Patent Application
20190060366
Hematopoietic stem cell (HSC) transplantation is currently the only curative treatment modality for a number of stem cell disorders, including both malignant and non-malignant hematologic conditions. Yet, despite the fact that hematopoietic transplant is the only curative option for patients having such stem cell disorders, transplant-related morbidity and mortality remains high, and only a fraction of the patients that could benefit from an HSC transplant actually receive one.
Sources of HSCs for transplantation include the bone marrow itself, umbilical cord blood, and mobilized peripheral blood. Under steady state conditions, HSCs and hematopoietic progenitor cells (HPCs) normally reside within the bone marrow niches, while the mature cells produced by these populations of HSCs and HPCs ultimately exit the bone marrow and enter the peripheral blood. Considerable evidence over the last several decades, however, clearly demonstrates that HSCs and HPCs (collectively referred to as “HSPCs”) also exit the bone marrow niche and traffic to the peripheral blood and we now know that this natural egress into the periphery can be enhanced, allowing for “mobilization” of these cells from the bone marrow to the peripheral blood. Mobilized adult HSCs and HPCs are widely used for autologous and allogeneic transplantation and have improved patient outcomes when compared to bone marrow grafts.
The hematopoietic growth factor, granulocyte-colony stimulating factor (G-CSF) is widely used clinically to mobilize HSC and HPC for transplantation. G-CSF-mobilized peripheral blood stem cells (PBSCs) are associated with more rapid engraftment, shorter hospital stays, and in some circumstances, superior overall survival compared to bone marrow grafts, though the use of G-CSF-mobilized grafts over bone marrow in some allogeneic settings is under scrutiny.
While successful, G-CSF mobilization regimens involve repeated subcutaneous injections and are often associated with morbidity from bone pain (an often severe and debilitating complication), nausea, headache, and fatigue. These can be lifestyle disruptive in normal volunteers and particularly distressing for patients who are enduring the rigors of cancer chemotherapy. In a small population of normal donors, G-CSF has also been associated with serious toxicity, including enlargement of the spleen and splenic rupture, and the pro-coagulant effects of G-CSF can increase the risk of myocardial infarction and cerebral ischemia in high-risk individuals. Despite its success for most patients and donors, poor mobilization in response to G-CSF occurs in 15% of normal, healthy donors, and often those who do achieve sufficient numbers of CD34+ cells require more than one apheresis procedure. Repeated, prolonged sessions of apheresis are particularly common among autologous donors, which is particularly troubling for them given their ongoing ordeals associated with their underlying cancer and its treatment. Up to 60% of patients that fail to mobilize an optimal CD34+ cell dose for autologous transplantation often requiring tandem cycles of high dose chemotherapy. This is particularly an issue for patients with lymphoma and multiple myeloma, who often require extended aphereses and comprise the largest group of transplant recipients.
The availability of alternative methods for mobilizing HSPC could have high impact on the foregoing obstacles associated with HSC transplantation. Needed are novel therapeutics and methods that are capable of enhancing graft acquisition and hematopoietic recovery and engraftment. Also needed are highly engraftable cells that may be used to treat stem cell and/or progenitor cell disorders, such as malignant and non-malignant hematologic diseases.
There remains a need for novel compositions, methods and therapies that are capable of reducing hematopoietic stem cell (HSC) transplant-related morbidity and mortality and enhancing engraftment of transplanted HSCs in subjects in need of a stem cell transplant. The present inventions are directed toward further solutions to address these unmet needs, in addition to having other desirable characteristics. Accordingly, disclosed herein is an isolated, highly engraftable hematopoietic stem cell (heHSC), as well as related methods of preparing such heHSCs and related methods of using such heHSCs for the treatment of stem cell and/or progenitor cell disorders and other diseases for which a stem cell transplant may be indicated.
In certain aspects, the present inventions are directed to an isolated, heHSC, wherein the heHSC is Sca-1+ and c-kit+ and is negative for Lineage markers (e.g., B221−, CD3−, Gr-1−, Mac-1−, TER119−) (e.g., a Sca-1+, c-kit+ and Lin− (SKL) cell). In certain aspects, the isolated heHSC is CD48−. In certain aspects the heHSC is not naturally occurring, i.e., differs from a naturally occurring HSC in one or more ways including but not limited to functionality (e.g., engraftability) and gene expression. In certain aspects, the isolated heHSC is CD150+. In certain aspects, the isolated heHSC is a Signaling lymphocytic activation molecule (SLAM) SKL cell, which is CD150+, CD48−, Sca-1+, c-kit+ and lineage negative. In certain aspects, the isolated heHSC does not express an immunophenotypic means of identifying human hematopoietic stem cells (e.g., the isolated heHSC does not express antigens, markers or other characteristics that may be useful for distinguishing such heHSC from other cell types). In some embodiments, the isolated heHSC comprises a unique transcriptome relative to hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or any combination thereof. For example, in some aspects, the isolated heHSCs disclosed herein are characterized based on their differential expression of one or more of the genes selected from the group consisting of Fos, CD93, Fosb, Dusp1, Jun, Dusp6, Cdk1, Fignl1, Plk2, Rsad2, Sgk1, Sdc1, Serpine2, Spp1, Cdca8, Nrp1, Mcam, Pbk, Akr1cl and Cyp11a1 (e.g., relative to the expression of one or more genes by hematopoietic stem cells mobilized using G-CSF). In some embodiments, the isolated heHSC expresses osteopontin (e.g., the heHSC is OPN+). In some embodiments, the isolated heHSC expresses CD93 (e.g., the heHSC is CD93+) than an HSC obtained from a subject subjected to a conventional mobilization regimen. In some embodiments, the isolated heHSC does not express CD34 or is CD34−. In some embodiments, the isolated heHSC is CD93+ and CD34−. In some embodiments, the heHSC is a non-native or non-naturally occurring cell, i.e., possesses one or more genotypic or phenotypic characteristics not present in native or naturally occurring HSC. In some embodiments, the isolated heHSC is from in a population of cells not present in a non-treated host and/or a host treated with a conventional mobilization regimen (e.g., a cell population with a different gene expression profile or a different phenotype profile). In some embodiments, the heHSC is from in a population of heHSC with a higher proportion of CD93+ cells than a HSC population obtained from a host treated with a conventional mobilization regimen.
Conventional procedures using G-CSF are known in the art. See Schmitt, M et al. “Mobilization of PBSC for Allogeneic Transplantation by the Use of the G-CSF Biosimilar XM02 in Healthy Donors.” Bone Marrow Transplantation 48.7 (2013): 922-925. PMC. Web. 24 Feb. 2017, incorporated herein by reference.
As used herein, “differentially expresses”, when used in reference to a cell population means an expression that is at least 10% higher than or lower than a reference value (e.g., an heHSC population differentially expresses CD93 from an HSC population obtained by a conventional immobilization technique if the heHSC population expresses at least 10% more or less CD93). As used herein, “differentially expresses,” when used in reference to a cell, means that the cell has a different expression pattern of one or more phenotypes than a reference cell.
In certain aspects of the present inventions, the isolated heHSCs disclosed herein may be transformed to express a polynucleotide (e.g., an exogenous polynucleotide). For example, in certain embodiments, an isolated heHSC is transformed with an expression vector to express a polynucleotide (e.g., an exogenous polynucleotide). In some embodiments, the expression vector comprises a viral vector selected from the group consisting of a retrovirus, a herpes simplex, an adenovirus, a lentivirus, and an adeno-associated virus. In some embodiments, the isolated heHSC is transfected with an expression vector that comprises the polynucleotide. In some embodiments, the polynucleotide comprises an exogenous polynucleotide.
Also disclosed herein is the use of isolated heHSCs to deliver an exogenous polynucleotide to a subject in need thereof. For example, the isolated heHSCs disclosed herein may be transformed to express an exogenous polynucleotide and, upon engraftment in the subject's tissues (e.g., bone marrow tissues), the engrafted heHSC expresses the exogenous polynucleotide, thereby delivering the expression product (e.g., a protein, enzyme or amino acid) to the subject.
Also disclosed herein are methods of transforming an isolated heHSC, wherein such methods comprise a step of contacting the heHSC with an expression vector under conditions sufficient for the vector to integrate into the heHSC genome. In yet other embodiments, the isolated heHSC of the present inventions are genetically modified to shut off expression of an endogenous polynucleotide.
In certain embodiments, the isolated heHSC is substantially pure (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 98%, 99% or more pure). In certain aspects, the isolated heHSC is non-quiescent.
Also disclosed herein are methods of preparing an isolated, heHSC. For example, in some embodiments, the isolated heHSC disclosed herein is prepared by contacting a hematopoietic stem cell and/or a progenitor cell with at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof. In some embodiments, the isolated heHSC disclosed herein is prepared by contacting a hematopoietic stem cell and/or a progenitor cell with at least one CXCR2 agonist and at least one CXCR4 antagonist. In some embodiments, such contacting is performed in vivo, for example by administering GROβ or an analog or derivative thereof and plerixafor or an analog or derivative thereof to a human subject. In some embodiments, such contacting is performed in vitro. In some in vivo embodiments, such contacting mobilizes an amount of circulating peripheral blood stem cells in the subject sufficient to harvest a cell dose of between about 1×106/kg body weight and 10×106/kg body weight in a single apheresis session. In some in vivo embodiments, such contacting mobilizes an amount of circulating peripheral blood stem cells in the subject sufficient to harvest a cell dose of between about 2×106/kg body weight and 8×106/kg body weight in a single apheresis session. In some in vivo embodiments, such contacting mobilizes an amount of circulating peripheral blood stem cells in the subject sufficient to harvest a cell dose of between about 3×106/kg body weight and 6×106/kg body weight in a single apheresis session. In some in vitro embodiments, isolated HSC are contacted with sufficient amount of at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof to obtain between 1×106 and 1.2×109 heHSC cells.
