GFP + populations (histograms on the right) and the GFP- (histograms on the left) were first analyzed for the expression of CD45 (Y axis) and CD31 (X axis).
  • GFP + populations (histograms on the right) surrounded by a circle correspond to a new negative hematopoietic precursor for CD31, CD34 and CD45 (CD31-CD34-CD45-), which appears in the initial stages of hematopoietic differentiation and that gradually disappears in the late stages of differentiation (days 15 and 22)
  • Panel B shows the same experimental test performed in panel A but using the pLVTHM vector that expresses GFP through the constitutive promoter
  • Panel C shows the same experimental test performed on panel A and B but using the WE vector.
  • Figure 5 AWE vectors identify a new population with hemogenic capacity and negative CD31.
  • Pluripotent cells transduced with the AWE vector were incubated in the middle of ethical hematopopy differentiation. On day 1 1, said cells were isolated and stained with anti-CD45 and anti-CD31 antibodies (upper histogram). Negative cells for CD31 and CD45 were separated into two populations; positive for the expression of GFP (histogram on the right) and negative for the expression of GFP (histogram on the left) for the expression of GFP. The histograms below (right and left) show the enrichment of separate populations in GFP positive cells.
  • transduction refers to the entry of a viral vector into a cell and the expression (eg, transcription and / or translation) of the sequences it carries in its genome.
  • transfection refers to the introduction of non-viral genetic material (plasmids) and the expression of the sequences it carries.
  • transgene is understood as any nucleic acid sequence that is inserted into the genome of an organism and that comes from a different organism. Therefore, a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or a fragment or part thereof, a genomic sequence, a regulatory element and the like. They can be marker sequences (internal or surface) or gene sequences for replacement or complementation for a native gene in the target cell.
  • the term "marker” or “biomarker” is understood as a protein or a fragment thereof, or the sequence encoding said protein or fragment, which distinguishes a cell (or group of cells) from another cell (or group of cells).
  • the fluorescent protein eGFP T has been used as a marker in the present invention
  • pluripotent stem cell / s / pluripotent cell / s refers to those cells that have the ability to self-renew by mitotic divisions or to continue the path of differentiation for which It is programmed and, therefore, produce cells of one or more mature, functional and fully differentiated tissues.
  • Pluripotent stem cells cannot form a complete organism, but any other type of cell corresponding to the three embryonic lineages (endoderm, ectoderm and mesoderm). as well as the germinal and sack vite linen. They can therefore form cell lineages.
  • Sources of pluripotent stem cells for the purposes of the present invention are known pluripotent induced cells (iPSs), both human and non-human embryonic stem cells, bone marrow stem cells, umbilical cord blood stem cells, peripheral blood and body fat stem cells.
  • iPSs pluripotent induced cells
  • the pluripotent stem cells described in the present invention are not limited to those described herein, any of those known for the same purpose may be used.
  • multipotent stem cells are those that can only generate cells of their same layer or lineage of embryonic origin (for example: a bone marrow mesenchymal stem cell, having nature mesodermal, will give rise to cells of that layer such as myocytes, adipocytes or osteocytes, among others).
  • iPSs for the purposes of the present invention is understood as a specific type of pluripotent stem cells, artificially derived from a non-pluripotent cell, usually a somatic adult cell.
  • the iPSs have the same capacity for differentiation and tissue formation as embryonic stem cells, in terms of the expression of certain genes and proteins, in the chromatin methylation patterns, in the formation of embryoid bodies, in the formation of teratomas and in the viable formation of chimeras.
  • embryonic stem cell ESC
  • ESC embryonic stem cell
  • HPC hematopoietic progenitor cell
  • hematopoietic progenitors derived from pluripotent or multipotent cells will be used to refer to HPC cells (hematopoietic progenitor cells) obtained by transducing pluripotent cells, as for example, and not limited to , ESCs, iPSs, bone marrow stem cells, umbilical cord blood stem cells, peripheral blood stem cells and / or body fat stem cells, with the vectors described in the present invention and subsequently differentiated in vitro to stem cells of Ethical hematopoy lineage.
  • HPC cells hematopoietic progenitor cells obtained by transducing pluripotent cells, as for example, and not limited to , ESCs, iPSs, bone marrow stem cells, umbilical cord blood stem cells, peripheral blood stem cells and / or body fat stem cells, with the vectors described in the present invention and subsequently differentiated in vitro to stem cells of Ethical hematopoy lineage.
