Generation of multi-lineage hematopoietic precursor cells by genetic programming

Abstract

The present disclosure relates generally to methods and compositions for providing multi-lineage hematopoietic precursor cells from pluripotent stem cells (PSCs). The PSCs comprise expression constructs encoding ETS / ERG gene, GATA2, and HOXA9. Methods for providing hematopoietic stem cells capable of long-term engraftment in a mammal (e.g., human) are also provided. Further provided are therapeutic compositions, including the provided hematopoietic stem cells and precursors of hematopoietic cells, and methods of using them to treat subjects.

Description

[0001] This application claims priority to U.S. Provisional Application No. 62 / 244,101, filed October 20, 2015, and U.S. Provisional Application No. 62 / 404,470, filed October 5, 2016, the entire contents of which are incorporated herein by reference. Background of the Invention

[0002] 1. Field of Invention

[0003] This invention generally relates to the fields of molecular biology, stem cells, and differentiated cells. More specifically, it relates to programming pluripotent stem cells (PSCs) into specific cell lineages, particularly hematopoietic cells and hematopoietic cell precursors.

[0004] 2. Description of related technologies

[0005] Hematopoietic stem cells (HSCs) are the only cells with the ability to self-renew, differentiate into all blood cell types, and rebuild the entire hematopoietic system after transplantation. These cells, along with their terminally differentiated derivatives such as erythrocytes, platelets, granulocytes, and lymphoid cells, have well-established therapeutic applications in treating various blood disorders and, more recently, cancer. However, the limited availability of HSCs from HLA-matched living donors significantly restricts their widespread use in clinics. Therefore, considerable effort has been made to derive HSCs from alternative cell types.

[0006] One approach to deriving HSCs from alternative cell types is to transdifferentiate non-HSC somatic cell types into HSCs. Various transgenic combinations have been used to transdifferentiate mouse fibroblasts into non-implantable hematopoietic progenitor cells (Pereira et al., 2013; Batta et al., 2014). However, this approach is limited by the number of initial primary cells and low transdifferentiation efficiency. Therefore, there is a lack of methods to provide an unlimited supply of hematopoietic progenitor cells with long-term engraftment potential.

[0007] Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can proliferate indefinitely in vitro while retaining their potential to differentiate into all somatic cell types. Therefore, human ESCs and iPSCs potentially provide an unlimited supply of patient-specific HSCs and functional blood cells for both in vitro and in vivo applications. The in vitro differentiation of human PSCs into hematopoietic lineage cells encapsulates normal in vivo development, including the mesodermal induction phase and the specialization of pluripotent hematopoietic precursors. Consequently, numerous methods have been developed to differentiate human PSCs into hematopoietic lineages through forward programming.

[0008] However, there is currently no method to robustly generate HSCs capable of efficient myeloid and lymphoid differentiation and long-term engraftment from PSCs, primarily due to the complex nature of hematopoietic individual development. In one approach, overexpression of transgenes such as HoxB4 and Lhx2 for hematopoietic differentiation in mouse PSCs has been shown to produce engraftable HSC-like cells (Kyba et al., 2002; Kitajima et al., 2011). However, these transgenes failed to generate HSCs from human PSCs. In another approach, Doulatov et al. reported that a combination of ERG, HOXA9, RORA, SOX4, and MYB transgenes in human PSCs could generate hematopoietic progenitor cells capable of both myeloid and erythrocyte differentiation (Doulatov et al., 2013). However, this method only produced cells capable of short-term engraftment dependent on sustained transgene expression. Therefore, while genetic programming has proven to be a very promising approach, a method is lacking that allows for the robust in vitro generation of hematopoietic progenitor cells capable of lymphoid and myeloid potential and long-term engraftment from human PSCs. Invention Overview

[0010] Embodiments of this disclosure provide methods for efficiently programming human pluripotent stem cells into multi-lineage hematopoietic progenitor cells. In a first embodiment, an in vitro method for generating hematopoietic progenitor cells (HPCs) from pluripotent stem cells is provided, comprising providing pluripotent stem cells (PSCs) containing an expression construct encoding at least one hematopoietic progenitor programming gene, wherein the hematopoietic progenitor programming gene includes the ETS / ERG gene, GATA2, and HOXA9, and culturing the pluripotent stem cells under conditions expressing the hematopoietic progenitor programming gene, thereby generating hematopoietic progenitor cells. In some aspects, HPCs are capable of differentiating into myeloid and lymphoid lineages. In some aspects, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some aspects, the pluripotent stem cells are human pluripotent stem cells.

[0011] In some respects, the expression construct is based on transposon or free organism expression constructs. In some respects, hematopoietic precursor programming genes are under the control of a single promoter. In certain respects, the single promoter is an inducible promoter. In a specific respect, the inducible promoter is the tetracycline-inducible promoter.

[0012] In a further aspect, methods for generating hematopoietic progenitor cells (HPCs) include culturing HPCs under conditions that prevent the expression of hematopoietic progenitor programming genes. In some aspects, HPCs are cultured in the absence of stromal cells. In some aspects, HPCs are cultured in serum-free or defined culture media. In some aspects, HPCs can be differentiated into two or more cell types selected from: plasma cells, natural killer cells, macrophages, mast cells, megakaryocytes, erythrocytes, granulocytes, lymphocytes, monocytes, leukocytes, and platelets. In some aspects, the lymphocytes are B lymphocytes and / or T lymphocytes.