In some embodiments, the at least one CXCR2 agonist comprises GROβ or an analog or derivative thereof. In some embodiments the at least one CXCR2 agonist comprises GROβ-Δ4 or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises plerixafor (AMD-3100) or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises ALT1188, ALT1187, ALT1128, ALT1228, or TG-0054 or an analog or derivative thereof. In some embodiments, the CXCR4 antagonist comprises at least one inhibitor described in Debnath B, et al., “Small Molecule Inhibitors of CXCR4,” Theranostics 2013; 3(1):47-75, incorporated herein by reference. In some embodiments, the α9β1 integrin/VLA-4 antagonist is N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine (BOP) or an analog or derivative thereof (e.g., R-BC154). In some embodiments, the VLA-4 antagonist is BIO 5192, Natalizumab, firategrast, or an analog or derivative thereof. In still other embodiments, the at least one CXCR2 agonist is GROβ or an analog or derivative thereof and the at least one CXCR4 antagonist is plerixafor or an analog or derivative thereof. In some embodiments, a Gro-beta analog or derivative is the desamino Gro-beta protein (also known as MIP-2alpha), which comprises the amino acid sequence of mature gro-S protein truncated at its N terminus between amino acid positions 2 and 8, as described in PCT International Application Publication WO/1994/029341, incorporated herein by reference in its entirety. In other embodiments, the Gro-beta analog or derivative is the dimeric modified Gro-beta protein described in U.S. Pat. No. 6,413,510, incorporated herein by reference in its entirety. In some embodiments, the Gro-beta analog or derivative is SB-251353, a Gro-beta analog involved in directing movement of stem cells and other leukocytes, as described by Bensinger et al. (Bone Marrow Transplantation (2009), 43, 181-195, incorporated by reference herein).
The isolated heHSCs disclosed herein are characterized by their enhanced ability to engraft in a target tissue of a subject (e.g., the bone marrow tissue of a subject). Accordingly, in some embodiments upon administration or transplant of the heHSC in a subject such heHSC demonstrates increased engrafting ability, for example, relative to engraftment of the same quantity of hematopoietic stem cells that are contacted or mobilized with granulocyte colony-stimulating factor (G-CSF), chemotherapeutic agents (e.g., mobilizing chemotherapeutic agents), or any combinations thereof. In certain embodiments, such engrafting ability is increased by at least about two-fold, three-fold, four-fold, five-fold, six-fold, or more.
In some embodiments, the heHSC is a non-native cell, i.e., possesses one or more genotypic or phenotypic characteristics not present in native HSC. In some embodiments, the isolated heHSC is from in a population of cells not present in a non-treated host and/or a host treated with a conventional mobilization regimen (e.g., a cell population with a different gene expression profile or a different phenotype profile). In some embodiments, the heHSC is from in a population of heHSC with a higher proportion of CD93+ cells than a HSC population obtained from a host treated with a conventional mobilization regimen.
The isolated heHSCs disclosed herein are also characterized by their ability to produce or cause improved or increased donor chimerism following their engraftment. In some embodiments, upon engraftment of the heHSCs in a subject the heHSCs demonstrate increased donor chimerism, for example, relative to the donor chimerism observed following engraftment of the same quantity of hematopoietic stem cells contacted or mobilized with G-CSF, chemotherapeutic agents (e.g., mobilizing chemotherapeutic agents), or any combinations thereof. In certain embodiments, such donor chimerism is increased by at least about two fold, three-fold, four-fold, five-fold, six-fold, or more. In some embodiments, such donor chimerism is at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more.
In certain aspects, the present inventions are directed to methods of treating a stem cell or progenitor cell disorder. Such methods comprise a step of administering an isolated heHSC (e.g., a SLAM SKL heHSC) to a subject in need thereof, wherein the administered heHSC engrafts in the subject's tissues (e.g., the subject's bone marrow compartment), thereby treating the stem cell or progenitor cell disorder. In some embodiments, the methods described herein comprise administering a population of cells comprising at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% heHSC cells.
In certain aspects, upon engraftment in a subject, the engrafted heHSCs demonstrate enhanced hematopoietic function relative to engraftment of the same quantity of hematopoietic stem cells contacted or mobilized with G-CSF, chemotherapeutic agents (e.g., mobilizing chemotherapeutic agents), or any combinations thereof. In some embodiments, upon engraftment in a subject the engrafted heHSCs demonstrate an enhanced CD34+ number relative to engraftment of the same quantity of hematopoietic stem cells contacted or mobilized with G-CSF, chemotherapeutic agents, or any combinations thereof. In certain embodiments, upon engraftment in a subject the engrafted heHSCs demonstrate enhanced hematopoietic function relative to engraftment of the same quantity of hematopoietic stem cells contacted or mobilized with granulocyte colony-stimulating factor (G-CSF), chemotherapeutic agents, or any combinations thereof.
In some embodiments, the subject (e.g., a human subject) is conditioned for engraftment prior to administering the isolated heHSCs disclosed herein. In some embodiments, the subject (e.g., a human subject) exhibits poor mobilization in response to a conventional mobilization regimen, such as G-CSF.
Also disclosed herein are methods of treating a stem cell and/or progenitor cell disorder in a subject, the method comprising: (a) depleting an endogenous hematopoietic stem cell or progenitor cell population in a bone marrow compartment of the subject; and (b) administering an isolated, non-native heHSC to the subject, wherein the heHSC is Sca-1+, c-kit+ and Lin− (SKL), and where the administered heHSC engrafts in the bone marrow compartment of the subject. In certain embodiments, the heHSC is a SLAM SKL heHSC.
The heHSCs disclosed herein may be used for the treatment of stem cell and/or progenitor cell disorders or any diseases for which a stem cell transplant may be indicted. In some embodiments, such a stem cell or progenitor cell disorder is a malignant hematologic disease. For example, in some embodiments, the malignant hematologic disease may be selected from the group consisting of acute lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, diffuse large B-cell non-Hodgkin's lymphoma, mantle cell lymphoma, lymphoblastic lymphoma, Burkitt's lymphoma, follicular B-cell non-Hodgkin's lymphoma, lymphocyte predominant nodular Hodgkin's lymphoma, multiple myeloma, and juvenile myelomonocytic leukemia. In some embodiments, the stem cell or progenitor cell disorder is a non-malignant disease. For example, in some embodiments the non-malignant disease may be selected from the group consisting of myelofibrosis, myelodysplastic syndrome, amyloidosis, severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, immune cytopenias, systemic sclerosis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Crohn's disorder, chronic inflammatory demyelinating polyradiculoneuropathy, human immunodeficiency virus (HIV), Fanconi anemia, sickle cell disorder, beta thalassemia major, Hurler's syndrome (MPS-IH), adrenoleukodystrophy, metachromatic leukodystrophy, familial erythrophagocytic lymphohistiocytosis and other histiocytic disorders, severe combined immunodeficiency (SCID), and Wiskott-Aldrich syndrome.
Also disclosed herein is an isolated, non-native heHSC, wherein the heHSC is Sca-1+, c-kit+ and Lin− (SKL); wherein the heHSC is prepared by mobilizing hematopoietic stem cells and/or progenitor cells from a bone marrow compartment of a subject to a peripheral compartment of the subject by administering at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof to the subject, and isolating the mobilized hematopoietic stem cells and/or progenitor cells from the peripheral compartment of the subject. In some embodiments, the isolated heHSC does not express CD48 or is CD48−. In some embodiments, the isolated heHSC expresses CD150 or is CD150+. In some embodiments, the isolated heHSC expresses CD93 or is CD93+. In certain aspects, the isolated heHSC does not express an immunophenotypic means of identifying human hematopoietic stem cells. In some embodiments the heHSC is a SLAM SKL heHSC. In some embodiments, the at least one CXCR2 agonist comprises GROβ or an analog or derivative thereof. In some embodiments the at least one CXCR2 agonist comprises GROβ-Δ4 or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises plerixafor (AMD-3100) or an analog or derivative thereof. In still other embodiments, the at least one CXCR2 agonist is GROβ or an analog or derivative thereof and the at least one CXCR4 antagonist is plerixafor or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises ALT1188, ALT1187, ALT1128, ALT1228, or TG-0054. In some embodiments, the α9β1 integrin/VLA-4 antagonist is N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine (BOP) or an analog or derivative thereof (e.g., R-BC154). In some embodiments, the VLA-4 antagonist is BIO 5192 or Natalizumab, or an analog or derivative thereof.