  • hemogenic progenitors in general, which can be differentiated to hematopoietic and endothelial lineage cells, as well as populations of hemato-restricted progenitors, which will specifically differentiate to hematopoietic lineage cells .
  • hematopoietic differentiation or “differentiation towards hematopoietic lineage” refers to the process in which pluripotent or multipotent cells are transformed into a type of hematopoietic cell.
  • the hematopoietic differentiation process is described as a hierarchy of progenitor cells, in which each successive stage is distinguished from the next by a characteristic phenotype. Therefore, the relationships between parents and their progeny, which define the beginning of the irreversible differentiation of said parents towards a specific hematopoietic lineage, is determined primarily by plasma membrane markers. The expression or not of these markers distinguishes the different parents during hematopoietic maturation and differentiation as shown in the examples section of the present invention.
  • the expression profile of surface markers that present the cellular elements belonging to the hematopoietic system and that allows them to be distinguished from each other, can be analyzed by flow cytometry.
  • hemato-restricted used in the present invention refers to a cell population that differs exclusively from hematopoietic lineage cells, for example, they can be any of the precursors of erythrocytes, platelets, granulocytes, monocytes or lymphocytes.
  • embryonic body is understood as those non-embryonic biological structures formed by aggregates of embryonic cells, with the three germ layers (endoderm, mesoderm and ectoderm) and which can reproduce many of the processes that They occur in the early stages of embryonic development.
  • hemangioblast or "hemogenic progenitor” is understood as the precursor embryonic mesodermal cells of the vascular endothelium and hematopoietic cells.
  • vector refers to a nucleic acid molecule that allows the expression of exogenous genetic material in a host cell.
  • the term "host cell” refers to those cells in which a vector has integrated and expressed its genetic material.
  • the cell is eukaryotic
  • the term also includes the entire progeny of the host cell. It is understood that the entire progeny may not be identical to the parental cell since there may be differentiation processes and / or mutations that occur during replication.
  • the term "lentivirus” refers to a group (or scientific genus) of retroviruses that in nature give rise to slowly developing diseases, due to their low ability to incorporate into the genome of the host cell Modified lentiviral genomes are useful as viral vectors for the insertion of a nucleic acid sequence in a cell.
  • transduction with lentiviral vectors is the ability to maintain a sustained expression of or of the transgenes that it incorporates into its genome. Therefore, the present invention employs lentiviral vectors to provide long-term expression of the transgene of interest in the target cell.
  • these vectors have a lentiviral spine.
  • the phrase "has a lentiviral spine" means that the nucleic acid molecule included in the virus particles that make up the vectors is based on a lentiviral genome.
  • the lentivirus vectors of the present invention are vectors in which a nucleic acid molecule contained in virus particles contains a lentiviral genome derived from the signal packaging sequence (non-coding sequence required for encapsidation of lentiviral RNA strands during the formation of viral particles).
  • lentiviruses are: the human immunodeficiency virus (HIV) (for example, HIV-1 or HIV 2); simian immunodeficiency virus (VIS); the feline immunodeficiency virus (IVF); the virus similar to the Maedi-Visna virus (EV1); equine infectious anemia virus (IEA), and caprine encephalitis arthritis virus (CAEV).
  • promoter refers to a set of control nucleic acid sequences that direct the transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the transcription initiation site.
  • a promoter may also optionally include an enhancer or repressor element.
  • a "constitutive promoter” is a promoter that is continuously active and is not subject to regulation by external signals or molecules. In contrast, the activity of an "inducible promoter” is regulated by an external signal or a molecule (for example, a transcription factor).
  • the term “medicament”, as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals.
  • the term "gene therapy”, as used herein refers to a general method for treating a pathological condition by inserting an exogenous nucleic acid into a suitable cell (s).
  • the nucleic acid is introduced into the cell, so that its functionality is maintained, for example, maintaining the ability to express a particular polypeptide.
  • the insertion of exogenous nucleic acid results is the expression of a therapeutically effective amount of a particular polypeptide.
  • the cells of the invention can be positive for certain phenotypic markers and negative for others.
