Patent Application
20180325947
Hematopoietic stem cells (HSC) are a unique and rare population of cells that have the ability to reconstitute the whole hematopoietic system and to undergo self-renewal for the maintenance of their population. Gene therapy (GT), which involves transferring a gene into HSCs, offers an attractive treatment strategy for curing hematopoietic disorders. However, GT requires ex vivo manipulation and culture of HSCs largely results in a large amount of HSC loss as they differentiate into hematopoietic progenitor cells (HPCs).
Currently, the numbers of human HSCs that repopulate after autologous transplants are a major limitation to effective gene transfer. Myelo-ablative conditioning is therefore often required to destroy resident HSCs, giving an engraftment advantage to the limited numbers of genetically-manipulated HSCs following ex vivo manipulation.
There is a lack of a good experimental model of human HSCs other than non-human primate models, which have limitations with respect to availability, the numbers of animals that can be transplanted, and high costs.
Immune deficient mice, specifically the NOD/SCID/IL-2Rgnull (NSG mice), can robustly engraft human CD34+ HSC/P, giving rise to B, Myeloid and T cell progeny, and have been used to study human hematopoiesis in vivo. However, most studies in NSG mice use umbilical cord blood CD34+ HSC/P that engraft readily but do not mimic bone marrow (BM) or mobilized peripheral blood (MPB) derived HSC/P kinetics or engraftment. The latter are primarily used for gene therapy.
Thus, development of a robust model of human HSC (not HPC) engraftment that can be used to test HSC maintenance during ex vivo manipulation will greatly advance the field of gene therapy.
The present disclosure is based, at least in part, on the unexpected discovery that inhibiting activation of p38 mitogen-activated protein kinase (MAPK) in stem cells, for example, using a p38 MAPK inhibitor such as doramapimod, successfully reduced the loss of stem cells during in vitro or ex vivo culture wherein the stem cells have undergone genetic manipulations that induce a DNA double strand break, such as vector integration, thereby increasing engraftment of the cultured stem cells in vivo.
Accordingly, one aspect of the present disclosure features a method for preparing stem cells such as hematopoietic stem cells (HSCs) having enhanced engraftment activity. The method comprises culturing stem cells (e.g., HSCs) that have undergone a genetic manipulation in a culture medium comprising an effective amount of a p38 MAPK inhibitor. The genetic manipulation induces a DNA double strand break in the stem cells. For example, the stem cells that are amenable to the methods described herein can be non-cycling or non-dividing stems cells (resting cells).
In another aspect, the present disclosure provides a method for preparing human stem cells such as hematopoietic stem cells (HSCs) having enhanced engraftment capacity, the method comprising: culturing human adult stem cells (e.g., human adult HSCs) in a medium that comprises an effective amount of a p38 MAPK inhibitor. In some examples, the human adult stem cells (e.g., human adult HSCs) are non-cycling or non-dividing human adult stem cells.
Any of the methods described herein may further comprise, prior to the culturing step, genetically manipulating the stem cells. The genetic manipulation may comprise transducing the stem cells with an integrating vector or performing genome editing in the stem cells. The genome editing may involve use of, e.g., but not limited to CRISPR-Cas9 systems, zinc finger nucleases (ZFN), and/or transcription activator-like effector-based nucleases (TALEN). In some examples, the genetic manipulation may be performed prior to a cell division cycle. Examples of an integrating vector include, but are not limited to viral vectors such as lentiviral vector and retroviral vectors.
In any of the methods described herein, the method may further comprise administering or transplanting to a subject in need thereof the HSCs that have been cultured in the presence of a p38 MAPK inhibitor. The HSCs may be cultured for 1 to 7 days prior to their administration or transplantation into the subject.
Further, the present disclosure provides a method of treating a hematopoietic disorder in a subject in need of the treatment. The method comprises: (a) providing HSCs that have been genetically manipulated and cultured in a medium comprising an effective amount of a p38 for at least 24 hours or longer, (b) transplanting a first population of the HSCs to the subject after (a), and optionally (c) transplanting a second population of the HSCs to the subject after (b).
In any methods described herein, the stem cells (e.g., HSCs) can be derived from bone marrow, peripheral blood cells, and/or umbilical cord blood of a suitable source (e.g., human). The stem cells (e.g., HSCs) can be allogeneic stem cells (e.g., HSCs) or autologous stem cells (e.g., HSCs).
In any aspects described herein, the p38 MAPK inhibitor can be a protein, a nucleic acid, a small molecule, or a combination thereof. In some examples, the p38 MAPK inhibitor can be a p38 MAPK blocking agent (e.g., a small molecule that binds p38-α and blocks p38 MAPK signaling). Examples of the p39 MAPK inhibitor include, but are not limited to doramapimod (e.g., BIRB-796), ralimetinib (e.g., LY2228820 dimesylate), aminopyridine-based, ATP-competitive inhibitors of p38 MAPK (e.g., Vx702), pyridinyl imidazole inhibitors (e.g., SB203580), and any combinations thereof.
In any of the methods described herein, the amount of the p38 MAPK inhibitor can be effective to increase the proportion of stem cells (e.g., HSCs) in the G0 quiescent phase and to decrease the proportion of the stem cells (e.g., HSCs) in the S-G2-M phase before the first cell division cycle (e.g., 24 hours); to delay the transition of stem cells from G0 quiescent phase to S phase; to reduce loss of long term repopulating potential (LTRP) in the stem cells; to reduce the myeloid skewing phenotype in the stem cells; and/or to promote engraftment of the HSCs transplanted to the subject.
In any of the methods described herein, the subject can be a human subject. In some embodiments, the subject is a human patient having a hematopoietic disorder.
Also described herein is a method for assessing engraftment of human hematopoietic stem cells (HSCs), the method comprising: (i) obtaining HSCs from a human subject; (ii) transplanting the HSCs to an immune deficient animal; and (iii) measuring the level of CD45+ cells in the mobilized peripheral blood of the animal, which is in reverse correlation to human HSC engraftment. In some examples, the immune deficient animal can be an immune deficient mouse, such as an NSG mouse.
In some embodiments, the HSCs can be human adult HSCs, which may be obtained from the bone marrow or peripheral blood cells of a human subject. When desirable, the HSCs can be cultured ex vivo prior to step (ii). In some examples, the HSCs have undergone a genetic manipulation that induces a DNA double strand break in the ex vivo culture. The genetic manipulation can be transduction of an integrating vector, for example, a viral vector such as a retroviral vector or a lentiviral vector. When the HSCs have been transduced by a lentiviral vector, the HSCs can be transplanted to the animal after 18-96 hours of ex vivo culture. When the HSCs have been transduced by a retroviral vector, the HSCs can be transplanted to the animal after 72-96 hours of ex vivo culture. The genetic manipulation may comprise performing genome editing in the stem cells, e.g., using any methods known in the art including, e.g., CRISPR-Cas9 systems, zinc finger nucleases (ZFN), and/or transcription activator-like effector-based nucleases (TALEN).