In some embodiments, the isolated heHSC comprises a unique transcriptome relative to hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or any combination thereof. For example, in some aspects, the isolated heHSCs disclosed herein are characterized based on their differential expression of one or more of the genes selected from the group consisting of Fos, CD93, Fosb, Dusp1, Jun, Dusp6, Cdk1, Fignl1, Plk2, Rsad2, Sgk1, Sdc1, Serpine2, Spp1, Cdca8, Nrp1, Mcam, Pbk, Akr1cl and Cyp11a1, relative to, for example the expression of one or more genes in HSCs mobilized using G-CSF. In certain aspects, the isolated heHSC is non-quiescent. In some embodiments, the isolated heHSC is OPN+(e.g., the isolated heHSC express osteopontin). In some embodiments, the isolated heHSC differentially expresses CD93 (e.g., the heHSC is CD93+). In some embodiments, the isolated heHSC does not express CD34 or is CD34−. In some embodiments, the isolated heHSC is CD93+ and CD34−.
In certain aspects of the present inventions, the isolated heHSCs disclosed herein are transformed to express a polynucleotide (e.g., an isolated heHSC may be transformed with an expression vector to express an exogenous polynucleotide). In some embodiments, the expression vector comprises a viral vector selected from the group consisting of a retrovirus, a herpes simplex, a lentivirus, an adenovirus, and an adeno-associated virus. In some embodiments, the isolated heHSC is transfected with an expression vector that comprises the polynucleotide. In some embodiments, the polynucleotide comprises an exogenous polynucleotide.
Also disclosed herein is the use of the isolated heHSC to effect or otherwise facilitate the delivery of an exogenous polynucleotide to a subject in need thereof. For example, the isolated heHSC disclosed herein may be transformed to express an exogenous polynucleotide and, upon engraftment in the subject's tissues (e.g., bone marrow tissues), the engrafted heHSC expresses the exogenous polynucleotide, thereby delivering the expression product of the exogenous polynucleotide (e.g., a protein or amino acid) to the subject.
In some embodiments, also disclosed herein are methods of transforming an isolated heHSC, wherein such methods comprise a step of contacting the heHSC with an expression vector under conditions sufficient for the vector to integrate into the heHSC genome. In yet other embodiments, the isolated heHSC of the present inventions are genetically modified to shut off expression of an endogenous polynucleotide.
In certain embodiments, the isolated heHSC is substantially pure.
The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present disclosure relates to a non-native, highly engraftable hematopoietic stem cell (heHSC) that is useful in connection with stem cell transplantation and the treatment of stem cell and/or progenitor cell disorders. Disclosed herein are isolated, non-native heHSCs, methods of their use and manufacture, and kits that comprise such heHSCs for use in connection with stem cell transplantation or the treatment of stem cell and/or progenitor cell disorders. The heHSCs disclosed herein are useful, for example, for transplantation and/or engraftment in a subject in connection with the treatment of any disease requiring stem cell transplantation.
The work described herein relates to the surprising discovery that heHSCs that are prepared by contacting or mobilizing with a combination of a CXCR2 agonist (e.g., GROβ) and a CXCR4 antagonist (e.g., plerixafor) exhibit superior engrafting ability, for example, superior engrafting ability relative to HSCs or peripheral blood stem cells (PBSCs) that are mobilized using traditional mobilizing regimens (e.g., granulocyte-colony stimulating factor (G-CSF) or chemotherapeutic agents). Accordingly, certain aspects of the present inventions relate to non-native, isolated heHSCs that are prepared by contacting or mobilizing hematopoietic stem cells and/or progenitor cells using a combination of one or more CXCR2 agonists (e.g., GROβ) and one or more CXCR4 antagonists (e.g., plerixafor). An exemplary method of mobilizing hematopoietic stem cells and/or progenitor cells in a subject comprises administering to the subject a combination of at least one CXCR2 agonist and at least one CXCR4 antagonist in amounts sufficient to mobilize such hematopoietic stem cells and/or progenitor cells into the subject's peripheral blood. The isolated heHSCs disclosed herein and the related methods of their preparation by mobilizing hematopoietic stem cells and/or progenitor cells have a variety of useful applications, for example for the treatment of stem cell and/or progenitor cell disorders.
In some embodiments, aspects of the present inventions relate to non-native, isolated heHSCs that are prepared by contacting or mobilizing hematopoietic stem cells and/or progenitor cells using a combination of at least one CXCR2 agonist (e.g., GROβ) and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof.
As used herein, the term “mobilizing” refers to the act of inducing the migration of hematopoietic stem cells and/or progenitor cells (e.g., heHSCs) from a first location (e.g., the stem cell niche or bone marrow tissues of a subject) to a second location (e.g., the peripheral blood or an organ, such as the spleen, of a subject). For example, in certain embodiments, the non-native, isolated heHSCs disclosed herein may be prepared by mobilizing hematopoietic stem cells and/or progenitor cells from the stem cell niche of a human subject into the subject's peripheral tissue by administering to the subject a combination of one or more CXCR2 agonists (e.g., GROβ) and one or more CXCR4 antagonists (e.g., plerixafor), following which the mobilized heHSCs may be harvested or isolated (e.g., by apheresis), as further described herein. With regard to the heHSCs disclosed herein, the term “isolated” means that the heHSC is substantially free of other cell types or cellular materials with which may be present when the heHSC is isolated from a treated subject. In some embodiments, an isolated heHSC or an isolated population of heHSCs is a substantially pure population of heHSCs, for example, as compared to the heterogeneous population from which the cells were isolated or enriched from (e.g., substantially pure as compared to the population of mobilized cells). In some embodiments, the heHSCs are enriched from a biological sample that is obtained from a subject following treatment with a combination of a CXCR2 agonist (e.g., GROβ) and a CXCR4 antagonist (e.g., plerixafor). In one embodiment, the mobilized and harvested heHSCs disclosed herein may be used in connection with an allogeneic or an autologous transplant. The terms “enriching” or “enriched” are used interchangeably herein and mean that the yield (fraction) of heHSCs is increased by at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more over the fraction of mobilized cells.
As used herein with respect to a population of heHSCs, term “substantially pure”, refers to a population of heHSCs that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, and still more preferably at least about 99% pure with respect to the cells making up a total population of mobilized cells. Recast, the terms “substantially pure” or “essentially purified”, with regard to a population of heHSCs, refers to a population of cells that contain fewer than about 20%, more preferably fewer than about 15%, 12%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are not heHSCs as defined by the terms herein. In some embodiments, the present invention encompasses methods to expand a population of heHSCs, wherein the expanded population of heHSCs is a substantially pure population.
While certain embodiments disclosed herein contemplate the in vivo preparation of the heHSCs by mobilizing hematopoietic stem cells and/or progenitor cells, it should be understood that the present inventions are not limited to such in vivo methods. Rather, also contemplated are in vitro methods of preparing heHSCs, for example by contacting hematopoietic stem cells and/or progenitor cells with a combination of a CXCR2 agonist (e.g., GROβ) and a CXCR4 antagonist (e.g., plerixafor), VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof. As used herein, the term “contacting” means bringing two or more moieties together, or within close proximity of one another such that the moieties may interact with each other. For example, in one embodiment of the present invention, a hematopoietic stem cell and/or a progenitor cell is contacted with a CXCR2 agonist and/or a CXCR4 antagonist to produce and/or mobilize a heHSC.
Contemplated CXCR2 agonists include any compounds or agents that are capable of activating the CXCR2 receptor (e.g., the human CXCR2 receptor). Exemplary CXCR2 agonists include chemokines, cytokines, biologic agents, antibodies and small organic molecules. For example, contemplated chemokines acting via the CXCR2 receptor include without limitation GROβ, GROα, GROγ, GCP-2 (granulocyte chemo-attractant protein 2), IL-8, NAP-2 (neutrophil activating peptide 2), ENA-78 (epithelial-cell derived neutrophil activating protein 78), and modified forms of any of the foregoing. In some embodiments, the CXCR2 agonist is selected from the group of compounds or agents consisting of small organic or inorganic molecules; oligosaccharides; polysaccharides; biological macromolecules selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; and any combination thereof.
In certain aspects, the CXCR2 agonist comprises GROβ.
In some embodiments, the at least one CXCR2 agonist is the chemokine GROβ or an analog or derivative thereof. An exemplary form of GROβ is the human GROβ polypeptide (GenBank Accession: AAP13104; SEQ ID NO: 1). In certain aspects, an exemplary form of GROβ is the human GROβ (UniProt ID No. P19875; SEQ ID NO: 2).
An exemplary GROβ analog or derivative is the desamino GROβ protein (also known as MIP-2alpha), which comprises the amino acid sequence of mature gro-S protein truncated at its N terminus between amino acid positions 2 and 8, as described in PCT International Application Publication WO/1994/029341, the contents of which are incorporated herein by reference in their entirety. Another GROβ analog or derivative is the dimeric modified GROβ protein described in U.S. Pat. No. 6,413,510, the contents of which are incorporated herein by reference in their entirety. Still another exemplary GROβ analog or derivative is SB-251353, a GROβ analog involved in directing movement of stem cells and other leukocytes, as described by Bensinger, et al., Bone Marrow Transplantation (2009), 43, 181-195, the entire contents of which are incorporated by reference herein.
In some embodiments of the present inventions, the at least one CXCR2 agonist is or comprises GROβ-Δ4 (e.g., SEQ ID NO: 3) or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist is selected from the group consisting of GROβ or an analog or derivative thereof and GROβ-Δ4 or an analog or derivative thereof.