[0013] In some respects, pluripotent stem cells are cultured for approximately four to ten days under conditions that enable the expression of hematopoietic precursor programming genes.

[0014] In some respects, HPCs express one or more hematopoietic precursor markers. In some respects, the hematopoietic precursor markers are selected from CD43, CD33, CD34, CD45, CD235a, and CD41a. In certain respects, one or more hematopoietic precursor markers are selected from CD43, CD45, and CD34. In some respects, HPCs are immature HPCs. In some respects, immature HPCs express CD34 and CD43. In some respects, at least 50% of HPCs are immature HPCs, for example, at least 55, 60, 65, 70, 75, 80, 90, 95, 96, 97, 98, or 99% of HPCs. In a further respects, at least 70% of HPCs are immature HPCs. In a further respects, at least 90% of HPCs are immature HPCs.

[0015] In some respects, the ETS / ERG gene is ERG (a homolog of the v-ets erythropoietin E26 oncogene), ETV2 (ets variant 2), FLI-1 (Friend leukemia virus integration 1), ELK3 (a protein containing the ETS domain), ETS1 (C-ets-1), or ETS2 (C-ets-2). In certain respects, the ETS / ERG gene is either ERG or ETV2.

[0016] In some respects, hematopoietic precursor programming genes include ERG, GATA2, and HOXA9. In other respects, hematopoietic precursor programming genes include ETV2, GATA2, and HOXA9.

[0017] In some respects, hematopoietic progenitor programming genes are fused with target sequences. For example, the target sequence is NUP98 or its homologous domain. In some respects, hematopoietic progenitor programming genes include ERG, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10. In other respects, hematopoietic progenitor programming genes include ETV2, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10.

[0018] In a further aspect, the PSC containing at least one expression construct encoding a hematopoietic precursor programming gene also contains at least one additional expression construct encoding one or more hematopoietic stem cell programming genes.

[0019] In a further aspect, the in vitro method for generating hematopoietic progenitor cells from pluripotent stem cells further includes culturing HPCs under conditions expressing one or more hematopoietic stem cell programming genes, thereby generating hematopoietic stem cells (HSCs) capable of long-term implantation in mammals. In some aspects, the mammal is a human. In some aspects, the one or more hematopoietic stem cell programming genes are selected from BCL2, BEND4, BMI1, CIITA, EGR3, ETV6, EZH1, EZH2, FOXL1, HIF3A, HLF, HMGA2, HOXA9, HOXA10, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXB3, HOXB6, HSF5, KLF2, KLF4, MECOM, MEIS1, MIR29A, MIR29B1, MS I2,MYB,MYCN,NKX2-3,NR4A2,PEG3,PRDM12,PRDM16,RBAK,RUNX1,RUNX3,SETBP1,SOX17,SOX8,TFEC,ZBTB14,ZB TB20, ZMAT1, ZNF131, ZNF134, ZNF136, ZNF256, ZNF26, ZNF300, ZNF337, ZNF350, ZNF414, ZNF662, ZNF667 and ZNF682. In some aspects, the one or more hematopoietic stem cell programming genes are selected from HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, RBAK, HOXA6, HOXB6, HOXA7, ZNF300, ZNF682, and MSI2. In specific aspects, the one or more hematopoietic stem cell programming genes are selected from HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, and RBAK.

[0020] In some respects, the expression of one or more hematopoietic stem cell programming genes is constitutive in HPC. In other respects, the expression of one or more hematopoietic stem cell programming genes is largely silenced in pluripotent stem cells.

[0021] In some respects, hematopoietic stem cell programming genes are fused with target sequences. In specific respects, the target sequence is NUP98 or its homologous domain.

[0022] In another embodiment, an in vitro method for generating hematopoietic progenitor cells from pluripotent stem cells is provided, comprising providing pluripotent stem cells (PSCs) containing expression constructs encoding ERG, GATA2, and HOXA9 under the control of a single promoter and culturing the pluripotent stem cells under conditions expressing ERG, GATA2, and HOXA9 to generate hematopoietic progenitor cells (HPCs).

[0023] In yet another embodiment, an in vitro method is provided for generating hematopoietic stem cells (HSCs) from pluripotent stem cells, the method comprising providing pluripotent stem cells (PSCs) containing an expression construct encoding ERG, GATA2, and HOXA9 under the control of a single promoter and at least a second expression construct encoding one or more hematopoietic stem cell programming genes, culturing the pluripotent stem cells under conditions expressing ERG, GATA2, HOXA9, and one or more hematopoietic stem cell programming genes, thereby generating HSCs capable of long-term implantation in mammals.