Also within the scope of the present disclosure is a composition for use in promoting engraftment of stem cells (e.g., HSCs) in a subject who is in need for a stem cell (e.g., HSC) transplantation. The composition comprises any of the p38 MAPK inhibitors described herein and stem cells such as hematopoietic stem cells. The composition may further comprise a cell culture medium. The subject can be a human patient having a hematopoietic disorder.
The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Hematopoietic stem cells (HSCs) are desirable targets for gene therapy for various inherited hematological diseases including, e.g., hemoglobinopathies. However, gene transfer into HSCs typically involves ex vivo manipulation and culture, which results in a large amount of HSC loss with their differentiation to hematopoietic progenitor cells (HPCs), making these cells less competent at engraftment in vivo. Currently, the limited number of HSCs that repopulate after autologous transplant is a major limitation to effective gene transfer. Thus, large transduced HSCs at myelo-ablative conditioning to destroy resident HSCs are currently required to provide an engraftment advantage. However, the source of HSCs is limited and myelo-ablative conditioning could induce adverse effects or complications in patients who are sensitive to such procedure. Accordingly, there is a need to develop methods and compositions for preparing hematopoietic stem cells (HSCs) for increased engraftment in subjects who are in need of a HSC transplantation.
The present disclosure is based, at least in part, on the unexpected discovery that extended ex vivo culture and gene manipulation induce a DNA damage response (DDR) in hematopoietic stem cells (HSCs) during ex vivo culture, which is mediated by activation of p38 MAPK stress signaling. For example, such HSCs after extended ex vivo culture and gene manipulation showed increased DNA double strand breaks (for example, increased γ-H2AX signaling), increased DNA damage response (for example, increased 53BP1 signaling), loss of long term repopulating potential, increased myeloid lineage bias, and/or increased phosphorylation level of p38 MAPK in the HSCs. Further, it was discovered that blocking the p38 MAPK signaling (e.g., via an anti-p38 MAPK inhibitor) significantly decreased DNA double strand breaks and/or DNA damage response that occurred in HSCs with both increasing time in culture and with transduction, retained long germ repopulating potential, reduced myeloid lineage skewing in the HSCs, and/or increased engraftment of HSCs transplanted in vivo. Thus, p38 MAPK inhibitors would be expected to enhance the engraftment capacity of HSCs cultured ex vivo via blocking the p38 MAPK stress signaling and reducing, e.g., DNA double strand breaks.
Accordingly, in some aspects, the present disclosure provides ex vivo cell culture methods for preserving the stemness of stem cells such as HSCs in the presence of one or more p38 MAPK inhibitors, which suppresses at least DNA damage response due to extended ex vivo culture and genetic manipulation. Such stem cells may have undergone a manipulation that causes a DNA double-strand break, for example, transduced by a vector that is capable of integrating into the genome of the stem cells, or induced by genome editing. Genome editing methods are generally classified based on the type of endonuclease that is involved in generating double stranded breaks in the target nucleic acid. Examples of genome editing methods include, e.g., use of zinc finger nucleases (ZFN), transcription activator-like effector-based nuclease (TALEN), meganucleases, and/or CRISPR/Cas systems. Without wishing to be bound by any particular theory, ex vivo manipulation of HSCs may activate stress signaling, resulting in their commitment to hematopoietic progenitor cells (HPCs) at the expense of HSC self-renewal. Events that involve DNA double-strand breaks, (e.g., vector integration events) may exacerbate HSC loss and the present discovery showed that blocking the p38 MAPK signaling pathway would rescue HSC loss in ex vivo culturing. Accordingly, described herein are also ex vivo methods and compositions for preparing stem cells such as HSCs having enhanced engraftment activity using one or more p38 MAPK inhibitors. The methods and compositions described herein promote engraftment of HSCs in a subject (e.g., a human patient) after HSC transplantation.
p38 mitogen-activated protein kinases (MAPKs) are a class of mitogen-activated protein kinases that are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and/or osmotic shock. The p38 MAPK family includes four members, p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12/ERK6), and p38-δ (MAPK13/SAPK4), which are involved in a signaling cascade that controls cellular response to cytokine and stress. Inhibitors for any of the p38 MAPK members can be used in the ex vivo culturing methods described herein. In some examples, the inhibitors used herein are specific to one of the members, for example, specific to p38-α, p38-β, p38-γ, or p38-δ. In other examples, the p38 MAPK inhibitors are universal to two or more members of the p38 MAPK family. In one example, the inhibitors used herein are specific or selective to p38-α (MAPK14).
As used herein, the term “p38 MAPK” refers to a p38 MAPK polypeptide having the same or similar bioactivity of a wild-type p38 MAPK. Ap38 MAPK polypeptide may have an amino acid sequence that is at least 70% or more (including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to that of a wild-type p38 MAPK, and is capable of trigger p38α signaling pathway.
The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Wild-type p38 MAPK sequences (e.g., sequences of p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12/ERK6), and p38-δ (MAPK13/SAPK4)) of various species are available on the world wide web from the NCBI, including human, mouse, and rat. For example, the nucleotide sequence encoding an isoform of human p38-α (MAPK14) is available at NCBI under Accession No. NM_001315 and its corresponding amino acid sequence is under Accession No. NP_001306.
As used herein, the term “p38 MAPK inhibitor” refers to a molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a p38 MAPK protein. Suitable inhibitor molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides, etc. Methods for identifying inhibitors of a polypeptide can comprise contacting a polypeptide with a candidate p38 MAPK inhibitor molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
A p38 MAPK inhibitor can be a molecule of any type that interferes with the signaling associated with at least one or more p38 MAPK family members (e.g., p38-α (MAPK14), p38-β (MAPK11), p38-γ (MAPK12/ERK6), and p38-δ (MAPK13/SAPK4)) in a cell, for example, either by decreasing transcription or translation of p38 MAPK-encoding nucleic acid, or by inhibiting or blocking p38 MAPK polypeptide activity, or both. In some examples, a p38 MAPK inhibitor is an agent that interferes with the signaling associated with p38-α (MAPK). Examples of p38 MAPK inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, p38 MAPK-specific aptamers, anti-p38 MAPK antibodies, p38 MAPK-binding fragments of anti-p38 MAPK antibodies, p38 MAPK-binding small molecules, p38 MAPK-binding peptides, and other polypeptides that specifically bind p38 MAPK (including, but not limited to, p38 MAPK-binding fragments of one or more p38 MAPK ligands, optionally fused to one or more additional domains), such that the interaction between the p38 MAPK inhibitor and p38 MAPK results in a reduction or cessation of P38 MAPK activity or expression. It will be understood by one of ordinary skill in the art that in some instances, a p38 MAPK inhibitor can antagonize or neutralize one p38 MAPK activity without affecting another p38 MAPK activity. For example, a desirable p38 MAPK inhibitor for use in certain of the methods herein is a p38 MAPK inhibitor that binds p38-α and blocks p38 MAPK signaling, e.g., without affecting or minimally affecting any of the other p38 MAPK interactions, for example, binding p38-β, p38-γ, and/or p38-δ.
In some embodiments, p38 MAPK inhibitors used for the methods described herein are cell-permeable.