Contemplated CXCR4 antagonists include any compounds or agents that are capable of blocking the CXCR4 receptor or preventing its activation. For example, contemplated are compounds and agents that block or otherwise interfere with the binding or interaction of the CXCR4 receptor with such receptor's ligand. Also contemplated are compounds or agents that block the downstream effects of the activated CXCR4 receptor. In some embodiments, the CXCR4 antagonist is selected from the group of compounds or agents consisting of small organic or inorganic molecules; oligosaccharides; polysaccharides; biological macromolecules selected from the group consisting of peptides, proteins, peptide analogs and derivatives; peptidomimetics; nucleic acids selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, and aptamers; and any combination thereof.
In some embodiments of the present inventions, the at least one CXCR4 antagonist is plerixafor (formerly known as AMD-3100), the structure of which is depicted below (I), or an analog or derivative thereof.
In some embodiments, the at least one CXCR4 antagonist is MOZOBIL® or an analog or derivative thereof. Exemplary analogs of plerixafor include, but are not limited to, AMD11070, AMD3465, KRH-3955, T-140, and 4F-benzoyl-TN14003, as depicted below (II-VI, respectively) and described by De Clercq, Pharmacol Ther. (2010) 128(3):509-18, the contents of which are incorporated by reference herein in their entirety.
In some embodiments, the at least one CXCR4 antagonist comprises ALT1188, ALT1187, ALT1128, ALT1228, or TG-0054 or an analog or derivative thereof. In some embodiments, the CXCR4 antagonist comprises at least one inhibitor described in Debnath B, et al., “Small Molecule Inhibitors of CXCR4,” Theranostics 2013; 3(1):47-75, incorporated herein by reference.
In some embodiments, non-native, isolated heHSCs are prepared by contacting or mobilizing hematopoietic stem cells and/or progenitor cells using a combination of at least one CXCR2 agonist (e.g., GROβ) and at least one α9β1 integrin/VLA-4 antagonist. In some embodiments, the α9β1 integrin/VLA-4 antagonist is N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine (BOP) or an analog or derivative thereof (e.g., R-BC154). In some embodiments, non-native, isolated heHSCs are prepared by contacting or mobilizing hematopoietic stem cells and/or progenitor cells using a combination of at least one CXCR2 agonist (e.g., GROβ) and at least one VLA-4 antagonist. In some embodiments, the VLA-4 antagonist is BIO 5192, Natalizumab, or an analog or derivative thereof.
In some embodiments, the at least one CXCR2 agonist is or comprises GROβ or an analog or derivative thereof, and the at least one CXCR4 antagonist is or comprises plerixafor (AMD-3100) or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist is selected from the group consisting of GROβ-Δ4 or an analog or derivative thereof and the at least one CXCR4 antagonist is selected from the group consisting of plerixafor or an analog or derivative thereof.
The combination of at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof may be administered directly to a subject in combination or, in certain aspects, may be administered independently. For example, the at least one CXCR2 agonist and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof can be, but need not be, administered (e.g., administered intravenously) to a subject at the same time. In one embodiment, the at least one CXCR2 agonist is administered in one or more doses, followed by the administration of the at least one CXCR4 antagonist in one or more doses.
In addition to inducing a faster mobilization (e.g., about two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, twelve-fold, fifteen-fold, twenty-fold or more faster relative to traditional mobilization regimens that are performed using, for example, G-CSF or, alternatively, within one hour, within 45 minutes, within 30 minutes, within 15 minutes within 10 minutes, within 5 minutes or faster) and producing a greater quantity of mobilized stem cells (e.g., heHSCs), the combination of at least one CXCR2 agonist (e.g., GROB-Δ4 or an analog or derivative thereof) and at least one CXCR4 antagonist (e.g., plerixafor or an analog or derivative thereof), VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof mobilizes a non-native stem cell that is characterized by its enhanced engrafting ability and its unique genetic signatures, as illustrated in
One such marker, CD93, is expressed in hematopoietic cells at the apex of hematopoiesis. These early hematopoietic CD93 expressing cells in humans may also be negative for CD34. heHSC populations generated upon treatment with combination of at least one CXCR2 agonist and at least one CXCR4 antagonist which also exhibit CD93 expression are indicative of early lineage stem cells and may serve to support improved transplantation and/or engraftment.
Similarly, in certain embodiments, stem cells that are mobilized using the combination of at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof may be characterized by improved function. In particular, the engrafting ability of the heHSCs mobilized using the combination of at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof is surprisingly increased or enhanced relative to the engrafting ability of stem cells or PBSCs that are mobilized following the contacting of hematopoietic stem cells and/or progenitor cells with traditional mobilizing agents, such as G-CSF.
In certain aspects, the heHSCs are characterized by their increased or enhanced engrafting ability relative to stem cells or PBSCs that are mobilized following the contacting of hematopoietic stem cells and/or progenitor cells with one or more chemotherapeutic agents (e.g., chemotherapeutic mobilization agents). Exemplary chemotherapeutic agents include paclitaxel, etoposide, vinblastine, doxorubicin, bleomycin, methotrexate, 5-fluorouracil, 6-thioguanine, cytarabine, cyclophosphamide, cisplatinum and combinations thereof. In certain aspects, such chemotherapeutic agents mobilize hematopoietic stem cells and/or progenitor cells. For example, such a chemotherapeutic mobilization agent may comprise EPO. In some embodiments, such a chemotherapeutic mobilization agent is or comprises stem cell factor. In some embodiments, such a chemotherapeutic mobilization agent is or comprises TPO. In still other embodiments, such a chemotherapeutic mobilization agent is or comprises parathyroid hormone.
As used herein, the term “hematopoietic stem cells” or “HSC” refers to stem cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells). Stem cells are defined by their ability to form multiple cell types (multipotency) and their ability to self-renew. Hematopoietic stem cells can be identified, for example by cell surface markers such as CD34−, CD133+, CD48−, CD150+, CD244−, cKit+, Sca1+, and lack of lineage markers (negative for B220, CD3, CD4, CD8, Mac1, Gr1, and Ter119, among others).
As used herein, the term “hematopoietic progenitor cells” encompasses pluripotent cells which are committed to the hematopoietic cell lineage, generally do not self-renew, and are capable of differentiating into several cell types of the hematopoietic system, such as granulocytes, monocytes, erythrocytes, megakaryocytes, B-cells and T-cells, including, but not limited to, short term hematopoietic stem cells (ST-HSCs), multi-potent progenitor cells (MPPs), common myeloid progenitor cells (CMPs), granulocyte-monocyte progenitor cells (GMPs), megakaryocyte-erythrocyte progenitor cells (MEPs), and committed lymphoid progenitor cells (CLPs). The presence of hematopoietic progenitor cells can be determined functionally as colony forming unit cells (CFU-Cs) in complete methylcellulose assays, or phenotypically through the detection of cell surface markers (e.g., CD45−, CD34+, Ter119−, CD16/32, CD127, cKit, Sca1) using assays known to those of skill in the art.
In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise SKL cells. In certain aspects, the mobilized hematopoietic stem cells and/or progenitor cells comprise SKL SLAM cells. In certain aspects, the mobilized hematopoietic stem cells and/or progenitor cells exhibit a SLAM (Signaling lymphocyte activation molecule) expression pattern which is CD150+, CD48−. A SLAM expression pattern (SLAM code) is an expression pattern of specific markers (SLAM markers) that are used to identify subpopulations of hematopoietic stem cells and multipotent progenitors. See Oguro, et al. (2013) “SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors,” Cell Stem Cell, 13(1), 102-116, and references cited therein.
In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise CD34−, CD133+ cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise common myeloid progenitor cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise granulocyte/monocyte progenitor cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise megakaryocyte/erythroid progenitor cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise committed lymphoid progenitor cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise a combination of common myeloid progenitor cells, granulocyte/monocyte progenitor cells, megakaryocyte/erythroid progenitor cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise CD150-, CD48−, CD244+ cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise CD150-, CD48+, CD244+ cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise Sca-1−, c-kit+, Lin−, CD34+, CD16/32mid cells. In some embodiments, the mobilized hematopoietic stem cells and/or progenitor cells comprise Sca-1−, c-kit+, Lin−, CD34−, CD16/32low cells. In some embodiments, the isolated heHSC does not express an immunophenotypic means of identifying human hematopoietic stem cells.
In some embodiments, the isolated heHSCs disclosed herein comprise a unique transcriptome relative to hematopoietic stem cells contacted with G-CSF, a chemotherapeutic agent, or a combination thereof. For example, in certain aspects, the isolated heHSCs disclosed herein are characterized based on their differential expression of one or more of the genes identified in
The heHSCs disclosed herein are prepared by mobilizing or contacting hematopoietic stem cells and/or progenitor cells with a combination of a CXCR2 agonist and a CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof. As used herein, the terms “highly engraftable hematopoietic stem cell” and “heHSC” refer to the isolated population or fraction of stem cells or PBSCs that are, for example, mobilized from the stem cell niche or bone marrow of a subject into the peripheral blood or organs of the subject following the administration of one or more CXCR2 agonists (e.g., GROβ or an analog or derivative thereof) and one or more CXCR4 antagonists (e.g., plerixafor or an analog or derivative thereof), VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof. In certain aspects, such heHSCs are substantially pure.