[0024] In a further embodiment, an expression construct encoding a hematopoietic precursor programming gene is provided, wherein the programming gene includes the ETS / ERG gene, GATA2, and HOXA9. In some aspects, the construct is a transposon- or free-body expression construct. In some aspects, the hematopoietic precursor programming gene is under the control of a single promoter. In some aspects, the single promoter is an inducible promoter. In a particular aspect, the inducible promoter is a tetracycline-inducible promoter. In some aspects, the ETS / ERG gene is ERG (a homolog of the v-ets erythropoietin E26 oncogene), ETV2 (variant 2), FLI-1 (Friend leukemia virus integration 1), ELK3 (a protein containing the ETS domain), ETS1 (C-ets-1), or ETS2 (C-ets-2). In a specific aspect, the ETS / ERG gene is ERG or ETV2. In some aspects, the hematopoietic precursor programming gene includes ERG, GATA2, and HOXA9. In other respects, hematopoietic progenitor programming genes include ETV2, GATA2, and HOXA9. In some respects, hematopoietic progenitor programming genes are fused with target sequences. In certain respects, the target sequence is NUP98 or its homologous domain. In some respects, hematopoietic progenitor programming genes include ERG, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10. In other respects, hematopoietic progenitor programming genes include ETV2, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10.

[0025] In another embodiment, a cell containing an expression construct encoding a hematopoietic precursor programming gene is provided, wherein the programming gene includes the ETS / ERG gene, GATA2, and HOXA9.

[0026] In yet another embodiment, an expression construct encoding one or more hematopoietic stem cell programming genes is provided. Specifically, the one or more hematopoietic stem cell programming genes are selected from BCL2, BEND4, BMI1, CIITA, EGR3, ETV6, EZH1, EZH2, FOXL1, HIF3A, HLF, HMGA2, HOXA9, HOXA10, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXB3, HOXB6, HSF5, KLF2, KLF4, MECOM, MEIS1, MIR29A, MIR29B1, MS I2,MYB,MYCN,NKX2-3,NR4A2,PEG3,PRDM12,PRDM16,RBAK,RUNX1,RUNX3,SETBP1,SOX17,SOX8,TFEC,ZBTB14,ZB TB20, ZMAT1, ZNF131, ZNF134, ZNF136, ZNF256, ZNF26, ZNF300, ZNF337, ZNF350, ZNF414, ZNF662, ZNF667 and ZNF682. In some aspects, the one or more hematopoietic stem cell programming genes are selected from HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, RBAK, HOXA6, HOXB6, HOXA7, ZNF300, ZNF682, and MSI2. In specific aspects, the one or more hematopoietic stem cell programming genes are selected from HMGA2, MYCN, NR4A2, SOX17, TFEC, MEIS1, HOXA4, ZNF414, KLF4, ZNF131, BCL2, ETV6, ZNF350, and RBAK. In some aspects, the one or more hematopoietic stem cell programming genes are under the control of a cytomegalovirus (CMV) promoter. In some aspects, the hematopoietic stem cell programming genes are fused with a target sequence. In one specific aspect, the target sequence is NUP98 or its homologous domain.

[0027] In another embodiment, a cell comprising an expression construct encoding one or more hematopoietic stem cell programming genes is provided.

[0028] In yet another implementation, hematopoietic stem cells differentiated in vitro from human pluripotent stem cells are provided, which can be transplanted into the bone marrow of mammals and produce differentiated human blood cells.

[0029] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while preferred embodiments of the invention have been indicated, the detailed description and specific examples are given by way of example only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description.

[0030] Brief description of the attached figures

[0031] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments given herein.

[0032] Figure 1A-1F Linked co-expression of ETV2 / ERG, GATA2, and HOXA9 effectively programmed human PSCs into immature CD34+ hematopoietic progenitor cells. (A) Test configuration of the programming genes ETV2 / ERG, GATA2, and HOXA9 is shown. (B) Test configuration of E+G, EG, and EGH-inducible gene expression in human PSCs modified to constitutively express rtTET protein is shown in relation to doxycycline (DOX)-inducible gene expression. (C) Absolute cell counts in cultures induced on day 8 in the ETV2 and ERG-based gene configuration. (D) Expansion and differentiation potential of DOX-induced cells co-cultured with MS5 stromal cells on day 8 is shown. (E) Total CD43 after 2 weeks of co-culture with MS5 stromal cells. + and immature CD43 + CD34 + Absolute cell count. (F) Multilineage colony-forming potential was detected in EGH-induced cells after 2 weeks of co-culture with MS5 matrix.

[0033] Figure 2 Transgenic expression following induction using the CMV promoter (pCMV). The pCMV-EGFP and pTight-EG constructs were introduced into human PSCs expressing rtTET using the PiggyBac expression vector. CD43 was observed after DOX-induced hematopoietic programming. + EGFP expression in cells.

[0034] Figures 3A-3C Screening for EGH-complementary transgenes that improve CD133 expression in amplified and conferred primitive hematopoietic progenitor cells. (A) EGH-induced cells with constitutive HOXA10 expression (pCMV) showed CD43 expression associated with the most primitive stem cell-like cells after 2 weeks of co-culture with MS5 matrix. + CD34 + CD133 +A smaller population of cells. (B) Designed for improving the original CD43 + CD34 + and CD43 + CD34 + CD133 + A schematic diagram of a screening model for EGH-complementary genes to detect cell yield. (C) shows the screening results for genes that have a positive effect on EGH-induced progenitor cell expansion. Score = P34 × P133 × (P34 / P45), where the P-yield of each subset is calculated as the internal EGH control and 34-CD43. + CD34 + 133-CD43 + CD34 + CD133 + 45-CD43 / 45 + CD34 - The fraction of cells.