  • “Positive” means that the cell expresses the marker. To consider that the marker is expressed, it must be present at a “detectable level”. In this report, “detectable level” means that the marker can be detected by one of the standard methodologies, such as PCR, blotting or FACS.
  • a gene is considered to be expressed by a cell of the invention if it can be reasonably detected after 20 cycles, preferably 25 cycles, and more preferably 30 cycles of PCR, which corresponds to a level of expression in the cell of at least 100 copies per cell. It is considered that a marker is not expressed by a cell of the invention, if the expression cannot be detected at a level of about 10-20 copies per cell.
  • the cell may be weakly positive for a given marker.
  • naturally expressed means that the cells have not been manipulated by recombinant technology, in any way. This is, for example, that the cells have not been artificially induced. to express these markers or to modulate the expression of these markers by the introduction into the cells of exogenous material, such as the introduction of heterologous promoters, or other sequences operatively linked to any of the endogenous genes, or by the introduction of exogenous genes.
  • the term "therapeutically effective amount” refers to an amount of a composition that produces a desired therapeutic effect, and either temporary or permanent, to prevent, treat or improve a condition or relieve symptoms or signs related to a pathological condition. Therefore, a therapeutically effective amount of a composition is also sufficient to cause a pharmacological effect. A therapeutically effective amount of a composition does not have to cause permanent improvement or improvement of symptoms or signs. The exact therapeutically effective amount is an amount of the composition at which the most effective results are produced in terms of the efficacy of the treatment of a given pathology.
  • This amount will vary depending on different factors, among others, to the characteristics of the therapeutic compounds themselves (activity, pharmacokinetics, pharmacodynamics and bioavailability), the physiological condition of the subject (age, sex, type of disease and grade, physical condition In general, the response to a given dose, and the type of medication), and the type of cell in which a nucleic acid is inserted, an expert in clinical and pharmacology can determine a therapeutically effective amount through routine experimentation, is that is, by controlling the subject's response to the administration of a compound and adjusting the dose accordingly.
  • the present invention faces the technical problem of providing a method capable of helping to know the existing mechanisms that lead the pluripotent or multipotent cells to differentiate to the different hematopoietic lineages, this method being capable of Identify early hematopoietic progenitors, as well as study the processes that lead these early progenitors to differentiate in different hematopoietic lineages and their use in clinical settings.
  • Figure 1A shows the construction scheme of these lentiviral vectors.
  • the lentiviral vector WE contains a 500 bp fragment of the WAS proximal promoter (SEQ ID No. 1) that directs the expression of the selected transgene in the embodiments described in the present invention, eGFP, as described in Martin , Toscano et al. 2005; Arabic, Frecha et al.
  • the lentiviral vector AWE contains a 387 bp fragment of the alternative WAS promoter immediately "upstream" of the 500 bp proximal promoter present in the WE vector (see SEQ ID No 2 where the sequence of the alternative promoter linked to the sequence of the proximal promoter is shown ), as described in Martin, Toscano et al. 2005; Arabic, Frecha et al. 2008.
  • the CE and pLVTHM vectors which contain the CMV and EFl-a constitutive promoters respectively, were used as positive controls.
  • the hESCs transduced with the WE and AWE lentiviral vectors containing respectively 1.4 and 1.3 g.c / c were differentiated to the hematopoietic lineage.
  • the expression of eGFP, CD45 and CD33 in said cells was determined after 22 days of induced hematopoietic differentiation (Figure 1C).
  • the WE and AWE vectors were only able to express the eGFP transgene in CD45 positive (CD45 +) and CD33 positive (CD33 +) cells.
  • 92.2% (AWE) and 90% (WE) of the hESCs that were eGFP + were also (CD45 + CD33 +), said extreme indicating almost complete specificity of expression in ethical hematopoy cells.
  • the expression of the transgene or eGFP marker present in the AWE lentiviral vector was analyzed, in hESCs cells transduced with said AWE vector and differentiated to other lineages that were not the hematopoietic lineage, for example to the neural lineage.
  • the hESCs were cultured in suspension in low adhesion culture plates in MSC-CM medium.
  • the hESCs aggregates were cultured in a chemically defined neuronal medium for another 3 days.
  • the neuronal precursors were analyzed by flow cytometry.