In some embodiments, the isolated heHSCs disclosed herein are immunophenotypically unique relative to cells or stem cells mobilized using traditional mobilization regimens (e.g., stem cells mobilized using G-CSF). For example, as illustrated in
In some embodiments, a population of cells (i.e., a cell population comprising or consisting of heHSC) isolated by the methods disclosed herein (e.g., by contacting cells with a combination of at least one CXCR2 agonist (e.g., GROβ) and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof) has an increased or decreased proportion of cells exhibiting one or more cell surface markers or one or more expression profiles disclosed herein as compared to cells isolated by conventional methods. The one or more cell surface markers or cell expression profiles may be increased or decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the one or more cell surface marker is CD93. In some embodiments, after performing the methods disclosed herein, an obtained cell population may be assayed to determine whether the prevalence of one or more cell surface markers or cell expression profiles has increased or decreased to determine whether the obtained cell population is suitable as heHSC for transplantation. In some embodiments, the obtained cell population is assayed to determine if at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells are CD93+. Any suitable assay (e.g., FACS analysis) may be used for the determination.
In some embodiments, the obtained cell population may be further enriched for a desired cell surface marker or gene expression pattern to obtain a desired heHSC population for transplantation. In some embodiments, the obtained cell population may be enriched for CD93+ cells or CD93+ and CD34− cells. In some embodiments, the cell population may be enriched by about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold or more. In some embodiments, the cell population may be enriched to contain at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of cells containing a desired cell surface marker or cell expression pattern (e.g., enriched for CD93+ cells or CD93+/CD34− cells). Any suitable procedure (e.g., FACS sorting) may be used for the enrichment. In some embodiments, the isolated heHSCs disclosed herein are not immunophenotypically unique relative to cells or stem cells mobilized using traditional mobilization regimens (e.g., stem cells mobilized using G-CSF). Such isolated heHSC may be functionally unique relative to cells or stem cells mobilized using traditional mobilization regimens.
Upon mobilization, which in certain instances may occur within 15-30 minutes of having administered a CXCR2 agonist and a CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof, the mobilized heHSCs can be harvested or isolated (e.g., via apheresis) as disclosed herein and are useful for subsequent transplantation in a subject in need thereof. For example, such mobilized heHSCs may be harvested or isolated for autologous transplantation into a subject or for allogeneic transplantation into a recipient subject. In some instances, the harvesting or isolation of the mobilized hematopoietic stem cells and/or progenitor cells can be initiated within as little as 15 minutes following the administration of the at least one CXCR2 agonist and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof. In some embodiments, the harvesting or isolating procedure can begin in as little as 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 47 minutes, 52 minutes, 58 minutes, or an hour after administration of the at least one CXCR2 agonist and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof.
The disclosure contemplates the use of any suitable method of harvesting and/or collecting mobilized hematopoietic stem cells and/or progenitor cells to prepare the isolated heHSCs disclosed herein. In some embodiments harvesting the mobilized hematopoietic stem cells and/or progenitor cells comprises apheresis. In some embodiments, the combination of at least one CXCR2 agonist (e.g., GROβ or GROβ-Δ4) and at least one CXCR4 antagonist (e.g., plerixafor), VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof rapidly and efficiently mobilizes mobilized hematopoietic stem cells and/or progenitor cells, and exhibits increased efficiencies compared to traditional mobilizing regimens. As a result, in some embodiments an apheresis procedure may be performed on the same day that the at least one CXCR2 agonist and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof are administered to the subject. In other words, harvesting mobilized heHSCs from a subject (e.g., a donor) via apheresis can be performed on the same day that the mobilization agents are administered to the subject (e.g., during a single visit to a healthcare facility). In some embodiments, an apheresis procedure may be performed on the same day that at least one CXCR2 agonist (e.g., GROβ or GROβ-Δ4) and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof is administered to the subject.
In some embodiments, administration of the at least one CXCR2 agonist (e.g., GROβ or GROβ-Δ4) and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof mobilizes an amount of hematopoietic stem cells and/or progenitor cells in the subject to harvest a heHSC cell dose of between about 1×106/kg body weight and 10×106/kg body weight in a single apheresis session. In some embodiments, a single session of apheresis collects enough heHSCs for a cell dose of between about 1×106/kg and 10×106/kg of the recipient's body weight. In some embodiments, administration of the at least one CXCR2 agonist (e.g., GROβ or GROβ-Δ4) and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof mobilizes an amount of hematopoietic stem cells and/or progenitor cells in the subject to harvest enough heHSCs for a cell dose of between about 2×106/kg body weight and 8×106/kg body weight in a single apheresis session. In some embodiments, a single session of apheresis collects enough heHSCs for a cell dose of between about 2×106/kg and 8×106/kg of the recipient's body weight. In some embodiments, administration of the at least one CXCR2 agonist (e.g., GROβ or GROβ-Δ4) and the at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof mobilizes an amount of hematopoietic stem cells and/or progenitor cells in the subject to harvest a heHSC cell dose of between about 3×106/kg body weight and 6×106/kg body weight in a single apheresis session. In some embodiments, a single session of apheresis collects enough heHSCs for a cell dose of between about 1×106/kg and 10×106/kg of the recipient's body weight.
Following harvesting, the isolated heHSCs disclosed herein may be administered to or transplanted in the donor subject (e.g., an autologous transplant), or alternatively may be donated to a different subject in need thereof (e.g., allogeneic transplant). In certain aspects, the administration or transplant of the isolated heHsCs occurs following or in combination with radiation or chemotherapy.
The mobilized heHSC disclosed herein are characterized by their increased engrafting ability (e.g., a two-fold increased engrafting ability), which makes such heHSCs suitable for use in connection with gene therapy. For example, where genetic manipulation of cells is associated with a corresponding reduction in their engrafting ability and, due to the improved or enhanced engrafting ability of the heHSCs disclosed herein, such heHSCs are rendered more tolerant to genetic manipulation, following which only limited reductions in their engrafting ability may be observed.
Gene therapy can be used to transform a heHSC, modify a heHSC to replace a gene product, to treat disease, or to improve engraftment of the heHSC following implantation into a subject. For example, in certain embodiments, the heHSCs disclosed herein may be transformed with an expression vector (e.g., a viral vector selected from the group consisting of a retrovirus, a herpes simplex, a lentivirus, an adenovirus, and an adeno-associated virus). In some embodiments, the isolated heHSC is transformed or transfected with an expression vector that comprises a polynucleotide. In some embodiments, the polynucleotide comprises an exogenous polynucleotide. In some embodiments, the expression product of a polynucleotide is a protein that is not endogenously expressed or is under expressed by the subject's cells.
As used herein, the term “transform” means to introduce into a heHSC an exogenous polynucleotide (e.g., a nucleic acid or nucleic acid analog) which replicates within that heHSC, that encodes a gene product (e.g., an amino acid, polypeptide sequence, protein or enzyme) which is expressed in that heHSC, and/or that is integrated into the genome of that heHSC so as to affect the expression of a genetic locus within the genome. The term “transform” is used to embrace all of the various methods of introducing such polynucleotides (e.g., nucleic acids or nucleic acid analogs), including, but not limited to the methods referred to in the art as transformation, transfection, transduction, or gene transfer, and including techniques such as microinjection, DEAE-dextran-mediated endocytosis, calcium phosphate coprecipitation, electroporation, liposome-mediated transfection, ballistic injection, viral-mediated transfection, and the like.
In some embodiments, also disclosed herein are methods of transforming an isolated heHSC, wherein such methods comprise a step of contacting the heHSC with an expression vector under conditions sufficient for the vector to integrate into the heHSC genome. In yet other embodiments, the isolated heHSC of the present inventions are genetically modified to shut off expression of an endogenous polynucleotide.
As used herein, the term “vector” means any genetic construct, such as for example, a plasmid, phage, transposon, cosmid, chromosome, virus and/or virion, which is capable transferring nucleic acids between cells. Vectors may be capable of one or more of replication, expression, and insertion or integration, but need not possess each of these capabilities. Thus, the term includes cloning, expression, homologous recombination, and knock-out vectors.
In certain aspects, prior to engraftment, a mobilized hematopoietic stem cell and/or progenitor cell can be manipulated to express one or more desired polynucleotides or gene products (e.g., one or more of a polypeptide, amino acid sequence protein and/or enzyme). Gene therapy can be used to either modify a mobilized hematopoietic stem cell and/or progenitor cell to replace a polynucleotide or gene product or to add or knockdown a gene product. In some embodiments the genetic engineering is done, for example, to treat disease, following which the genetically engineered heHSC would be transplanted and engraft into a subject. For example, a mobilized heHSC may be manipulated to express one or more polynucleotides or genes that would enhance the engrafting ability of the transplanted heHSC.
Techniques for transfecting cells are known in the art. In an exemplary embodiment, gene therapy can be used to insert a polynucleotide (e.g., DNA) into a mobilized hematopoietic stem cell from a patient or subject with a genetic defect to correct such genetic defect, following which the corrected or genetically engineered mobilized hematopoietic stem cell may be transplanted into a subject.
In some other embodiments, the heHSCs disclosed herein can be used as carriers for gene therapy.