[0035] Figures 4A-4B EGH-induced lymphoid differentiation potential of cells. (A) Schematic diagram of a screening model designed to detect genes that improve lymphoid cell development in EGH-induced cells. (B) Flow cytometry analysis of 4-week T / NK and B cell differentiation cultures.

[0036] Figures 5A-5C EGH-induced cell engraftment potential. (A) Schematic diagram designed to detect a screening model for EGH-complementary genes capable of hematopoietic transplantation in immunocompromised mice. (B) Showing artificial blood CD45 in peripheral blood and bone marrow of NBSGW mice 12 weeks after injection. + Cellular detection. (C) Shows artificial blood CD43 / 45 in peripheral blood and bone marrow of NBSGW mice 12 weeks after injection. + Cell detection.

[0037] Figure 6 : A graph showing the ratio of transgene expression in implanted cells to injected cells. The corresponding implantation / injection expression ratio indicates whether the transgene has been enriched (positive) or depleted (negative) after cell transplantation.

[0038] Description of the illustrative implementation scheme

[0039] This disclosure overcomes several major problems of the prior art by providing methods and compositions for generating multilineage hematopoietic progenitor cells from pluripotent stem cells (PSCs). In particular, multilineage hematopoietic progenitor cells can be programmed to be hematopoietic stem cells capable of long-term engraftment. The inventors have discovered that one way to achieve multilineage hematopoietic progenitor cells is to transfect PSCs with one or more expression vectors that achieve the expression of at least three specific genes, the expression of which regulates the “forward programming” of PSCs into multilineage hematopoietic progenitor cells. Notably, the methods of this disclosure are applicable to any type of pluripotent stem cell, including, for example, embryonic stem cells or induced pluripotent stem cells. In particular, multilineage hematopoietic progenitor cells have the potential to efficiently differentiate into myeloid and lymphoid lineage cells.

[0040] Preferably, the hematopoietic programming gene is ETV2 or ERG, GATA2, and HOXA9. In certain aspects, the hematopoietic programming gene is under the control of a single promoter (e.g., an inducible promoter). Typically, the hematopoietic programming gene is expressed only for a sufficient period of time to positively program PSCs into hematopoietic progenitor cells.

[0041] After the formation of immature multilineage hematopoietic progenitors, the inventors have discovered that, in order to enable long-term engraftment of hematopoietic stem cells, one or more additional steps are preferably taken. In one method, PSCs are transfected with one or more additional expression constructs encoding one or more hematopoietic stem cell programming genes, the expression of which enables the multilineage hematopoietic progenitors to be stably engrafted in vivo. In some aspects, the hematopoietic stem cell programming genes for long-term engraftment are expressed in immature hematopoietic progenitor cells but not in PSCs.

[0042] Therefore, the methods of this disclosure provide an unlimited number of multi-lineage hematopoietic precursors and hematopoietic stem cells for a wide range of applications, such as stable in vivo transplantation of hematopoietic precursors, in vitro screening of compounds, and elucidation of the mechanisms of blood diseases and injuries.

[0043] I. Definition

[0044] As used herein, "substantially free of" with respect to a particular component is used to indicate that the particular component was not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any accidental contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition in which the amount of the particular component is undetectable by standard analytical methods is preferred.

[0045] As used herein, "an" or "a" may mean one or more. As used in the claims, when used with the word "comprising," the word "an" or "a" may mean one or more.

[0046] The use of the term "or" in the claims is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definition of "and / or" referring only to alternatives. As used herein, "another" may mean at least a second or more.

[0047] Throughout this application, the term “about” is used to indicate that the value includes inherent variations in the error of the method or apparatus used to determine the value, or variations that exist among study subjects.

[0048] When used in conjunction with proteins, genes, nucleic acids, or polynucleotides in cells or organisms, the term "exogenous" refers to proteins, genes, nucleic acids, or polynucleotides that have been introduced into cells or organisms by artificial or natural means; or, in relation to cells, the term refers to cells isolated and subsequently introduced into other cells or organisms by artificial or natural means. Exogenous nucleic acids can originate from different organisms or cells, or they can be one or more additional copies of nucleic acids that are naturally present in an organism or cell. Exogenous cells may originate from different organisms or may originate from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid located at a different chromosomal position than it does in its natural cell, or its flanks are nucleic acid sequences different from those found in nature.

[0049] An "expression construct" or "expression cassette" is a nucleic acid molecule that directs transcription. An expression construct includes at least one or more transcriptional regulatory elements (e.g., promoters, enhancers, or equivalent structures) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as transcription termination signals, may also be included.

[0050] A “carrier” or “construction” (sometimes referred to as a gene delivery system or gene transfer “medium”) is a complex of macromolecules or molecules containing polynucleotides to be delivered to host cells in vitro or in vivo.

[0051] Plasmids are a common type of vector; they are extrachromosomal DNA molecules that are separate from chromosomal DNA and can replicate independently of chromosomal DNA. In some cases, they are circular or double-stranded.