In some embodiments, a p38 MAPK inhibitor is an agent that directly or indirectly inhibits or reduces DNA double strand breaks and/or DNA damage response in genetically manipulated stem cells such as HSCs (e.g., by using an integrating vector such as viral vectors or performing genome editing, e.g., which involves use of zinc finger nucleases (ZFN), transcription activator-like effector-based nuclease (TALEN), meganucleases, and/or CRISPR/Cas systems), wherein the DNA double strand breaks and/or DNA damage response are mediated by one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, p38-δ, and any combinations thereof). Accordingly, a p38 MAPK inhibitor can target the p38 MAPK (e.g., p38-α, p38-β, p38-γ, p38-δ, and any combinations thereof) or any of p38 MAPK's upstream molecules. Examples of p38 MAPK inhibitors include, without limitations, anti-p38-α molecules, anti-p38-β molecules, anti-p38-γ molecules, anti-p38-δ molecules, and any combinations thereof. A p38 MAPK inhibitor can be a protein, a peptide, a peptidomimetic, an aptamer, a nucleic acid, an antibody, a small molecule, or any combinations thereof.
A p38 MAPK inhibitor can be a molecule (e.g., an antibody, an aptamer, or a small molecule) that interferes with the binding of one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, p38-δ, and any combinations thereof). Alternatively, the p38 MAPK inhibitor can be a molecule (e.g., a inhibitory polynucleotide or oligonucleotide such as interfering RNA or antisense oligonucleotide) that suppresses transcription and/or translation of one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, p38-δ, and any combinations thereof), thereby reducing the mRNA/protein level of this enzyme. The p38 MAPK inhibitor as described herein may reduce the P38 MAPK signaling in stem cells or HSCs (e.g., during ex vivo culture after genetic manipulation) by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above. The inhibitory activity of such an inhibitor against p38 MAPK can be determined by conventional methods, e.g., measuring the phosphorylation level of p-p38, for example, using protein assays such as ELISA or Western blot.
In some embodiments, the p38 MAPK inhibitor is an antibody that specifically binds to one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, p38-δ, and any combinations thereof) and neutralizes its activity to activate p38 MAPK signaling pathway. As used herein, the term “antibody” as includes but is not limited to polyclonal, monoclonal, humanized, chimeric, Fab fragments, Fv fragments, F(ab′) fragments and F(ab′)2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
Antibodies can be made by the skilled person using methods and commercially available services and kits known in the art. Methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (Sep. 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (Jul. 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (Aug. 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (Feb. 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (Sep. 30, 2013)).
Antibodies may be produced by standard techniques, for example by immunization with the appropriate polypeptide or portion(s) thereof, or by using a phage display library. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc) is immunized with an immunogenic polypeptide bearing a desired epitope(s), optionally haptenized to another polypeptide. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the desired epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography or any other method known in the art. Techniques for producing and processing polyclonal antisera are well known in the art.
A p38 MAPK inhibitor specifically binds to one member of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) if the inhibitor binds to the specific member of p38 MAPK with a greater affinity than for an irrelevant polypeptide. In some embodiments, the inhibitor binds to one member of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) with at least 5, or at least 10 or at least 50 times greater affinity than for the irrelevant polypeptide. In some embodiments, the inhibitor binds to one member of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) with at least 100, or at least 1,000, or at least 10,000 times greater affinity than for the irrelevant polypeptide. Such binding may be determined by methods well known in the art, such surface plasmon resonance such as a Biacore® system. In some embodiments, the inhibitor has an affinity (as measured by a dissociation constant, KD) for a specific member of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) of at least 10−7 M, 10−8 M, 10−9 M, 10−10 M, or 10−11 M.
In some embodiments, the p38 MAPK inhibitor is a small molecule, such as a small organic molecule, which typically has a molecular weight less than 5,000 kDa. Suitable small molecules include those that bind to one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) or a fragment thereof, and may be identified by methods such as screening large libraries of compounds (Beck-Sickinger & Weber (2001) Combinational Strategies in Biology and Chemistry (John Wiley & Sons, Chichester, Sussex); by structure-activity relationship by nuclear magnetic resonance (Shuker et al (1996) “Discovering high-affinity ligands for proteins: SAR by NMR. Science 274: 1531-1534); encoded self-assembling chemical libraries Melkko et al (2004) “Encoded self-assembling chemical libraries.” Nature Biotechnol. 22: 568-574); DNA-templated chemistry (Gartner et al (2004) “DNA-tem plated organic synthesis and selection of a library of macrocycles. Science 305: 1601-1605); dynamic combinatorial chemistry (Ramstrom & Lehn (2002) “Drug discovery by dynamic combinatorial libraries.” Nature Rev. Drug Discov. 1: 26-36); tethering (Arkin & Wells (2004) “Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature Rev. Drug Discov. 3: 301-317); and speed screen (Muckenschnabel et al (2004) “SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands.” Anal. Biochem. 324: 241-249). Typically, small molecules will have a dissociation constant for P38 MAPK in the nanomolar range.
Examples of small molecule p38 MAPK inhibitors for use in the ex vivo culturing method described herein are provided in Table 1 below:
Exemplary p38 MAPK inhibitors also include doramapimod (e.g., BIRB-796), ralimetinib (e.g., LY2228820 dimesylate), aminopyridine-based, ATP-competitive inhibitors of p38 MAPK (e.g., Vx702), pyridinyl imidazole inhibitors (e.g., SB203580), and any combinations thereof.
Other p38 MAPK inhibitors are well known in the art, for example, those described in U.S. Pat. Nos. 7,169,779, 6,635,644, 6,608,060, 6,632,945, 6,528,508, 6,509,363 (Heterocyclic inhibitors of p38), U.S. Pat. Nos. 6,147,080, 6,800,626, 6,093,742, 6,949,560 (Imidazo-substituted compounds), U.S. Pat. No. 6,852,740 (Pyrazole derivatives), U.S. Pat. No. 6,630,485, 6,759,410 (3,4-dihydro-(1 h)-quinazolin-2ones), U.S. Pat. No. 6,696,471 (Aminopyrrole compounds), U.S. Pat. No. 6,696,443 (Piperidine/piperazine-type inhibitors), U.S. Pat. No. 6,509,361 (1,5-diaryl substituted pyrazoles), U.S. Pat. No. 6,444,696 (pyrazole derivatives), and PCT patent publications WO2000017175, WO2000017204, WO1996021654, WO1999000357, WO1999064400, the relevant teachings of each of which is incorporated by reference herein. Other p38 MAPK inhibitors as described in Xing “Clinical candidates of small molecule p38 MAPK inhibitors for inflammatory diseases” (2015) MAP Kinase 4: 5508 may also be used for the ex vivo methods and compositions described herein.