In some embodiments, the isolated heHSCs and the related methods of mobilizing such heHSCs are useful for treating subjects that have demonstrated poor mobilization in response to a conventional hematopoietic stem cell and/or progenitor cell mobilization regimen (e.g., subjects that have failed to mobilize a sufficient numbers of stem cells following a mobilization regimen comprising or consisting of G-CSF). For example, such heHSCs and the related methods disclosed herein may be used to enhance hematopoietic stem cell and/or progenitor cell mobilization in individuals exhibiting stem cell and/or progenitor cell mobilopathy. Accordingly, in certain embodiments, any of the methods and compositions disclosed herein may be suitable for use in mobilizing hematopoietic stem cell and/or progenitor stem cells in a subject having an underlying disease that impairs egress of such hematopoietic stem cells and/or progenitor stem cells from bone marrow and into the peripheral circulation, including, for example, subjects that have or are at risk of developing diabetic stem cell mobilopathy. In certain aspects, subjects that have failed to mobilize a sufficient number of hematopoietic stem cells and/or progenitor cells in response to a mobilization regimen comprising G-CSF (e.g., subjects that have failed to mobilize a sufficient number of stem cells about five days after receiving a G-CSF mobilization regimen) are candidates for mobilization using the methods and compositions disclosed herein. In certain embodiments, the isolated heHSCs may be administered to a subject exhibiting mobilopathy for the treatment of a stem cell or progenitor cell disorder.
As used herein to describe a mobilization regimen, the term “conventional” generally refers to those mobilization regimens that have traditionally been used to mobilize stem cells. For example, conventional mobilization regimens include those comprising or consisting of G-CSF and that have historically been used to mobilize stem cells from the bone marrow compartment. Such convention mobilization regimens are frequently associated with poor mobilization results, which may often occur over an extended period of time (e.g., over about 5 days), and subjecting the patient to repeated and prolonged apheresis procedures.
In addition to being phenotypically unique relative to stem cells mobilized using traditional mobilization regimens, the heHSCs disclosed herein are characterized by their improved functional properties. For example, in certain embodiments, the heHSCs disclosed herein are characterized by their improved engrafting ability. Accordingly, certain aspects of the methods disclosed herein comprise administering or otherwise transplanting the isolated, non-native heHSCs to a subject in need, such that the administered heHSCs engraft in the tissues (e.g., the bone marrow tissue) of the recipient subject. As used herein, the terms “engrafting” and “engraftment” refer to placing or administration of the heHSCs into an animal (e.g., by injection), wherein following such placement or administration, the heHSCs persist in vivo. Engraftment may be readily measured by the ability of the transplanted heHSCs to, for example, contribute to the ongoing blood cell formation or by assessing donor chimerism following the transplant of such heHSCs.
Successful stem cell transplantation depends on the ability to engraft sufficient quantities of transplanted stem cells in the tissues of the subject (e.g., the bone marrow tissues of the subject). The heHSCs disclosed herein are characterized by their improved engrafting ability and accordingly, certain aspects of the present invention relate to methods of treating stem cell and/or progenitor cell disorders or other diseases requiring transplantation of hematopoietic stem cells and/or progenitor cells by administering to a subject the non-native, isolated heHSCs disclosed herein.
The heHSCs disclosed herein are also characterized by their ability to achieve enhanced or improved donor chimerism following their engraftment in the tissues of a subject. For example, as illustrated in
In certain aspects, the heHSCs disclosed herein are also characterized by their ability to achieve an enhanced or improved CD34+ number upon engraftment in a subject. For example, such engrafted heHSCs demonstrate an enhanced or improved CD34+ number relative to an engraftment of the same quantity of hematopoietic stem cells contacted with G-CSF or one or more chemotherapeutic agents described herein. In some embodiments, such CD34+ number is increased by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, 150%, 200%, 300%, or more relative to, for example, the CD34+ number observed following engraftment of a G-CSF-mobilized stem cell. In some embodiments, such CD34+ number is increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or more relative to, for example, the CD34+ number observed following engraftment of a G-CSF-mobilized stem cell.
In some embodiments, also disclosed herein are methods of treating a stem cell or progenitor cell disorder or a disease requiring transplantation of stem cells, the methods comprising administering the isolated, non-native heHSCs to a subject, wherein the administered heHSCs engrafts in the subject's tissues (e.g., the subject's bone marrow compartment), thereby treating the stem cell or progenitor cell disorder.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a stem cell disorder, progenitor cell disorder or any disease requiring stem cell transplantation, generally refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” also includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally effective if one or more symptoms or clinical markers of the condition or disease are reduced. Alternatively, treatment is effective if the progression of a condition is reduced or halted. That is, treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized state of, for example, a condition, disease, or disorder described herein, or delaying or slowing onset of a condition, disease, or disorder described herein, and an increased lifespan as compared to that expected in the absence of treatment.
As used herein, the term “administering,” generally refers to the placement of the heHSCs described herein into a subject (e.g., the parenteral placement of heHSCs into a subject) by a method or route which results in delivery of such heHSCs to an intended target tissue or site of action (e.g., the bone marrow tissue of a subject). In certain aspects, the term “administering” refers to the placement of at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof to a subject to mobilize hematopoietic stem cells and/or progenitor cells from, for example, the subject's bone marrow tissues and into the subject's peripheral tissues (e.g., mobilizing such hematopoietic stem cells and/or progenitor cells out of the bone marrow compartment and into one or more of the peripheral compartments, such as the peripheral blood compartment).
The isolated, non-native heHSCs disclosed herein are useful for the treatment of any disease, disorder, condition, or complication associated with a disease, disorder, or condition, in which transplantation of hematopoietic stem cells and/or progenitor cells is desirable. In some embodiments, the present inventions relate to methods of treating diseases that require peripheral blood stem cell transplantation. In some embodiments, the disclosure provides method of treating stem cell disorders and progenitor cell disorders in a subject in need of such treatment. Examples of such stem cell and progenitor disorders include hematological malignancies and non-malignant hematological diseases.
In some embodiments, the disease, stem cell disorder or progenitor cell disorder is a hematological malignancy. Exemplary hematological malignancies which can be treated with the heHSCs and methods described herein include, but are not limited to, acute lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, diffuse large B-cell non-Hodgkin's lymphoma, mantle cell lymphoma, lymphoblastic lymphoma, Burkitt's lymphoma, follicular B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, lymphocyte predominant nodular Hodgkin's lymphoma, multiple myeloma, and juvenile myelomonocytic leukemia.
In some embodiments, the disease, stem cell disorder or progenitor cell disorder is a non-malignant disorder. Exemplary non-malignant diseases which can be treated with the methods and heHSCs described herein include, but are not limited to, myelofibrosis, myelodysplastic syndrome, amyloidosis, severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, immune cytopenias, systemic sclerosis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Crohn's disease, chronic inflammatory demyelinating polyradiculoneuropathy, human immunodeficiency virus (HIV), Fanconi anemia, sickle cell disease, beta thalassemia major, Hurler's syndrome (MPS-IH), adrenoleukodystrophy, metachromatic leukodystrophy, familial erythrophagocytic lymphohistiocytosis and other histiocytic disorders, severe combined immunodeficiency (SCID), and Wiskott-Aldrich syndrome.
As used herein, the term “subject” means any human or animal. In certain aspects, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing (e.g., all of the above), but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal (e.g., a primate or human). In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, and is not limited to these examples. Mammals other than humans can be advantageously used, for example, as subjects that represent animal models of, for example, a hematological malignancy. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female.
In certain embodiments, a subject can be one who has been previously diagnosed with or otherwise identified as suffering from or having a condition, disease, stem cell disorder or progenitor cell disorder described herein in need of treatment (e.g., of a hematological malignancy or non-malignant disease described herein) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. Rather, a subject can include one who exhibits one or more risk factors for a condition or one or more complications related to a condition.
A “subject in need” of treatment for a particular condition (e.g., a stem cell or progenitor cell disorder) can be a subject having that condition, diagnosed as having that condition, or at increased risk of developing that condition relative to a given reference population. In some embodiments, the methods of treatment described herein comprise selecting a subject diagnosed with, suspected of having, or at risk of developing a hematological malignancy, for example a hematological malignancy described herein. In some embodiments, the methods described herein comprise selecting a subject diagnosed with, suspected of having, or at risk of developing a non-malignant disease, for example a non-malignant disease described herein.
In other aspects of the invention, heHSC described herein may be produced by obtaining a HSC cell population by any conventional method disclosed in the art and enriching the HSC cell population for one or more cell surface markers or gene expression profiles for heHSC disclosed herein. In some embodiments, the obtained HSC cell population is enriched for CD93+ cells. In some embodiments, the HSC cell population is enriched for CD93+/CD34− cells. In some embodiments, the HSC cell population is enriched by about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold or more. In some embodiments, the cell population may be enriched to contain at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of cells containing a desired cell surface marker or cell expression pattern (e.g., enriched for CD93+ cells or CD93+/CD34− cells). Any suitable procedure (e.g., FACS sorting) may be used for the enrichment.
Some aspects of the invention are directed towards a method of making an HSC product comprising: i) contacting hematopoietic stem cells and/or progenitor cells with at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof to produce a candidate product; ii) providing a target expression profile for an heHSC product; iii) determining whether the candidate product meets the target expression profile of an heHSC product; and iv) releasing the candidate product as an heHSC product if the candidate product meets the target expression profile of an heHSC product.
In some embodiments, the target expression profile comprises Sca-1+, c-kit+ and Lin− (SKL) cells. In some embodiments, the target expression profile comprises CD48− cells. In some embodiments, the target expression profile comprises CD150+ cells. In some embodiments, the target expression profile comprises CD93+ cells. In some embodiments, the target expression profile comprises CD34− cells. In some embodiments, the target expression profile comprises OPN+ cells.