  • the pLVTHM vector expressing eGFP through the constitutive promoter EFl- ⁇ was used as a positive control.
  • hESCs transduced with the AWE lentiviral vector and differentiated to a neuronal phenotype were unable to express eGFP.
  • Figure 2 shows the correlation between eGFP and CD45 expression levels in different hematopoietic differentiation experiments of hESCs transduced with the WE and AWE lentiviral vectors. As can be seen in this figure, the greater the hematopoietic differentiation (greater expression of CD45), the greater the expression of GFP when the AWE and WE vectors are used.
  • operably or operatively linked we refer to a first nucleic acid sequence that is operably linked to a second nucleic acid sequence when the first nucleic acid sequence has a functional relationship with the second nucleic acid sequence.
  • the promoter is operatively linked to the marker gene if the promoter affects the transcription or expression of said gene.
  • the authors of the present invention analyzed the expression pattern of the WE and AWE vectors described in the present invention at different times of hematopoietic differentiation.
  • the pluripotential cells transduced with said lentiviral vectors as described in the present invention were analyzed on days 5, 10, 15 and 22 during the hematopoietic differentiation process.
  • the expression of the eGFP transgene present in the AWE and WE vectors began to be observed from day 10 ( Figure 4 A and C, central histogram day 10), progressively increasing on successive days of differentiation until days 10-15 ( Figure 4 A and C, central histograms days 10, 15 and 22).
  • the phenotypic analysis, obtained by flow cytometry, of the eGFP + cells derived from the pluripotential / AWE and WE cells at early days of GFP appearance (day 10) showed that these vectors mark a sub-population very specifically (CD45-CD31 + CD34dim) of hemogenic precursors restricted to the hematopoietic lineage (population shown in the lower histogram of Figure 4A day 10), as well as to hematopoietic precursors (CD45 + CD34 +) (population shown in the upper histogram of Figure 4A day 10).
  • the cell populations (CD31-GFP +) were separated and ( CD31-GFP-) using FACS-sorting ( Figure 5A). Once separated, they were incubated in the culture medium that favors hematopoietic differentiation, previously described. Only cells expressing GFP gave rise to cells (CD31 + CD45 +) after 10 days in this differentiation medium ( Figure 5B).
  • a first aspect of the present invention relates to a method for the identification and / or isolation of hematopoietic progenitor cells (hereinafter "method of the invention") comprising the following steps:
  • a pluripotent or multipotent stem cell or a group of pluripotent or multipotent stem cells with a nucleic acid molecule capable of integrating into the genome of said cell comprising a first nucleic acid sequence that is operably linked to a second sequence of nucleic acid, where the first nucleic acid sequence comprises the promoter sequence SEQ ID No 1 (proximal promoter) and where the second nucleic acid sequence comprises a marker gene;
  • the first nucleic acid sequence comprises SEQ ID No 2 (alternative promoter).
  • the nucleic acid molecule capable of integrating into the cell genome is SEQ ID No 3 (WE vector) or alternatively the nucleic acid molecule capable of integrating into the genome of the cell. cell is SEQ ID No 4 (AWE vector).
  • the present invention describes the use of vectors, specifically lentiviral vectors, that are specific to hematopoietic tissue by being under the control of the was gene promoter.
  • hematopoietic cells derived from pluripotent or multipotent cells can be specifically labeled by, as used in the present invention, the use of marker genes, whether surface or internal, such as the green protein marker gene fluorescence (GFP or eGFP), or the nerve growth factor surface marker (NGF) gene, any other gene can be used marker known in the state of the art for the same purpose.
  • GFP green protein marker gene fluorescence
  • NGF nerve growth factor surface marker
  • the expression of the marker gene thanks to the presence of the WAS promoter, begins in the early stages of hematopoietic development, which allows identifying early hematopoietic progenitors and monitoring them "/ ' « v / ' vo "thereof and from its differentiation process, under different conditions, towards the different lineages of ethical hematopoy cells.
  • the present invention provides a method for the identification and / or isolation of HPCs (hematopoietic progenitor cells) derived from pluripotent or multipotent cells.
  • said hematopoietic progenitor cells that are identified and isolated by the WAS gene are obtained from pluripotent stem cells.