In some embodiments, the p38 MAPK inhibitor is an interfering RNA such as a small interfering RNA (siRNA) short hairpin RNA (shRNA). In some embodiments, the p38 MAPK inhibitor is a small interfering RNA (siRNA) that binds to the mRNA of one or more family members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) and blocks its translation or degrades the mRNA via RNA interference. Exemplary small interfering RNAs are described by Hannon et al. Nature, 418 (6894): 244-51 (2002); Brummelkamp et al., Science 21, 21 (2002); and Sui et al., Proc. Natl Acad. Sci. USA 99, 5515-5520 (2002). RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing in animals initiated by double-stranded (dsRNA) that is homologous in sequence to the silenced gene. siRNAs are generally RNA duplexes with each strand being 20-25 (such as 19-21) base pairs in length. In some embodiments, the p38 MAPK inhibitor is a short hairpin RNA (shRNA) that is complementary to a p38 MAPK nucleic acid (e.g., a p38 MAPK mRNA). An shRNA typically contains of a stem of 19-29 base pairs, a loop of at least 4 nucleotides (nt), and optionally a dinucleotide overhang at the 3′ end. Expression of shRNA in a subject can be obtained by delivery of a vector (e.g., a plasmid or viral or bacterial vectors) encoding the shRNA. siRNAs and shRNAs may be designed using any method known in the art or commercially available (see, e.g., products available from Dharmacon and Life Technologies). An siRNA may also comprise one or more chemical modifications, such as a base modification and/or a bond modification to at least improve its stability and binding affinity to the target mRNA.
In some embodiments, the p38 MAPK inhibitor is an antisense oligonucleotide that is complementary to a p38 MAPK nucleic acid (e.g., a p38 MAPK mRNA). Antisense oligonucleotides are generally single-stranded nucleic acids (either a DNA, RNA, or hybrid RNA-DNA molecule), which are complementary to a target nucleic acid sequence, such as a portion of a p38 MAPK mRNA. By binding to the target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed, thereby inhibiting the function or level of the target nucleic acid, such as by blocking the transcription, processing, poly(A) addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting mRNA degradation. In some embodiments, an antisense oligonucleotide is 10 to 40, 12 to 35, or 15 to 35 bases in length, or any integer in between. An antisense oligonucleotide can comprise one or more modified bases, such as 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), 5-Bromo dU, 5-Methyl dC, deoxyInosine, Locked Nucleic Acid (LNA), 5-Nitroindole, 2′-O-Methyl bases, Hydroxmethyl dC, 2′ Fluoro bases. An antisense oligonucleotide can comprise one or more modified bonds, such as a phosphorothioate bond.
In some embodiments, the p38 MAPK inhibitor is a ribozyme that is complementary to a p38 MAPK nucleic acid (e.g., a p38 MAPK mRNA) and cleaves the p38 MAPK nucleic acid. Ribozymes are RNA or RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. The ribozymes of the present disclosure may be synthetic ribozymes, such as those described in U.S. Pat. No. 5,254,678. These synthetic ribozymes have separate hybridizing regions and catalytic regions; therefore, the hybridizing regions can be designed to recognize a target sequence, such as a p38 MAPK sequence.
siRNAs, shRNAs, ribozymes, and antisense oligonucleotides as described herein may be complementary to a p38 MAPK nucleic acid (e.g., a p38 MAPK mRNA), or a portion thereof. It is to be understood that complementarity includes 100% complementarity but does not necessarily exclude mismatches at one or more locations, resulting in, e.g., at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% complementarity.
In some embodiments, the p38 MAPK inhibitor is a non-antibody peptide or protein. The peptide or protein may comprise an amino acid sequence that interferes with the p38 MAPK signaling. Proteins and peptides may be designed using any method known in the art, e.g., by screening libraries of proteins or peptides for binding to p38 MAPK or inhibition of p38 MAPK binding to a ligand, such as p38 MAPK.
The capability of a candidate compound, such as a small molecule, protein, or peptide, to bind to or interact with a p38 MAPK polypeptide or fragment thereof may be measured by any method of detecting/measuring a protein/protein interaction or other compound/protein interaction. Suitable methods include methods such as, for example, yeast two-hybrid interactions, co-purification, ELISA, co-immunoprecipitation and surface plasmon resonance methods. Thus, the candidate compound may be considered capable of binding to the polypeptide or fragment thereof if an interaction may be detected between the candidate compound and the polypeptide or fragment thereof by ELISA, co-immunoprecipitation or surface plasmon resonance methods or by a yeast two-hybrid interaction or co-purification method, all of which are known in the art. Screening assays which are capable of high throughput operation are also contemplated. Examples may include cell based assays and protein-protein binding assays.
Any of the p38 MAPK inhibitors, e.g., those described herein, can be used for preserving stemness of stem cells (e.g., hematopoietic stem cells) in ex vivo or in vitro culture. Stemness refers to the ability of unspecialized cells to renew themselves as unspecialized cells but still retain this ability to specialize to produce specific types of cells. The stem cell potential, or “stemness” of stem cells (e.g., hematopoietic stem cells) relies upon a combination of properties: quiescence, repopulation potential, self-renewal potential, and multi-lineage differentiation potential. Cell-cycle quiescence in stem cells (e.g., HSCs) maintains stemness by protecting cells from differentiation or senescence.
The present disclosure features ex vivo culturing methods for preserving the stemness of stem cells in cell cultures by culturing stem cells (e.g., HSCs) in the presence of one or more p38 MAPK inhibitors. The stem cell thus prepared can be used in treating suitable diseases via stem cell transplantation.
To perform the ex vivo culturing methods described herein, a suitable population of stem cells (e.g., pluripotent stem cells) can be obtained from a suitable source. In some instances the population of stem cells (e.g., HSCs) can be derived from a human subject, e.g., from the bone marrow cells, peripheral blood cells, and/or umbilical cord blood cells of the human subject, via a convention method. In some examples, the stem cells are adult stem cells (e.g., HSCs), which can be derived from the bone marrow or peripheral blood cells of a human adult. In some examples, the stem cell population is substantially free of umbilical stem cells.
In some embodiments, any of the stem cell populations described herein have undergone a genetic manipulation that causes a DNA damage, e.g., double strand breaks, dimerization or cross-linking, unpaired bases, modified bases, conversion of one base into another resulting in unpaired bases, chromatin unwinding or other modifications, etc. In some embodiments, any of the stem cell populations described herein have undergone a genetic manipulation that a double strand break. A genetic manipulation includes modifying, inserting, or deleting at least one of the genes in the stem cells (e.g., HSCs). Genetic manipulation may include transduction with a vector that is capable of being integrated into the cell genome.
A “vector”, as used herein is any nucleic acid vehicle (DNA or RNA) capable of facilitating the transfer of a nucleic acid molecule into stem cells (e.g., HSCs). In general, vectors include, but are not limited to, plasmids, phagemids, viral vectors, and other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of a target nucleotide sequence. Viral vectors include, but are not limited to vectors comprising nucleotide sequences derived from the genome of the following viruses: retrovirus; lentivirus; adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus. One can readily employ other vectors not named but known to the art.
Viral vectors may be based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a target nucleotide sequence. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are known in the art.
Other viral vectors include adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have also been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their preintegration complex (virus “shell”) can get through the intact membrane of the nucleus of the target cell. An exemplar lentiviral vector includes, but are not limited to a vector derived from HIV.