“The target expression profile” refers to a transcriptome and/or cell surface marker profile indicating the presence of heHSC cells or a certain percentage of heHSC cells in a cell population. In some embodiments, the target expression profile comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of cells in the candidate product or enriched candidate product having one or more cell surface markers. In some embodiments, the target expression profile can be a transcriptome profile of the candidate product or enriched candidate product indicating an heHSC product. In some embodiments, the transcriptome profile can be similar or substantially similar to the profiles shown in
In some embodiments, the contacting of the hematopoietic stem cells and/or progenitor cells with at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof is performed in vivo. In some embodiments, the contacting is performed in vitro.
In some embodiments, the at least one CXCR2 agonist comprises GROβ or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist comprises GROβ-Δ4 or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises plerixafor or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist is GROβ or an analog or derivative thereof, and wherein the at least one CXCR4 antagonist is plerixafor or an analog or derivative thereof.
In some embodiments of the invention, the heHSC product, upon transplant into a subject, demonstrates increased engrafting ability relative to engraftment of the same quantity of hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or a combination thereof. In some embodiments, the engrafting ability is increased by at least about two-fold. In certain embodiments, such engrafting ability is increased by at least about two-fold, three-fold, four-fold, five-fold, six-fold, or more.
In some embodiments of the invention, upon engraftment in a subject the heHSC product demonstrates increased donor chimerism relative to engraftment of the same quantity of hematopoietic stem cells contacted with G-CSF, a chemotherapeutic agent, or a combination thereof. In some embodiments, the donor chimerism is increased by at least about two fold. In certain embodiments, such donor chimerism is increased by at least about two-fold, three-fold, four-fold, five-fold, six-fold, or more. In some embodiments, donor chimerism is increased by at least about 50%.
In some embodiments, the heHSC product is non-quiescent.
In some embodiments, the method of making an HSC product additionally comprises a step of enriching the candidate product for one or more cell surface markers and/or one or more gene expression profiles. Any suitable method of enrichment may be employed. In some embodiments, the method is FACS.
In some embodiments, the heHSC product comprises a unique transcriptome relative to hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or a combination thereof. In some embodiments, the heHSC product differentially express one or more of genes selected from the group consisting of Fos, CD93, Fosb, Dusp1, Jun, Dusp6, Cdk1, Fignl1, Plk2, Rsad2, Sgk1, Sdc1, Serpine2, Spp1, Cdca8, Nrp1, Mcam, Pbk, Akr1cl and Cyp11a1, relative to one or more genes expressed by hematopoietic stem cells mobilized using G-CSF. In some embodiments, the heHSC product comprises at least a unique transcriptome or a unique phenotype as compared to a naturally occurring HSC.
In some aspects of the invention, the heHSC product is transformed to express a polynucleotide. In some embodiments, the heHSC product is transformed with an expression vector to express a polynucleotide. In some embodiments, the expression vector comprises a viral vector selected from the group consisting of a retrovirus, a herpes simplex, a lentivirus, an adenovirus, and an adeno-associated virus. In some embodiments, the heHSC product is transfected with an expression vector that comprises the polynucleotide. In some embodiments, polynucleotide comprises an exogenous polynucleotide.
In some embodiments, the heHSC product comprises at least 40% CD93+ cells. In some embodiments, the heHSC product comprises at least about 2×106 cells. In some embodiments, the hematopoietic stem cells and/or progenitor cells are human or mouse cells.
Another aspect of the invention is directed to a method of treating a stem cell or progenitor cell disorder comprising: i) contacting hematopoietic stem cells and/or progenitor cells with at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof to produce a candidate product; ii) providing a target expression profile for an heHSC product; iii) determining whether the candidate product meets the target expression profile of an heHSC product; and iv) administering the candidate product to a subject in need thereof if the candidate product meets the target expression profile of an heHSC product.
In some embodiments, the target expression profile comprises Sca-1+, c-kit+ and Lin− (SKL) cells. In some embodiments, the target expression profile comprises CD48− cells. In some embodiments, the target expression profile comprises CD150+ cells. In some embodiments, the target expression profile comprises CD93+ cells. In some embodiments, the target expression profile comprises CD34− cells. In some embodiments, the target expression profile comprises OPN+ cells.
“The target expression profile” refers to a transcriptome and/or cell surface marker profile indicating the presence of heHSC cells or a certain percentage of heHSC cells in a cell population. In some embodiments, the target expression profile comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of cells in the candidate product or enriched candidate product having one or more cell surface markers. In some embodiments, the target expression profile can be a transcriptome profile of the candidate product or enriched candidate product indicating an heHSC product. In some embodiments, the transcriptome profile can be similar or substantially similar to the profiles shown in
In some embodiments, the contacting of the hematopoietic stem cells and/or progenitor cells with at least one CXCR2 agonist and at least one CXCR4 antagonist, VLA-4 antagonist, α9β1 antagonist, α9β1 integrin/VLA-4 antagonist or combination thereof is performed in vivo. In some embodiments, the contacting is performed in vitro.
In some embodiments, the at least one CXCR2 agonist comprises GROβ or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist comprises GROβ-Δ4 or an analog or derivative thereof. In some embodiments, the at least one CXCR4 antagonist comprises plerixafor or an analog or derivative thereof. In some embodiments, the at least one CXCR2 agonist is GROβ or an analog or derivative thereof, and wherein the at least one CXCR4 antagonist is plerixafor or an analog or derivative thereof.
In some embodiments of the invention, the heHSC product, upon transplant into a subject, demonstrates increased engrafting ability relative to engraftment of the same quantity of hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or a combination thereof. In some embodiments, the engrafting ability is increased by at least about two-fold. In certain embodiments, such engrafting ability is increased by at least about two-fold, three-fold, four-fold, five-fold, six-fold, or more.
In some embodiments of the invention, upon engraftment in a subject the heHSC product demonstrates increased donor chimerism relative to engraftment of the same quantity of hematopoietic stem cells contacted with G-CSF, a chemotherapeutic agent, or a combination thereof. In some embodiments, the donor chimerism is increased by at least about two fold. In certain embodiments, such donor chimerism is increased by at least about two-fold, three-fold, four-fold, five-fold, six-fold, or more. In some embodiments, donor chimerism is increased by at least about 50%.
In some embodiments, the heHSC product is non-quiescent.
In some embodiments, the method of making an HSC product additionally comprises a step of enriching the candidate product for one or more cell surface markers and/or one or more gene expression profiles. Any suitable method of enrichment may be employed. In some embodiments, the method is FACS.
In some embodiments, the heHSC product comprises a unique transcriptome relative to hematopoietic stem cells contacted with granulocyte colony-stimulating factor (G-CSF), a chemotherapeutic agent, or a combination thereof. In some embodiments, the heHSC product differentially express one or more of genes selected from the group consisting of Fos, CD93, Fosb, Dusp1, Jun, Dusp6, Cdk1, Fignl1, Plk2, Rsad2, Sgk1, Sdc1, Serpine2, Spp1, Cdca8, Nrp1, Mcam, Pbk, Akr1cl and Cyp11a1, relative to one or more genes expressed by hematopoietic stem cells mobilized using G-CSF. In some embodiments, the heHSC product comprises at least a unique transcriptome or a unique phenotype as compared to a naturally occurring HSC.
In some aspects of the invention, the heHSC product is transformed to express a polynucleotide. In some embodiments, the heHSC product is transformed with an expression vector to express a polynucleotide. In some embodiments, the expression vector comprises a viral vector selected from the group consisting of a retrovirus, a herpes simplex, a lentivirus, an adenovirus, and an adeno-associated virus. In some embodiments, the heHSC product is transfected with an expression vector that comprises the polynucleotide. In some embodiments, polynucleotide comprises an exogenous polynucleotide.
In some embodiments, the heHSC product comprises at least 40% CD93+ cells. In some embodiments, the heHSC product comprises at least about 2×106 cells. In some embodiments, the hematopoietic stem cells and/or progenitor cells are human or mouse cells.
In some embodiments, the stem cell or progenitor cell disorder is a malignant hematologic disease. In some embodiments, the malignant hematologic disease is selected from the group consisting of acute lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, diffuse large B-cell non-Hodgkin's lymphoma, mantle cell lymphoma, lymphoblastic lymphoma, Burkitt's lymphoma, follicular B-cell non-Hodgkin's lymphoma, lymphocyte predominant nodular Hodgkin's lymphoma, multiple myeloma, and juvenile myelomonocytic leukemia. In some embodiments, the stem cell or progenitor cell disorder is a non-malignant disease. In some embodiments, the non-malignant disease is selected from the group consisting of myelofibrosis, myelodysplastic syndrome, amyloidosis, severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, immune cytopenias, systemic sclerosis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Crohn's disorder, chronic inflammatory demyelinating polyradiculoneuropathy, human immunodeficiency virus (HIV), Fanconi anemia, sickle cell disorder, beta thalassemia major, Hurler's syndrome (MPS-IH), adrenoleukodystrophy, metachromatic leukodystrophy, familial erythrophagocytic lymphohistiocytosis and other histiocytic disorders, severe combined immunodeficiency (SCID), and Wiskott-Aldrich syndrome.
In certain aspects, the heHSCs described herein can be provided in the form of a kit. For example, the kit may comprise one or more isolated, non-native heHSCs and informational or instructional materials relating to the use or administration of such heHSCs to a subject in need. In some embodiments, such kits may comprise at least one CXCR2 agonist, at least one CXCR4 antagonist and instructions for their administration to a subject to mobilize and/or harvest the hematopoietic stem cells and/or progenitor cells, thereby preparing the isolated heHSCs disclosed herein.