  • pluripotent stem cells are undifferentiated, immature and self-renewing cells capable of differentiating into cells that constitute tissues derived from any of the three embryonic layers, the ectoderm, the endoderm and the mesoderm, that will give rise to known tissues and structures in higher animals.
  • the identified or isolated progenitor cells are derived from induced human pluripotent cells (iPS) or embryonic stem cells, preferably non-human embryonic stem cells.
  • pluripotent stem cells can be obtained from non-viable triploid zygotes (WO03 / 075646).
  • pluripotent cells are human cells that are derived from adult tissue.
  • the nucleic acid molecule used in the method of the present invention can proceed from a method comprising the following steps: a) Transfect or transduce packaging cells with plasmids capable of producing viral vectors, preferably, lentivirals; Y
  • packaging cells are understood as those cells that have been modified to express the viral proteins necessary for the formation of the viral particle, since these have previously been removed from the viral genome for the construction of the vector.
  • the packaging cells are preferably 293T cells.
  • a preferred embodiment that in no way limits the present invention relates to a method for the identification and isolation of HPCs from iPS cells, comprising the following steps:
  • Hematopoietic parents must appear between days 10 and 15 identified by the expression of the marker gene and the expression of the following combinations of markers: CD34 + CD45 +, CD34 + CD45- and to a lesser extent CD34-CD45-.
  • the method for the identification and isolation of HPCs, described in the present invention is characterized in that the transduced or transfected pluripotent or multipotent cells are maintained in a specific culture medium supplemented with cell growth factors to induce their differentiation. at hematopoietic lineage for 10, 15 or 22 days depending on the hematopoietic population desired.
  • hematogenic hematogenic precursors restricted to hematopoietic lineage
  • HPCs that are CD45- hematopoietic precursors
  • at days 15-22 are obtained more differentiated hematopoietic lineage cells, preferably (CD31 + CD45 + CD34-), although hematopoietic progenitors (CD31 + CD45 + CD34 +) can also be detected, on the other hand, on day 22 differentiated hematopoietic cells (CD45 +) are obtained.
  • the method for the identification and / or isolation of HPCs, described in the present invention is characterized in that the marker gene is preferably GFP and / or eGFP and the characteristic hematopoietic markers present in said cells are preferably CD45 , CD31, CD33 and CD34 and / or any combination thereof.
  • the techniques used for such identification are preferably flow cytometry and qRT-PCR techniques, although any other technique used in the state of the art can be used for the same purpose.
  • the method for the identification and isolation of HPCs, described in the present invention is characterized in that the expression of the marker defines the population of hemogenic progenitor cells and hematopoietic cells.
  • expression of the marker and CD45 gene identifies the population of hematopoietic total cells (CD45 +); the expression of the marker gene and the absence of expression of CD31 and CD45 (CD31-CD45-) identifies a new hemogenic progenitor population, the expression of the marker gene and CD31 and the absence of expression of the CD45 marker (CD31 + CD45-) identifies to the population of hemogenic progenitors restricted to the hematopoietic lineage (shown in Figure 4 as CD34dim); expression of the marker gene and of the CD34 and CD45 markers (CD34 + CD45 +) identifies the population of hematopoietic precursors and the expression of the marker gene and CD33 and CD45 (CD33 + CD45 +) identifies the population of differentiated hem
  • Another aspect described in the present invention relates to identifiable and isolable HPC cells by the method described previously.
  • the HSCs identified and / or isolated according to the method described in the present invention are characterized in that they are derived from pluripotent cells that are selected from any of the following: iPSs, ESCs, bone marrow cells, blood cells peripheral, umbilical cord blood cells and / or body fat cells.
  • the HSCs come from mammals, preferably from man.
  • the HPCs identified and / or isolated at day 10 of hematopoietic differentiation are characterized in that they can be: a) hemogenic progenitors, of unknown origin so far (CD45-CD31 -CD34-); b) hemato-restricted hemogenic progenitors (CD45-CD34 + dim cells with the ability to give rise to hematopoiesis only); c) hematopoietic progenitors (CD45 + CD34 + cells that only give rise to hematopoietic lineage).
  • HPCs identified and isolated at day 10 and which are (GFP + CD31-) identify, as mentioned above, a new cell population of hemogenic progenitors of unknown origin so far.