Other vectors include non-viral plasmid vectors, which have been extensively described in the art and are well known to those of skill in the art. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 4th edition (Jun. 15, 2012). Exemplary plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA.
In some embodiments, the stem cells involved in the methods and/or compositions described herein (e.g., HSCs) have been genetically manipulated using a viral vector such as a retroviral vector or a lentiviral vector. Accordingly, in some embodiments, the stem cells involved in the methods and/or compositions described herein are gene-modified stem cells (e.g., HSCs).
The genetic manipulation (e.g., transduction) of the stem cells is preferably performed when the stem cells are resting cells (non-cycling), i.e., cells that are not dividing. Such resting cells may in the quiescent G0 phase. Phenotypes of resting HSCs are known in the art and can be used to identify such HSCs for the methods and/or compositions described herein. For example, HSC-enriched population is at least CD34+CD38−CD90+. Like all somatic cells, stem cells (e.g., HSCs) progress through the cell cycle, which is characterized by four phases: G1 (interphase), S (DNA synthesis phase), G2 (interphase) and M (mitosis phase). Stem cells (e.g., HSCs) that proceed past the restriction point in the G1 phase enter the S phase, whereas those that do not pass the restriction point remain undivided. These undivided cells can withdraw from the cell cycle and enter the G0 phase: a state in which cells are termed quiescent or dormant. Such resting cells in the G0 phase can either reversibly re-enter the cell cycle and divide or remain dormant, losing the potential to cycle and, in some cases, becoming senescent. Quiescence is thus a property that characterizes stem cells (e.g., HSCs) and allows them to maintain stemness of the cells. Without wishing to be bound by theory, when the stem cells (e.g., HSCs) are genetically manipulated or modified before they enter a cell cycle (e.g., when the cells are non-cycling) and are subsequently cultured in the presence of a p38 MAPK inhibitor, the p38 MAPK inhibitor delays the transition of the stem cells (e.g., HSCs) to S phase, which may likely allow for stem cells (e.g., HSCs) to repair any DNA damage, e.g., induced by the genetic manipulation, thus retaining the stemness of the stem cells (e.g., HSCs). This may be characterized by retained long term repopulating potential and/or a balanced lineage production. Accordingly, in some embodiments of the stem cells involved in the methods and/or compositions described herein, the stem cells (e.g., HSCs) are non-cycling or non-dividing cells, e.g., the stem cells are in the quiescent G0 phase.
Any of the stem cell populations described herein can be cultured in a suitable medium (e.g., cell culture medium) in the presence of an effective amount of one or more p38 MAPK inhibitors as those described herein for a suitable period of time, e.g., at least 18 hours, at least about 24 hour, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, or longer. In some embodiments, any of the stem cell populations described herein can be cultured in a suitable medium (e.g., cell culture medium) in the presence of an effective amount of one or more p38 MAPK inhibitors as those described herein for about 18 hours to about 7 days, or about 1 day to about 7 days, or about 2 days to about 7 days, or about 3 days to about 7 days, or longer.
An “effective amount,” “effective dose,” or an “amount effective to”, as used herein, refers to an amount of a p38 MAPK inhibitor as described herein that is effective in preserving at least one characteristic of the sternness (quiescence, repopulation potential, self-renewal potential, and multi-lineage differentiation potential) of stem cells, e.g., HSCS, and/or results in a desired clinical effect, such as increased engraftment of HSCs in a subject after HSC transplantation. This can be monitored by routine methods or can be monitored according to the method for assessing engraftment of HSCs described herein. Effective amounts vary, as recognized by those skilled in the art, depending on, for example, the potency of the p38 MAPK inhibitor used.
For example, the effective amount of a p38 MAPK inhibitor for culturing the stem cells (e.g., HSCs) in the methods described herein results in an increase in the proportion of stem cells (e.g., HSCs) in the G0 quiescent phase by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the proportion of stem cells (e.g., HSCs) in the G0 quiescent phase without culturing in the presence of a p38 MAPK inhibitor. In some embodiments, the effective amount of a p38 MAPK inhibitor results in an increase in the proportion of stem cells (e.g., HSCs) in the G0 quiescent phase by at least about 1.1-fold or more, including, e.g., at least about 2-fold at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold or more, as compared to the proportion of stem cells (e.g., HSCs) in the G0 quiescent phase without culturing in the presence of a p38 MAPK inhibitor.
In some embodiments, the effective amount of a p38 MAPK inhibitor used in the methods described herein results in a decrease in the proportion of the stem cells (e.g., HSCs) in the S-G2-M phase before the first cell division cycle (e.g., 24 hours) by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the proportion of stem cells (e.g., HSCs) in the S-G2-M phase before the first cell division cycle without culturing in the presence of a p38 MAPK inhibitor.
In some embodiments of the methods described herein, the stem cells (e.g., HSCs) are cultured in the presence of a p38 MAPK inhibitor in an amount effective to reduce DNA damage or DNA double strand break (e.g., as measured by the expression of γH2AX foci or 53 bp1) in the stem cells (e.g., HSCs) by at least about 20% or more, including, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the level of DNA damage or DNA double strand break measured in stem cells without culturing in the presence of a p38 MAPK inhibitor.
In some embodiments of the methods described herein, the stem cells (e.g., HSCs) are cultured in the presence of a p38 MAPK inhibitor in an amount effective to reduce loss of long term repopulating potential (LTRP) (e.g., as assessed in secondary transplant (2T)) by at least about 20% or more, including, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the LTRP when the cells are cultured without a p38 MAPK inhibitor. Examples 1-2 including
In some embodiments of the methods described herein, the stem cells (e.g., HSCs) are cultured in the presence of a p38 MAPK inhibitor in an amount effective to reduce myeloid skewing bias in the stem cells by at least about 20% or more, including, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the myeloid skewing bias when the cells are cultured without a p38 MAPK inhibitor. Examples 1-2 including
In some embodiments of the methods described herein, the effective amount of a p38 MAPK inhibitor for culturing the stem cells (e.g., HSCs) results in a decrease in the phosphorylation level of at least one or more (including, e.g., at least two, or at least three) members of p38 MAPK (e.g., p38-α, p38-β, p38-γ, or p38-δ) in the stem cells (e.g., HSCs), for example, by at least about 20%, including, for example, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more, as compared to the phosphorylation level of the corresponding member of p38 MAPK in the stem cells (e.g., HSCs) without the treatment of the p38 MAPK inhibitor.
In some embodiments of the methods described herein, the effective amount of a p38 MAPK inhibitor does not substantially increase the phosphorylation level of ERK or JNK in the stem cells (e.g., HSCs), for example, by no more than 20%, including, for example, no more than 10%, no more than 5%, no more than 3%, or lower, as compared to the phosphorylation level of ERK or JNK in the stem cells (e.g., HSCs) without the treatment of the p38 MAPK inhibitor.