[0052] The “ori” or “ori” is a DNA sequence (e.g., in lymphotropic herpesviruses) and / or site at or near the initiation of DNA synthesis in a plasmid that, when present in a cell, maintains linkage within the plasmid. For example, the ori of EBV includes the FR sequence (20 incomplete copies of a 30 bp repeat sequence), preferably the DS sequence; however, other sites in EBV bind EBNA-1, such as the Rep* sequence, which can substitute for DS as the origin of replication (Kirshmaier and Sugden, 1998). Therefore, the origin of replication of EBV includes the FR, DS, or Rep* sequence or any functionally equivalent sequence or synthetic combination derived therefrom through nucleic acid modification. For example, as specifically described in Lindner et al., 2008, this method can also utilize the genetically engineered origin of replication of EBV, such as through the insertion or mutation of individual elements.

[0053] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgenic" that "encodes" a specific protein is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed in vitro or in vivo and optionally translated into a gene product such as a polypeptide. The coding region can exist as cDNA, genomic DNA, or RNA. When existing as DNA, the nucleic acid molecule can be single-stranded (i.e., sense strand) or double-stranded. The boundaries of the coding region are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Genes can include, but are not limited to, cDNA derived from prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. The transcription termination sequence is typically located at the 3' end of the gene sequence.

[0054] The term "control element" refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, splice sites, etc., that collectively enable the replication, transcription, post-transcriptional processing, and translation of a coding sequence in recipient cells. Not all of these control elements are necessary, provided the selected coding sequence can be replicated, transcribed, and translated in a suitable host cell.

[0055] The term "promoter" is used in its general sense herein to refer to a nucleotide region containing a DNA regulatory sequence derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may contain genetic elements that regulatory proteins and molecules (such as RNA polymerase and other transcription factors) may bind to initiate specific transcription of a nucleic acid sequence. The phrases "operable location," "operable linkage," "under control," and "under transcriptional control" refer to a promoter being in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0056] An enhancer is a nucleic acid sequence that, when located near a promoter, imparts increased transcriptional activity to the promoter compared to when the enhancer domain is absent.

[0057] "Operationally linked" or "co-expressed" of nucleic acid molecules refers to the linkage of two or more nucleic acid molecules (e.g., nucleic acid molecules to be transcribed, promoters, and enhancer elements) in a manner that allows the nucleic acid molecules to be transcribed. "Operationally linked" or "co-expressed" of peptides and / or polypeptide molecules refers to the linkage of two or more peptides and / or polypeptide molecules in a manner that produces a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and / or polypeptide component of the fusion. The fusion polypeptide is preferably chimeric, i.e., composed of heterologous molecules.

[0058] "Homology" refers to the percentage of identity between two polynucleotides or two polypeptides. The correspondence between one sequence and another can be determined using techniques known in the art. For example, homology can be determined by direct comparison of sequence information between two polypeptide molecules using readily available computer programs. Alternatively, homology can be determined by polynucleotide hybridization under conditions that promote stable double-strand formation between homologous regions, followed by digestion with a single-strand-specific nuclease and determination of the size of the digested fragment. As determined using the methods described above, two DNA sequences or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match at a defined length in the molecule.

[0059] The term "cell" is used in its broadest sense herein, referring to a living organism that is the structural unit of a multicellular organism, surrounded by a membrane structure that isolates it from the outside, possesses the ability to self-replicate, and has genetic information and the mechanisms for expressing it. The cells used herein may be naturally occurring cells or artificially modified cells (e.g., fused cells, genetically modified cells, etc.).

[0060] The term "stem cell" as used herein refers to a cell that, under suitable conditions, can differentiate into a wide range of specialized cell types, while under other suitable conditions, it can self-renew and remain in a substantially undifferentiated pluripotent state. The term "stem cell" also includes pluripotent cells, multipotent cells, progenitor cells, and precursor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells derived from bone marrow tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming them into a pluripotent state through the expression of certain transcription factors associated with pluripotency; these cells are referred to as "induced pluripotent stem cells" or "iPSCs."

[0061] "Embryonic stem (ES) cells" herein refers to undifferentiated pluripotent stem cells, or those produced artificially (e.g., by nuclear transfer). In the context of this invention, the undifferentiated pluripotent stem cells are selected from embryonic stem cells (ESCs) derived from non-human organisms, established human embryonic stem cell lines, or induced pluripotent stem cells (iPSCs).

[0062] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells through the expression or induction of a combination of expression factors (referred to herein as reprogramming factors). iPSCs can be generated using postnatal, neonatal, juvenile, or adult somatic cells. In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed into pluripotent stem cells by expressing at least two, at least three, at least four, or at least five reprogramming factors.

[0063] "Pluripotent stem cells" are stem cells that have the potential to differentiate into all cells that make up one or more tissues or organs, or preferably any one of the three germ layers: endoderm (internal stomach lining, gastrointestinal tract, lung), mesoderm (muscle, bone, blood, urogenital organs), or ectoderm (epidermal tissue and nervous system).

[0064] As used herein, the term "somatic cell" refers to any cell that is not a germ cell (such as an egg, sperm, etc.) and does not directly transfer its DNA to the next generation. Somatic cells typically have limited or no pluripotency. Somatic cells used herein can be naturally occurring or genetically modified.