  • the hematopoietic cells identified and isolated on differentiation day according to the method described in the present invention are characterized in that they are mostly differentiated hematopoietic cells (CD31 + CD45 + CD34-), although hematopoietic progenitors can still be detected ( CD31 + CD45 + CD34 +).
  • the hematopoietic cells identified and isolated at day 22 of differentiation according to the method described in the present invention are characterized in that they are mostly CD33 + myeloid cells.
  • Another aspect described in the present invention relates to the use of HPCs, identified and isolated according to the method described in the present invention, for the manufacture of a medicament.
  • Another aspect described in the present invention relates to the use of HPCs identified and isolated according to the method described in the present invention, for the preparation of a pharmaceutical composition for the treatment of hematopoietic pathologies, preferably primary immunodeficiencies and autoimmune diseases. .
  • HPCs identified and isolated according to the method described in the present invention, for use as a medicament.
  • HPCs identified and isolated according to the method described in the present invention, for use in the treatment of diseases of a hematopoietic nature.
  • compositions comprising a therapeutically effective amount of the identified and isolated HPCs according to the method described in the present invention.
  • the pharmaceutical composition of the invention may also contain, when necessary, other compounds to increase, control or otherwise direct the desired therapeutic effect of the cells.
  • Said compounds comprise, among others, auxiliary substances or pharmaceutically acceptable substances, such as, excipients, buffering agents, surfactants, co-solvents, preservatives, etc.
  • metal chelators to stabilize the cell suspension.
  • the stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, and so on.
  • Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions.
  • suitable pharmaceutical carriers or excipients are described, for example, in "Remington's Pharmaceutical Sciences” by EW Martin. Additional information on these vehicles can be found in any pharmaceutical technology manual (Galenic Pharmacy).
  • Another aspect described in the present invention relates to said pharmaceutical compositions for use in the treatment of pathologies of a hematopoietic nature, preferably primary immunodeficiencies and autoimmune diseases.
  • Another aspect described in the present invention relates to methods of treating pathologies of a hematopoietic nature characterized in that it comprises the administration of a therapeutically effective amount of the HPCs or of the pharmaceutical composition described in the present invention, to a patient.
  • Example 1 Materials and Methods used to carry out the present invention: 1.1 Lines and culture media. 293T cells were grown in Dulbecco's Modified Eagle Medium medium
  • Figure 1A The construction scheme of the lentiviral vectors used in the present invention is shown in Figure 1A.
  • the lentiviral vector WE contains a 500 bp fragment of the proximal was promoter that directs the expression of the selected transgene in the embodiments described in the present invention, eGFP, as described in: Martin, Toscano et al. .
  • the AWE lentiviral vector contains a 387 bp fragment of the alternative was immediately "upstream" promoter of the 500 bp wasal promoter present in the WE vector, as described in: Martin, Toscano et al. . 2005; Samsung, Frecha et al. 2008. All vectors share the self-activating region "self inactivated (SIN) lentiviral backbone" described by (Zufferey, Dull et al. 1998).
  • the eGFP transgene is expressed under the constitutive promoter EFl-ot (htt: / ' www .addgene. Org / 12247) and the EC vector, expresses the eGFP transgene under the control of the cytomegalovirus constituent promoter (CMV) .
  • CMV cytomegalovirus constituent promoter
  • Lentiviral vectors were produced by the co-transfection of 293T cells with three plasmids: (1) vector plasmid (WE, AWE, CE, and pLVTHM), (2) packaging plasmid (pCMVAR 8.91) and (3) plasmid VSV-G envelope (pMD2.G), as described in Toscano, Frecha et al. 2004.
  • the packaging and wrapping plasmids used were obtained from http: // www. addgene org / Didier Throne.
  • 293T cells were plated on amine-treated Petri dishes (Sarstedt, Newton, NC), to ensure exponential growth and 80% confluence.
  • Plasmids pCMVAR 8.91 and pMD2.G were resuspended in 1.5ml of Opti-MEM (Gibco) together with 60 ⁇ of Lipofectamine 2000 (Invitrogen) or 45 ⁇ TransIT 2020 (Mirus Bio LLC Madison, WI, USA) (proportions of plasmid 3 :twenty-one). This mixture was added to the cell culture, previously washed with Opti-MEM. Viral supernatants were collected, filtered through pores with a diameter of 0.45 ⁇ (Nalgene, Rochester, NY), concentrated by ultracentrifugation (Beckman Coulter) and resuspended in MSC-CM culture medium.