It is to be understood that the invention is not limited in its application to the details set forth in the description or as exemplified. The invention encompasses other embodiments and is capable of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
While certain agents, compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the methods and compositions of the invention and are not intended to limit the same.
The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, (e.g., in Markush group or similar format) it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.
To address the still remaining deficiencies in hematopoietic mobilization, the present inventors believe an effective alternative method is the use of rapid mobilizing agents that do not require multiple injections, that are more predictable in their peak mobilization kinetics, and that result in an enhanced CD34+ number and hematopoietic function upon transplant. One agent with potential is the CXCR2 agonist, GROβ. GROβ and GROβ-Δ4 (collectively referred to herein as “GROβ”) rapidly mobilize hematopoietic stem cells (HSC), including all classes of short-term progenitor cells as well as long-term repopulating cells. In mice, peak GROβ-induced mobilization occurs within 15-30 minutes of administration. Moreover, not only was the observed mobilization faster following GROβ administration, the present inventors believe that the stem cell quality was also greater, at least in view of the improved engrafting ability of the mobilized stem cells (e.g., the two-fold greater engrafting ability of the stem cells mobilized from the bone marrow compartment, relative to stem cells mobilized using, for example, a mobilization regimen comprising C-GSF) and the donor chimerism observed following engraftment of such mobilized stem cells.
To assess this, the present inventors mobilized large cohorts of mice (15-20 per group) with either G-CSF (125 ug/kg/day, five days) or with a combination of GROβ (2.5 mg/kg) and plerixafor (AMD-3100) (5 mg/kg), and then sorted the peripheral blood for highly purified SLAM SKL cells (CD150+, CD48−, Sca-1+, c-kit+, lineage negative)
In two separate experiments, the present inventors then competitively transplanted either (a) 190 SLAM SKL cells against 300,000 whole bone marrow competitors, or (b) 50 SLAM SKL cells against 300,000 whole bone marrow competitors. This experimental design allowed for a direct assessment of the engrafting ability of the mobilized SLAM SKL cells, independent of accessory cell populations (e.g., non-CD150+, CD48−, Sca-1+, c-kit+, lineage negative cells) that may have been mobilized, as well as normalized the HSC content so that the same number of HSCs from either the G-CSF-mobilized donors, or the GROβ plus plerixafor-mobilized donors, went into the irradiated recipients. As illustrated in
Over the last decade, there has been increasing evidence that the hematopoietic stem cell (HSC) pool is heterogeneous in function, with identification of HSCs with differing lineage outputs, kinetics of repopulation, length of life-span, and perhaps differences amongst HSCs contributing to homeostatic blood production from those that are the engraftable units in transplantation. To date, however, there are no reliable methods for prospectively isolating differing HSC populations to study heterogeneity. Rather, much of the available data has been acquired based on clonal tracking, single cell transplantation, etc.
Much like panning for gold, the present inventors can now use the differential mobilization properties of the mobilization regimen using GROβ and plerixafor and the regimen using G-CSF as a “biologic sieve” to isolate the heterogeneous HSC populations from the blood. These differential mobilization properties enabled the present inventors, and without destroying the cell, to prospectively isolate what is referred to herein as a highly engraftable HSC (heHSC) population for further functional analysis, and to prospectively isolate a differing HSC population with known, predictable function (the heHSCs) for further molecular characterization.
As a preliminary proof of concept and to demonstrate the feasibility of the approach described herein, SLAM SKL cells were sorted from large cohorts of mice that were treated or mobilized with either G-CSF, or with the combination of GROβ and plerixafor (AMD-3100), as described in Example 1.
In the present study, 200 cells were directly sorted into 5 uL TCL lysis buffer (Qiagen, #1031576). Library preparation was performed by the Smart-Seq2 protocol (Picelli et al., 2013) with subsequent RNA sequencing by Illumina NextSeq500. In addition to SLAM SKL cells from the G-CSF mobilized blood and the GROβ plus plerixafor mobilized blood, additional control samples were sequenced, including steady state bone marrow, bone marrow from the G-CSF-treated mice group, bone marrow from the GROβ plus plerixafor-treated mice, and a “drug spike” control, which consisted of G-CSF mobilized blood spiked with GROβ (350 ng/ml) plus AMD-3100 (10 ug/ml), concentrations based on prior PK data, for 15 minutes, with subsequent downstream processing for FACS sorting. This enabled the present inventors to directly compare the heHSCs from those that were isolated from G-CSF mobilized HSCs, HSCs from the bone marrow of treated and untreated mice, and a drug control to account for any direct effects the GROβ plus plerixafor may have had on the gene signatures that are not due to specific, differential mobilization effects. The RNASeq data was subsequently analyzed, as illustrated in
Surprisingly, as illustrated in
Hematopoietic stem cells (HSCs) are at the apex of lifelong blood cell production. Recent clonal analysis studies suggest that HSCs are heterogeneous in function and those that contribute to homeostatic production may be distinct from those that engraft during transplant. The present inventors developed a rapid mobilization regimen utilizing a unique CXCR2 agonist (an N-terminal truncated MIP-2a) and the CXCR4 antagonist AMD-3100. A single subcutaneous injection of both agents together resulted in rapid mobilization in mice with a peak progenitor cell content in blood reached within 15 minutes.
The observed mobilization was equivalent to a 5-day regimen of G-CSF and is the result of synergistic signaling, and was blocked in CXCR4 or CXCR2 knockout mice, confirming receptor and mechanism specificity and is caused by synergistic release of MMP-9 from neutrophils that was blocked in MMP-9 knockout mice, mice treated with an anti-MMP-9 antibody, TIMP-1 transgenic mice, or mice where neutrophils were depleted in vivo using anti-GR-1 antibody. In vivo confocal imaging of mice demonstrated that the mobilization regimen caused a rapid and transient increase in bone marrow vascular permeability, “opening the doorway” for hematopoietic egress to the peripheral blood.
Transplantation of 2×106 peripheral blood mononuclear cells (PBMCs) from the rapid regimen resulted in a 4 or 6 day quicker recovery of neutrophils and platelets, respectively, compared to a G-CSF mobilized graft (n=12 mice per group, P<0.01). In limiting dilution competitive transplants, the rapid regimen demonstrated a greater than 2-fold enhancement in competitiveness (n=30 mice/treatment group, 2 individual experiments, P<0.001). Additionally, in secondarily transplanted mice, competitiveness of the rapidly mobilized graft increased as measured by contribution to chimerism, while G-CSF mobilized grafts remained static (n=16 mice/group, P<0.01). Surprisingly, despite robust enhancement in both short and long-term engraftment by the rapidly mobilized graft, phenotypic analysis of the blood of mobilized mice for CD150+CD48− Sca-1+c-kit+ Lineage neg (SLAM SKL) cells, a highly purified HSC population, showed lower numbers of phenotypically defined HSCs than in the G-CSF group.
The foregoing data suggest that a unique subset of “highly engraftable” HSCs (heHSCs) are mobilized by the rapid regimen comprising an N-terminal truncated MIP-2a and AMD-3100, compared to G-CSF. However, as our earlier studies were performed using grafts that contained the total PBMC fraction (similar to the clinical apheresis product) the present inventors could not rule out the potential contribution of accessory cells to the enhanced engrafting ability of the heHSCs.
Following the conclusions set out in Example 3, in 3 independent experiments, the present inventors mobilized large cohorts of mice with the rapid regimen comprising an N-terminal truncated MIP-2a (2.5 mg/kg) and AMD-3100 (5 mg/kg), or G-CSF (125 ug/kg/day, fice days) and sorted SLAM SKL cells from the PBMC fraction and competitively transplanted equal numbers of SLAM SKL cells (190, or 50) from either the rapid regimen or G-CSF and tracked contribution to chimerism over 36 weeks. Remarkably, the heHSCs from the rapid regimen demonstrated a 2-fold enhancement in competitiveness compared to SLAM SKL cells from the G-CSF group (n=11 mice/group, P<0.0004). See
While appreciation for HSC heterogeneity has grown, methods are lacking for prospectively isolating differing HSC populations with known biologic function, to study molecular heterogeneity. The present inventors sought to use the differential mobilization properties of our rapid regimen and G-CSF to isolate the heterogeneous HSC populations from the blood. The present inventors again flow sorted SLAM SKL cells from mice mobilized with the rapid regimen or G-CSF and performed RNASeq analysis of the purified populations. The heHSCs mobilized by the rapid regimen had a unique transcriptomic signature compared to G-CSF mobilized or random HSCs acquired from bone marrow (P<0.000001). Strikingly, gene set enrichment analysis (GSEA) demonstrated that the heHSCs had a gene signature highly significantly clustered to that of fetal liver HSCs, further demonstrating the selective harvesting of a subset of highly engraftable stem cells. Our results mechanistically define a new mobilization strategy, that in a single day can mobilize a graft with superior engraftment properties compared to G-CSF, and selectively mobilize a novel population of heHSCs with an immature molecular phenotype capable of robust long-term engraftment.
This application claims the benefit of U.S. Provisional Application Ser. No. 62/413,821, filed Oct. 27, 2016 and U.S. Provisional Application No. 62/300,694, filed Feb. 26, 2016, the contents of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/19778 | 2/27/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62300694 | Feb 2016 | US | |
| 62413821 | Oct 2016 | US |