[0065] "Programming" is the process of altering the types of progeny a cell can produce. For example, a cell has been programmed to form at least one new cell type in culture or in vivo, compared to cells that would have formed under the same unprogrammed conditions. This means that, after sufficient proliferation, a measurable ratio of progeny with the phenotypic characteristics of the new cell type is observed, if such progeny could not have been formed substantially before programming; or, a measurably greater ratio of progeny with the characteristics of the new cell type compared to before programming. This process includes differentiation, dedifferentiation, and transdifferentiation.

[0066] Differentiation is the process by which less specialized cells become more specialized cell types. Dedifferentiation is a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as pluripotency or polymorphism. Transdifferentiation is the process of converting one differentiated cell type into another. Typically, transdifferentiation occurs through programming without intermediate pluripotency stages—that is, cells are directly programmed from one differentiated cell type to another. Under certain conditions, the proportion of offspring with characteristics of the new cell type can increase in priority to at least about 1%, 5%, 25%, or more.

[0067] The term "forward programming" refers to programming pluripotent or multipotent cells by providing them with one or more specific lineage-determining genes or gene products, as opposed to differentiated somatic cells that are not pluripotent. For example, forward programming can describe the process of programming ESCs or iPSCs into hematopoietic progenitor cells or other progenitor cells, or hematopoietic cells or other differentiated somatic cells.

[0068] The term "hematopoietic precursor programming gene" refers to a gene that, when expressed alone or in combination with another programming gene, can positively program pluripotent stem cells into hematopoietic precursor cells capable of producing lymphoid and myeloid lineage cells.

[0069] The term "hematopoietic stem cell programming gene" refers to a gene that, when expressed alone or in combination with another programming gene, can program hematopoietic precursor programming genes to engraft hematopoietic stem cells for long-term implantation.

[0070] As used herein, a “2A sequence” refers to a short peptide that allows for the co-expression of multiple proteins from a single vector. These small peptides can be introduced as linkers between two proteins, thereby allowing autonomous ribosome self-processing of multiple proteins (see, for example, deFelipe. Genetic Vaccines and Ther. 2:13 (2004); deFelipe et al., Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thoseea asigna virus (T2A), and porcine cheisenvirus-1 (P2A) described in U.S. Patent Publication No. 20070116690, which are incorporated herein by reference.

[0071] As used herein, the term “subject” or “subject in need” refers to a male or female mammal, preferably a human, of any age who requires a cell or tissue transplant. Typically, a subject requires a cell or tissue transplant (also referred to herein as a recipient) due to a condition or pathology suitable for treatment via cell or tissue transplantation or an undesirable condition, state or syndrome or physical, morphological or physiological abnormality.

[0072] In this document, "multi-lineage construct" refers to a construct that encodes at least three hematopoietic programming genes, including the ETS gene, homeobox genes, and hematopoietic development genes. An exemplary construct encodes either ETV2 or ERG, GATA2, and HOXA9.

[0073] As used in this article, the term “implantation” for hematopoietic stem cells or hematopoietic progenitor cells refers to cells introduced into a recipient located in the recipient’s bone marrow and that can provide long-term reconstruction of myeloid and lymphoid cell lineages in the recipient.

[0074] “Long-term engraftment” is defined herein as the stable transplantation of cells, such as hematopoietic progenitor cells provided by the methods herein, into a recipient, such that the transplanted cells persist in the host blood and / or bone marrow for more than 10 weeks, preferably more than 20 weeks. Furthermore, long-term engraftment can be characterized by the persistence of transplanted cells in consecutively transplanted mice.

[0075] II. Cells involved in hematopoietic cell programming

[0076] In some embodiments, methods and compositions for providing multilineage hematopoietic progenitor cells from pluripotent stem cells are disclosed. Pluripotent stem cells can be stem cells, including but not limited to induced pluripotent stem cells and embryonic stem cells.

[0077] The pluripotent stem cells used in the present invention for generating hematopoietic progenitor cells are characterized by their ability to self-renew through mitotic cell division and their ability to differentiate into multiple specialized cell types.

[0078] Specifically, the pluripotent stem cells used herein are human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) capable of long-term in vitro proliferation while retaining the potential to differentiate into all cell types in the body, including the hematopoietic progenitor cells of this disclosure. Therefore, these cells may provide an unlimited supply of patient-specific functional hematopoietic cells for drug development and therapeutic uses. Certain aspects of this disclosure provide multilineage hematopoietic progenitor cells by positively programming human PSCs, such as ESCs and iPSCs, through the expression of a combination of programming genes important for hematopoietic cell differentiation / function.

[0079] A. Embryonic stem cells

[0080] In some respects, pluripotent stem cells are equivalent to embryonic stem cells (ESCs). The pluripotency of ES cells, combined with genetic manipulation and selection, can be used for in vivo genetic analysis studies by generating transgenic, chimeric, and knockout mice.

[0081] Methods for generating mouse ES cells are well known. In one method, preimplantation blastocysts from mouse strain 129 are treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in a medium containing fetal bovine serum. Undifferentiated ES cell colonies are passaged on the mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to generate an ES cell population. In some methods, mouse ES cells can be cultured without a feeder layer by adding the cytokine leukemia suppressor factor (LIF) to a serum-containing medium (Smith, 2000). In other methods, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0082] Using the methods previously described (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000; U.S. Patent No. 5,843,780), ES cells can also be derived from other organisms, including rhesus monkeys and marmosets, as well as from established mouse and human cell lines. For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As another example, established mouse ES cell lines include the CGR8 cell line derived from the inner cell mass of 129 strain mouse embryos, and cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0083] ES stem cells can be detected by protein markers, including transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1.