  • the hESCs cells were dissociated for 1 minute at room temperature in the presence of collagenase IV. Subsequently, said hESCs were grown in culture plates treated with matrigel, to which the previously concentrated viral particles were added. During the infection procedure, the hESCs adhere to the surface of the culture plate. When the colonies were confluent they expanded. In some cases a second round of infection was necessary until a concentration of 0.6-1 vg / cel was obtained. 1.4. In vitro hematopoietic differentiation and analysis through the formation of embryoid bodies (EBs)
  • EBs embryoid bodies
  • Pluripotent cells differentiated in vitro to cells of the ethical hematopoy lineage Briefly, on day 0, the hESCs were treated with collagenase IV for 1 min and subsequently separated from the culture plate. Subsequently, these cells to culture dishes low adhesion treated with matrigel (Corning, NY) kept in culture overnight in the presence of culture medium KO-Dulbecco 's modified Eagle's medium (Invitrogen, USA) supplemented with 20 transferred % FBS, 1 mmol / L-glutamine, 0.1 mM non-essential amino acids and 0.1 mM ⁇ -mercaptoethanol to obtain embryoid bodies (EBs).
  • EBs embryoid bodies
  • the EBs obtained were centrifuged and maintained in culture in the same medium described above but supplemented with growth factors: bone morphogenic protein 4 (BMP-4) (25ng / ml), fetal tyrosine ligand 3 (Flt -3L) (300ng / ml), stem cell factor (SCF) (300ng / ml), interleukin 3 (IL-3) (10ng / ml), interleukin 6 (IL-6) (10ng / ml) and granulocyte colony stimulating factor (G-CSF) (50ng / ml) (Chadwick, Wang et al. 2003).
  • BMP-4 bone morphogenic protein 4
  • Flt -3L fetal tyrosine ligand 3
  • SCF stem cell factor
  • IL-3 interleukin 3
  • IL-6 interleukin 6
  • G-CSF granulocyte colony stimulating factor
  • EBs were dissociated by adding collagenase B (Roche Diagnostic, Basel, Switzerland) to the culture medium for 2 hours at 37 ° C followed by a 10 minute incubation at 37 ° C with Cell Dissociation Buffer (Gibco) at day 15 to analyze the formation of colony forming units (CFUs) and on days 10, 15 and 22 to perform flow cytometry analysis (FACS).
  • collagenase B Roche Diagnostic, Basel, Switzerland
  • the hESCs were resuspended in a buffer solution containing PBS + FBS + EDTA and subsequently filtered through a 70 ⁇ filter (Becton Dickinson, San Jose, CA). Once disintegrated, they were incubated in the presence of monoclonal antibodies conjugated with different fluorochromes: anti-CD31-phycoerythrin (PE), anti-CD33-PE, anti-CD34-PE-Cy7 (Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA ) and anti-CD45-APC (allophycocyanin) (Miltenyi). Live cells were identified by exclusion of 7-AAD (7-amino-actinomiacin D).
  • eGFP The expression of eGFP was also analyzed by flow cytometry with the FACS Canto II cytometer equipped with the FACS Diva analysis software (Becton Dickinson).
  • CFUs Colony Forming Units Test 20,000-35,000 cells were plated in H4434 methylcellulose (Stem Cell Technologies, Vancouver, Canada) supplemented with 30 U / ml EPO. The cells were incubated at 37 ° C and 5% C0 2 . Colonies were counted based on their morphological characteristics after 10-14 days.
  • the protocol was slightly modified from that described by Pankratz (Pankratz, M. T., et al. 2007).
  • the pluripotent cells, hESCs were grown in suspension as hEBs in MSC-CM for 4 days.
  • the hEBs were then cultured in a neural medium composed of DMEM / F 12, non-essential amino acids, 2 ⁇ g / ml of heparin, and the neural supplement N2 (Gibco) for an additional 3 days.
  • Early neural differentiation was evaluated on day 8 of culture by dissociation and staining with the anti-human antibody A2B5 (neural embryonic marker antigen) (Miltenyi Biotech) or its corresponding control isotype.