In some embodiments, the stem the stem cells (e.g., HSCs), prior to transplantation in vivo, are cultured in a medium comprising an effective amount of a p38 inhibitor described herein, wherein the effective amount results in an increase in subsequent engraftment of stem cells (e.g., HSCs) in vivo by at least about 10% or more, including, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to engraftment of stem cells (e.g., HSCs) without culturing the cells with a p38 MAPK inhibitor prior to transplantation. In some embodiments, the effective amount of a p38 MAPK inhibitor results in an increase in subsequent engraftment of stem cells (e.g., HSCs) by at least about 1.1-fold or more, including, e.g., at least about 2-fold at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold or more, as compared to engraftment of stem cells (e.g., HSCs) without culturing the cells with a p38 MAPK inhibitor prior to transplantation.
An effective dose of a p38 MAPK inhibitor for the methods described herein can be at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1 μM, at least about 2 μM, at least 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, or at least about 10 μM. In some embodiments, the effective dose of a p38 MAPK inhibitor for the methods described herein can be no more than 10 μM, no more than 9 μM, no more than 8 μM, no more than 7 μM, no more than 6 μM, no more than 5 μM, no more than 4 μM, no more than 3 μM, no more than 2 μM, no more than 1 μM, no more than 900 nM, no more than 800 nM, no more than 700 nM, no more than 600 nM, no more than 500 nM, no more than 400 nM, no more than 300 nM, no more than 200 nM, no more than 100 nM, no more than 50 nM, no more than 40 nM, no more than 30 nM, no more than 20 nM, or no more than 10 nM. Combinations of the above-referenced ranges are also possible. For example, an effective dose of a p38 MAPK inhibitor for the methods described herein can be about 30 nM to about 10 μM, or about 100 nM to about 5 μM, or about 400 nM to about 800 nM.
The stem cells can preserve their stemness when they are cultured in the presence of a p38 MAPK inhibitor according to the methods described herein. In some embodiments, the percentage of pluripotent stem cells after the ex vivo culturing process is at least 70% (e.g., 80%, 90%, 95%, 97%, or above) of that before the ex vivo culture. In other embodiments, less than 30% (e.g., less than 25%, 20%, 15%, 10%, or 5% or less) of the stem cells would differentiate, e.g., from pluripotent stem cells to multipotent stem cells, or from multipotent cells to specialized cells, during the ex vivo culture process described herein. The presence of different types of stem cells, e.g., pluripotent stem cells and multipotent cells, and specialized cells in the ex vivo culture can be monitored via a routine method, for example monitored by the presence of cell surface markers specific to a specific type of stem cells or specific to a specialized cell.
In some embodiments, adult HSCs are subjected to the ex vivo culturing process described herein, which involves the use of one or more p38 MAPK inhibitors. The percentage of HSCs after the culturing may be at least 70% (e.g., 80%, 90%, 95%, or higher) of that of HSCs prior to the culturing. Alternatively or in addition, the percentage of hematopoietic progenitor cells (HPCs) in the cells after the ex vivo culture may be lower than 30% (e.g., lower than 25%, 20%, 15%, 10%, or 5%).
The stem cells prepared by the ex vivo culturing methods described herein can be used in stem-cell therapy, which is the use of stem cells to treat or prevent a disease or condition, including, for example, neurodegenerative diseases and conditions, diabetes, heart disease, and other conditions. Examples of suitable conditions to be treated by stem cell therapy include, but are not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, Non-Hodgkin lymphoma, neuroblastoma, Ewing sarcoma, Myelodysplastic syndromes, Gliomas, and other solid tumors. Stem cell therapy can also be applied to non-malignant conditions such as thalassemia, aplastic anemia, Fanconi anemia, immune deficiency syndromes, or inborn errors of metabolism. In some embodiments, the HSCs prepared by the ex vivo culturing methods described herein can be used for transplantation in treatment of hematopoietic disorders, including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin lymphoma, and Non-Hodgkin lymphoma.
Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. In some instances, the HSCs can be autologous (the patient's own stem cells are cultured by the ex vivo culturing methods described herein and used for treating a disease). In other examples, the HSCs may be allogeneic (the stem cells come from a donor and is then cultured by the ex vivo culturing methods described herein). Such HSCs can be used for treating certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia. In these cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation.
In some examples, the HSCs described herein (e.g., human adult HSCs) can be genetically engineered to express a γ globin for use in treating anemia, such as sickle cell anemia and thalassemia. See, e.g., US20110294114 and WO2015/117027, the relevant teachings of each of which are incorporated by reference herein.
In any of the stem cell therapy described herein, suitable stem cells can be collected from the ex vivo culturing method described herein and mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
To perform the treatment methods described herein, an effective amount of the stem cells can be administered into a subject in need of the treatment. The stem cells may be autologous to the subject. Administration of autologous cells to a subject may result in reduced rejection of the stem cells as compared to administration of non-autologous cells. Alternatively, the stem cells are allogeneic cells. For example, allogeneic stem cells may be derived from a human donor and administered to a human recipient who is different from the donor.
In some embodiments, the stem cells can be co-used with a therapeutic agent for a target disease, such as those described herein. The efficacy of the stem cell therapy described herein may be assessed by any method known in the art and would be evident to a skilled medical professional. Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject.
Another aspect of the present disclosure features a method for assessing engraftment of human hematopoietic stem cells (HSCs), such as the human HSCs described herein, which can be prepared by any of the ex vivo culturing methods also described herein. Such HSCs can be obtained from a human subject who is transplanted with the HSCs and then transplanted into a suitable immune deficient animal, such as an immune deficient mouse (e.g., an NSG mouse). Other suitable immune deficient animals are also known in the art, for example, those provided by Charles River Laboratories (see Table 2 below):
After a suitable period of time, the mobilized peripheral blood of the recipient animal can be collected and the level of CD45+ cells therein can be measured by a conventional method, e.g., FACS. The level of CD45+ cells is in a reverse correlation to the rate of human HSC engraftment.
The present disclosure also provides kits or compositions for use in preserving the stemness of stem cells (e.g., HSCs) or increasing stem cell engraftment in a subject need thereof. Such kits or compositions can include one or more containers comprising a p38 MAPK inhibitor, and optionally, one or populations of stem cells (e.g., HSCs). The kits or compositions may further comprise a cell culture medium suitable for culturing stem cells (e.g., HSCs).
In some embodiments, the kit can comprise instructions for use in accordance with any of the methods described herein. The included instructions can comprise a description of culturing stem cells (e.g., HSCs) in a medium comprising an effective amount of a p38 MAPK inhibitor as described herein. The kit may further comprise a description of selecting specific stem cells, e.g., HSCs, based on identifying surface markers associated with specific stem cells (e.g., CD34+CD38−CD90+ for HSCs). In still other embodiments, the instructions comprise a description of administering the HSCs to an individual in need of the treatment.
The instructions relating to the use of a p38 MAPK inhibitor generally include information as to dosage, and dosing schedule for the intended treatment of stem cells (e.g., HSCs). The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
The label or package insert indicates that the composition is used for preserving the stemness of stem cells (e.g., HSCs). Instructions may be provided for practicing any of the methods described herein.
The kits of this invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture comprising contents of the kits described above.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.