[0084] B. Induced pluripotent stem cells

[0085] In other respects, the pluripotent stem cells used in this article are induced pluripotent stem (iPS) cells, commonly abbreviated as iPS cells or iPSCs. Pluripotency induction was initially achieved in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells with pluripotency-related transcription factors. Using iPSCs avoids most of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive therapy to prevent transplant rejection.

[0086] Besides germ cells, any cell type can be used as the starting point for iPSCs. For example, cell types can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of reprogrammed somatic cells (US Patent 8,741,648). There are no restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected; even undifferentiated progenitor cells (including adult stem cells) and ultimately differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein.

[0087] Somatic cells can be reprogrammed to generate induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art. Those skilled in the art can readily generate induced pluripotent stem cells, see, for example, published U.S. Patent Application No. 20090246875, published U.S. Patent Application No. 2010 / 0210014; published U.S. Patent Application No. 20120276636; U.S. Patent No. 8,058,065; U.S. Patent No. 8,129,187; U.S. Patent No. 8,268,620; PCT Publication No. WO 2007 / 069666 A1 and U.S. Patent No. 8,268,620, which are incorporated herein by reference. Typically, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0088] The mouse and human cDNA sequences of these nuclear reprogramming substances can be found in WO 2007 / 069666 and U.S. Patent 8,183,038, which are incorporated herein by reference. Methods for introducing one or more reprogramming substances or nucleic acids encoding such reprogramming substances are known in the art and disclosed, for example, in U.S. Patent Nos. 8,268,620, 8,691,574, 8,741,648, 8,546,140, ​​U.S. Patent Nos. 8,900,871 and 8,071,369, both of which are incorporated herein by reference.

[0089] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. As described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603, iPSCs can be used with various culture media and techniques for culturing pluripotent stem cells, more specifically embryonic stem cells. In the case of mouse cells, culture is carried out by adding leukemia inhibitory factor (LIF), a differentiation inhibitor, to a normal culture medium. In the case of human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for the culture and maintenance of iPSCs, as known to those skilled in the art, can be used in the method of the present invention.

[0090] In some embodiments, undefined conditions may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or on a medium already exposed to fibroblast feeder cells to maintain stem cells in an undifferentiated state. In some embodiments, cells are cultured in the coexistence of mouse embryonic fibroblasts (treated with radiation or antibiotics to terminate cell division) as feeder cells. Alternatively, established feeder cell-independent culture systems such as TESR may be used. TM Culture medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8 TM / Essential 8 TM Culture medium (Chen et al., 2011) was used to culture pluripotent cells and maintain them in a largely undifferentiated state.

[0091] Plasmids have been designed for multiple objectives, such as achieving regulated high copy numbers and avoiding potential causes of plasmid instability in bacteria, and providing plasmid selection means compatible with mammalian cells, including human cells. Particular attention has been paid to the dual requirements of plasmids for human cells. First, they are suitable for maintenance and fermentation in *E. coli*, allowing for the production and purification of large quantities of DNA. Second, they are safe and suitable for human patients and animals. The first requirement necessitates high copy number plasmids that can be selected during bacterial fermentation and maintained relatively easily. The second requirement demands attention to elements such as selectivity markers and other coding sequences. In some embodiments, plasmids encoding markers consist of: (1) a high copy number origin of replication, (2) a selectivity marker, such as, but not limited to, a neo gene selected for antibiotic use with kanamycin, (3) a transcription termination sequence, including a tyrosinase enhancer, and (4) a multiple cloning site for incorporation into various nucleic acid cassettes; and (5) a nucleic acid sequence encoding a marker operablely linked to a tyrosinase promoter. Many plasmid vectors are known in the art for inducing nucleic acids encoding proteins. These include, but are not limited to, the carriers disclosed in U.S. Patent Nos. 6,103,470, 7,598,364, 7,989,425, and 6,416,998, which are incorporated herein by reference.

[0092] Free gene delivery systems can be plasmids, Epstein-Barr virus (EBV)-based free vectors (US Patent 8,546,140), yeast-based vectors, adenovirus-based vectors, simian virus 40 (SV40)-based free vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors. Viral gene delivery systems can be RNA-based or DNA-based viral vectors (PCT / JP2009 / 062911).