  • A2B5 neural embryonic marker antigen
  • the hESC AND-1 cell line was transduced with the different vectors shown in Figure 1A (AWE, WE, CE and pLVTHM) and according to the previously mentioned procedures.
  • the vectors CE and pLVTHM were used, which contain the constitutive promoters CMV and EF l-ot, respectively.
  • HESCs transduced with lentiviral vectors WE and AWE containing respectively 1.4 and 1.3 gc / c were differentiated into hematopoietic lineage by, as mentioned above, the culture in the presence of KOt Dulbecco 's modified Eagle' s medium (Invitrogen, USA) supplemented with 20% FBS, 1 mmol / L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM ⁇ -mercaptoethanol and a cocktail of growth factors: BMP-4 (25ng / ml), Flt-3L (300ng / ml ), SCF (300ng / ml), IL-3 (10ng / ml), IL-6 (10ng / ml) and G-CSF (50ng / ml).
  • BMP-4 25ng / ml
  • Flt-3L 300ng / ml
  • SCF 300ng / ml
  • IL-3 10ng / m
  • eGFP, CD45 and CD33 The expression of eGFP, CD45 and CD33 in said cells was determined after 22 days of induced hematopoietic differentiation (Figure 1C). At this stage of differentiation (day 22) The WE and AWE vectors were only able to express the eGFP transgene in CD45 positive (CD45 +) and CD33 positive (CD33 +) cells. In total, 92.2% (AWE) and 90% (WE) of the hESCs that were eGFP +, were also (CD45 + CD33 +), said extreme indicating almost complete specificity of expression in ethical hematopoy cells.
  • AWE transgene or eGFP marker present in the lentiviral vector
  • hESCs aggregates were cultured in a chemically defined neuronal medium (NM): DMEM / F12, non-essential amino acids, 2 ⁇ g / ml heparin and the N2 neural supplement (Gibco) for another 3 days.
  • NM neuronal medium
  • the neuronal precursors were analyzed by flow cytometry.
  • pLVTHM that expresses eGFP through the constitutive promoter EF l-ot.
  • hESCs transduced with the AWE lentiviral vector and differentiated to a neuronal phenotype were unable to express eGFP.
  • Figure 2 shows the correlation between the expression levels of eGFP and CD45 in different hematopoietic differentiation experiments of hESCs transduced with the WE and AWE lentiviral vectors, as described in the present invention.
  • the greater hematopoietic differentiation greater expression of CD45
  • the expression is detected even if there are no hematopoietic cells (CD45 +).
  • Example 3 The AWE and WE vectors specifically mark hematopoietic progenitors at 10-15 day of differentiation.
  • the expression pattern of the WE and AWE vectors described in the present invention at different hematopoietic differentiation times was then analyzed.
  • the pluripotential cells transduced with said lentiviral vectors as described in the present invention were analyzed on days 5, 10, 15 and 22 during the hematopoietic differentiation process.
  • the expression of the eGFP transgene present in the AWE and WE vectors began to be observed from day 10 ( Figure 4 A and C, histogram central day 10), progressively increasing on successive days of differentiation until days 10-15 ( Figure 4 A and C, central histograms days 10, 15 and 22).
  • the cell populations (CD31-GFP +) were separated and ( CD31-GFP-) using FACS-sorting ( Figure 5A). Once separated, they were incubated in the culture medium that favors hematopoietic differentiation, previously described. Only cells expressing GFP gave rise to cells (CD31 + CD45 +) after 10 days in this differentiation medium ( Figure 5B).
  • SEQ ID No 1 Proximal 500 base pair promoter of the WAS gene:

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Abstract

La présente invention concerne l'utilisation de vecteurs lentiviraux spécifiques du tissu hématopoïétique qui, sous le contrôle du promoteur du gène was (syndrome de Wiskott-Aldrich) (AWE et WE), identifient de nouveaux progéniteurs hématopoïétiques (HPC) et de nouvelles sous-populations dans les progéniteurs déjà décrits dérivés de cellules souches pluripotentes. La présente invention concerne en outre les HPC isolées au moyen du procédé d'identification décrit, ainsi que leur utilisation dans le traitement de pathologies de nature hématopoïétique.
PCT/ES2013/070200 2012-03-26 2013-03-26 Vecteurs pour l'identification de la lignée hématopoïétique WO2013144409A2 (fr)

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