The goals of the present study were to develop a robust animal model of human HSC in a xenograft model that mimics human clinical trial data, to study the mechanism of HSC loss during ex vivo culture and gene transfer, and to use small molecule inhibitors of identified pathways to reverse the HSC loss during ex vivo culture and/or genetic manipulation.
Ex vivo culture of HSC may activate stress signaling (p38 MAPK) in HSC, resulting in either asymmetric HSC division or symmetric HPC commitment at the expense of HSC self-renewal. Vector integration events further instigates a DNA damage response that results in their differentiation to HPC. Thus, using a p38 MAPK inhibitor may reverse HSC loss during ex vivo culture and/or genetic manipulation (e.g., vector integration).
(i) Myelo-Ablative Conditioning
Two recent gene therapy trials exploring myelo-ablative conditioning have been performed (Table 3). Kang et al., Blood (2010); Cavazzana-Calvo et al., Nature (2010) About 4-70 million CD34+ cells were infused in the studies, and among them, the number of transduced CD34+ cells infused ranged from 1-30 million. When the numbers were extrapolated in terms of transduced HSCs, about 13,000 to 300,000 were infused, but this resulted in a long term engraftment of about 0-1%, showing tremendous loss.
(ii) Xenograft Model of Adult Human HSCs
A xenograft model was developed to study hHSC engraftment. In the xenograft model of adult human HSC, CD34+ cells were derived and isolated from fresh MPB. The fresh CD34+ cells were either directly transplanted into immunocompromised NSG mice or cultured with human cytokines and then transduced either with lentivirus or retrovirus. For the lentivirus (LV) vector, the cultured cells were transduced for 18-24 hours and harvested and injected into NSG mice after 18-24, 36-42, and 72-96 hours of ex vivo culture. For the retrovirus (RV) vector, the cells were injected into NSG mice after 72-96 hours of ex vivo culture. The multipotential human hematopoietic progeny (hCD45+) following huCD34+ transplant in NSG mice were then measured to confirm that the model human HSC engraftment from adult (mobilized peripheral blood) CD34+ HSC/P that mimics the results observed in gene therapy clinical trials. Human cell engraftment in the bone marrow of NSG mice at 6, 12, and 24 weeks post-primary transplant (1T), and 6 weeks post-secondary transplant (2T) of fresh/cultured human CD34+ cells. Cells were transduced with lentiviral (LV) or retroviral (RV) vector.
It was observed that increased time in culture leads to HSC division, and that a portion of the transduced HSCs undergo apoptosis. The increased time of ex vivo manipulation of human HSC does not affect engraftment in primary NSG mice, even at 6 months, but does lower secondary engraftment. Thus, increased time of ex vivo manipulation of human HSCs (hHSC) results in loss of long-term engraftment potential of hHSC in a xenograft model. The loss of secondary engraftment was found to correlate with HSPC and HSC division, and a portion of HSC (CD34+38−90+) with vector integration were observed via flow cytometry to undergo apoptosis. The apoptotic population has high GFP expression in flow cytometry studies, suggesting the high vector copies or increased double strand breaks (DSBs) that occur from vector integration are not well-tolerated by HSCs.
The loss of secondary engraftment was found to correlate with HSPC and HSC division, as extended cultures result in both HSPC and HSC division. In addition, p38 MAPK activation increases as HSCs progress through the cell cycle.
Next, the ex vivo cultured cells were subjected to flow cytometry for further characterization. It was observed that an increased time in culture showed an increase in p-p38 MAPK level in human HSCs. Human HSCs can be characterized as CD34+CD38lowCD90+CD45RA−CD49f+ There also appeared to be slight increase in ERK at 72 hours.
Flow cytometry also demonstrated that p38MAPK inhibitors can prevent HSC loss and may even retain HSC status through cell division during ex vivo cultures. Four different p38 inhibitors were shown to increase the percent of hHSCs ex vivo (
p38 inhibition was also shown to significantly increase the number of 2T mice (6 weeks post-secondary transplant) with any human cell engraftment while not affecting the overall human cell engraftment in the 1T mice (except for lentivirus-transduced cells cultured for 72 hours). For engraftment in 1T model, hCD45+ cell engraftment in bone marrow of 1T mice at 24 weeks following transplant of culture hCD34+ cells with or without p38 inhibitor BIRB-796 were assessed.
p38 inhibition, e.g., with BIRB-796, was also shown to maintain the G0 quiescent phase of both untransduced and transduced HSCs during the early time period of ex vivo culture. Ki67, a nuclear antigen associated with cell proliferation, was used to determine cell cycle status via flow cytometry; negative stains were considered to represent quiescent HSCs.
Data from 10 experiments have demonstrated that lentivirus and retrovirus transduce HSC and HPC comparably.
Primary transplant (1T) mice (n=8-16) were analyzed 24 weeks after their respective transplants with respect to HPC progeny. A multilineage analysis of the harvested cells demonstrated that genetic manipulation of CD34+ cells during ex vivo culture shows a loss of lymphoid potential and myeloid skewing of the long term in vivo progeny. p38MAPK inhibition resulted in long term repopulating ability, but does not revert the effect.
p38 inhibition maintains the quiescent phase of HSCs and reduces DNA damage response (DDR) early in culture. Cell cycle entry and gene manipulation induces a DDR in human HSCs that is reduced by inhibiting p38MAPK early in culture. DNA double-strand breaks in Human HSCs were assessed by γ-H2AX staining, a sensitive and specific indicator of DNA double-strand breaks.
HSC division in culture decreases the self-renewing potential of adult human HSC read out in secondary transplant. Some HSCs with a high level transduction undergo apoptosis.
Increased time in culture has been demonstrated to activate p38MAPK in human HSC, while the inhibition of p38MAPK during ex vivo cultures appears to maintain or increase the phenotypic HSC in vitro. Functionally, inhibition of p38MAPK during ex vivo manipulation and gene transfer retains human HSC engraftment in secondary transplant mice. p38 inhibition may exert its effect by maintaining HSC quiescence during ex vivo culture manipulation.
Hematopoietic stem cells (HSCs) are ideal targets for gene therapy for several inherited hematological diseases including hemoglobinopathies. Gene transfer into HSC involves in a 2-4 day ex-vivo culture, making these cells less competent at engraftment in vivo. This necessitates large transduced HSC (tHSC) doses and myelo-ablative conditioning to destroy resident HSCs. In this Example, the transduced human CD34+ HSPC were modeled with long term repopulating potential (LTRP) in NOD.LtSz-scid Il2rγ−/− (NSG) mice using primary and secondary transplants, with B, Myeloid and T cell reconstitution. Mobilized peripheral blood CD34+ cells (containing adult hematopoietic stem cells) were used, since these are the relevant targets for gene therapy and their engraftment kinetics vastly differ from cord blood CD34+ cells, which are commonly used in the NSG mouse model. Gene transfer into hHSPCs by lentivirus (LV) or γ-retrovirus vectors (RV) followed by engraftment into NSG mice revealed results similar to those seen in gene therapy trials, showing that extended time of ex-vivo manipulation of hCD34+ cells results in huge loss of the LTRP in secondary transplanted mice. Furthermore, genetically manipulated CD34+ cell progeny transduced in 3-4 day culture conditions showed relative loss of lymphoid potential at the expense of increased myeloid lineage skewing, which is specific to transduced cells. Mechanistically, extended ex vivo culture and gene manipulation induced a DNA damage response (DDR) in hHSCs as revealed by increased γ-H2AX and 53BP1 signaling that is associated with activation of p38MAPK; this effect was reversed by use of a p38 inhibitor, which helped retain the LTRP and engraftment in secondary transplanted NSG mice and partially reverted the myeloid skewing phenotype.
Gene transfer into HSCs requires cytokine rich culture conditions for therapeutically relevant gene transfer. Girard-Gagnepain, A., et al., Blood (2014) 124(8): p. 1221-31. In order to investigate the effect of ex vivo culture and genetic manipulation on the long term repopulating potential (LTRP) of adult human CD34+ hematopoietic stem/progenitor cells (HSPC), the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mouse model (Jackson Laboratory; Mouse Strain Datasheet—005557), also known as NOD/SCID/IL2rg null or NSG xenograft model, was adapted to an adult HSC model, since the majority of the studies in this model are based on umbilical cord blood-derived (CB) CD34+ cells; and adult CD34+ cells do not engraft sufficiently, or sustain a secondary graft at CD34+ cell doses used for CB CD34+ cells in this model (data not shown). One million CD34+ cells from G-CSF mobilized peripheral blood (MPB) were transplanted in irradiated NSG mice, and serial engraftment in the bone marrow (BM) of the animals at 6, 12 and 24-weeks was assessed [circulating human cells do not correlate with engraftment in BM (
Two protocols for lentivirus vector (LV) and γ-retrovirus vector (RV) gene transfer, commonly used for clinical transductions were used: MPB CD34+ HSPCs were transduced with (a) GFP-encoding LV for 18-24 hours (18-24 h) and either immediately transplanted, or kept in culture for an additional 12-16 h and transplanted at 36-42 h into irradiated NSG mice; (b) alternatively, CD34+ cells were pre-stimulated for 2 days ex vivo, and then transduced with a self-inactivating GFP-encoding RV at day 2 and 3, and transplanted into NSG mice between 72-96 h. Notably, gene editing also requires similar conditions, the former for gene disruption or knockout (24 h or 36 h) using the cellular NHEJ repair pathway, and the latter conditions for homology directed repair, that requires cycling HSC. As a control, NSG mice were transplanted with unmanipulated CD34+ HSPCs immediately following their isolation (0 h). Variations in ex vivo culture time or time of exposure to vectors were made in some experiments, to obtain appropriate controls (
Next, the multi-lineage reconstitution was analyzed at 6, 12 and 24 weeks in BM of 1T animals, both of the untransduced (GFP−) versus transduced (GFP+) progeny. Similar lineage proportions were observed (mostly B-myeloid in both the GFP+ and GFP− populations), comparable to the lineage pattern of unmanipulated CD34+ cell (0 h) derived graft at 6-weeks post 1T. However, by 12-weeks, the 72-96 h RV-gene modified population showed a significant increase in myeloid cells, which occurred at the expense of reduced B cell progeny (
Mechanisms for the loss of LTRP and myeloid-lineage biased progeny were investigated. RV only transduce dividing cells while LV can transduce both dividing and non-dividing cells, although LV also preferably transduce dividing cells. Lewis, P. F. and M. Emerman, J Virol (1994) 68(1): p. 510-6. Hence, it is assumed that the failure of RV trials was from preferential integration of transgenes into hematopoietic progenitor cells (HPCs) or multipotent progenitors (MPP) that comprise 99% of the CD34+ HSPC population, and poor transduction of the quiescent HSC population. Using the phenotype of human HSC identified by Dick and coworkers (Notta, F., et al., Science (2011) 333(6039): p. 218-21), the clinical transduction conditions were found to be optimized to transduce human HSC (CD34+CD38−CD90+CD45RA−CD49f+), (MPP, defined as CD34+CD38−CD90−CD45RA− (Id.; Huntsman, H. D., et al., Blood (2015) 126(13): p. 1631-3)) and total CD34+ cells comparably with RV and LV (
Among these p38 inhibitors (p38i), Birb-796, a highly selective p38α inhibitor (Pargellis, C., et al., Nat Struct Biol (2002) 9(4): p. 268-72), was used for further studies. p38i was able to significantly lower p-p38 levels in the HSC population to that seen in unmanipulated HSC at all the different time points in culture and transductions (
Remarkably, however, p38i at all time points was able to retain the secondary engraftment potential or long term repopulating potential (LTRP) to the levels observed after transplanting unmanipulated CD34+ cells (
With p38i exposure during ex vivo culture, the myeloid lineage skewing was restored to levels similar to untransduced cells or 0 h controls if HSPC were transduced within the first 24 h with LV, even if they were kept in prolonged cultures for up to 72-96 h. However, in the 72-96 h RV group, where myeloid skewing was most pronounced at the expense of the lymphoid lineage and CD34+ cells without p38i, there was some improvement in the skewing, but it was still not lineage balanced. Significant increase in myeloid progeny, with a reduction in B lymphoid cells and CD34+ cells was still observed, and there was significant loss of human HSPCs (
Hence, lineage skewing were specific to cells that were transduced when they were dividing. p38i was able to significantly revert this phenotype but only partially (
The mechanism by which p38 inhibition is able to retain the LTRP of cultured and transduced HSCs was next investigated. There was no significant change in the total number of CD34+ cells or their viability with or without p38i. The HSC enriched CD34+38− 90+ population showed no difference in apoptosis, ROS levels or transduction efficiency (
Since the transition of quiescent HSCs into cell cycle has been shown to trigger the DNA damage response (DDR) and repair pathway (Walter, D., et al., Nature (2015) 520(7548): p. 549-52), whether p38 activation that occurs with HSC division mediates the DDR pathway in ex vivo cultured and transduced HSPCs was examined. Indeed, increased γH2AX foci were found in the CD34+ CD38− CD90+ HSC enriched population that were cultured and transduced for either 36-42 hours or 72 hours; such DNA damage response was rescued by p38 inhibition (
The role of p38i on HSC cell cycle was next examined, since transition of quiescent HSCs into cell cycle has been shown to trigger the DDR pathway. Walter, D., et al., Nature (2015) 520(7548): p. 549-52. Nearly all HSC were in G0 phase when freshly isolated from MPB. By 24 h, nearly a third of them transitioned to the G1 phase. p38i significantly increased the proportion of HSC in the G0 quiescent phase, and decreased the proportion of HSC in the S-G2-M phase before the first HSC division (24 h). However, after 24 h, as HSC progressed through cell cycle, the effect of p38i was lost. Hence, the data suggests that when the transduction occurs at the first 24 hours (when HSC are non-cycling), p38i-mediated delay in transition to S phase (
In sum, these results can be utilized to develop a clinical gene therapy protocol to prevent the loss of gene-modified hHSCs and improve the overall engraftment.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/246,964 filed Oct. 27, 2015, the contents of which are incorporated by reference herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/59204 | 10/27/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62246964 | Oct 2015 | US |