[0093] C. Embryonic stem cells derived from somatic cell nuclear transfer

[0094] Pluripotent stem cells for generating hematopoietic progenitor cells can also be prepared via somatic cell nuclear transfer, in which the donor cell nucleus is transferred to a non-spindle oocyte. The stem cells generated by nuclear transfer are genetically identical to the donor cell nucleus. In one approach, the nucleus of a donor fibroblast derived from rhesus monkey skin fibroblasts is introduced into the cytoplasm of a mid-mature II rhesus macaque oocyte via electrofusion (Byrne et al., 2007); the fused oocytes are activated by exposure to inosine and then incubated until the blastocyst stage; the inner cell mass of selected blastocysts is then cultured to generate embryonic stem cell lines. These embryonic stem cell lines exhibit normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0095] III. Hematopoietic progenitor cell programming factors

[0096] A. Hematopoietic precursor programming factor

[0097] Certain aspects of this disclosure provide constructs encoding hematopoietic precursor programming genes for programming PSCs into multilineage hematopoietic precursor cells. The multilineage hematopoietic precursor cells of this disclosure can be directly generated from pluripotent stem cells by modifying PSCs to express at least three hematopoietic precursor programming genes, such as the ETS gene, hematopoietic development genes, and homoebox genes. These at least three hematopoietic precursor programming genes can be encoded by one or more multilineage constructs.

[0098] Hematopoietic precursor programming genes can be fused with sequences known in the art for the expansion of hematopoietic precursor cells (US Patent Publication No. US20080299095, incorporated herein by reference). An exemplary sequence is NUP98 or its homologous domain.

[0099] 1. ETS gene

[0100] The multilineage construct encodes at least one gene from the E26 transformation-specific (ETS) transcription factor family. All ETS family members are identified by the highly conserved DNA-binding domain, the ETS domain, a winged helix-turn-helix structure that binds to a DNA site with a central GGA (A / T) DNA sequence. In addition to its DNA-binding function, there is evidence that the ETS domain is also involved in protein-protein interactions. The ETS family is distributed throughout the body and is involved in a variety of functions, including regulating cell differentiation, cell cycle control, cell migration, cell proliferation, apoptosis (programmed cell death), and angiogenesis. Members of this gene family have been involved in the development of various tissues and cancer progression.

[0101] The ETS gene can be any gene from the 12 subfamilies of the ETS family, including ELF, ELG, ERG, ERF, ESE, ETS, PDEF, PEA3, ER71, SPI, TCF, and TEL. For example, ETS can be ERG (v-ets erythropoietin E26 oncogene homolog; accession number NM_001136154), ETV2 (ets variant 2; accession number NC_000019.10), FLI-1 (Friend leukemia virus integration 1; accession number NM_001167681), ELK3 (a protein containing the ETS domain; accession number NM_001303511), or ETS1 (C-ets-1; accession number NM_001143820). ETS2 (C-ets-2; accession number NM_001256295), E74-like factor 1 (ELF1; accession number M_001145353), E74-like factor 2 (ELF2; accession number NM_001276457), ETS-associated transcription factor (ELF4; accession number NM_001127197), ETS variant 3 (ETV3; accession number NM_001145312), or transcription factor PU.1 (SPI1; accession number NM_001080547). Specifically, the ETS gene can be an endothelial differentiation factor called ERG, also known as: transcription regulator ERG, ETS-associated transforming protein ERG, TMPRSS2-ERG prostate cancer specific, v-ets erythropoietin E26 oncogene-like, v-ets avian erythropoietin E26 oncogene-associated, or transforming protein ERG. In some implementations, the ETS gene is a specific isotype of ERG, such as ERG isotype 2 (ERG-2) (accession number NM_004449) or ERG isotype 3 (ERG-3) (accession number NM_001136154). In a specific implementation, the ETS gene is ETV2.

[0102] 2. Hematopoietic development genes

[0103] The multilineage construct also encodes at least one hematopoietic development gene. A hematopoietic development gene can be any gene that induces hematopoiesis. Non-restrictive examples of hematopoietic development genes include GFI1 (growth factor-independent 1 transcriptional repressor; accession number NM_001127215), GFI1B (growth factor-independent 1B transcriptional repressor; accession number NM_001135031), TAL1 (T-cell acute lymphoblastic leukemia; accession number NM_001287347), LYL1 (lymphoblastic leukemia-derived sequence 1; accession number NM_005583), LMO2 (LIM domain only 2 (rhombotin-like 1); accession number M_001142315), GATA2 (GATA-binding protein 2; accession number NM_001145661), or GATA3 (GATA-binding protein 3; accession number NM_001002295). In the specific implementation plan, the hematopoietic development gene is GATA2.

[0104] 3. Homeobox genes

[0105] In addition, the multi-lineage construct encodes at least one homeobox gene. The homeobox gene encodes a homeobox approximately 180 base pairs long, which encodes a protein domain that binds to DNA. The characteristic homeodomain protein fold consists of a 60-amino acid helical-turn-helix (HTH) structure, in which three α-helices are linked by a short loop region. The two N-terminal helices are antiparallel, and the longer C-terminal helix is ​​approximately perpendicular to the axis established by the first two. This third helix interacts directly with DNA through numerous hydrogen bonds and hydrophobic interactions, occurring between specific side chains within the major groove of the DNA and exposed bases and thymine methyl groups. Many homeodomain proteins induce cell differentiation by initiating a cascade of co-regulatory genes required to generate individual tissues and organs.

[0106] Homeobox genes can be any gene encoding a homeobox domain. For example, homeobox genes can be HOX genes such as HOXA9 (accession number NM_152739), HOXA10 (accession number NM_018951), HOXA3 (accession number NM_030661), HOXA4 (accession number NM_002141), HOXA5 (accession number NM_019102), HOXA6 (accession number NM_024014), HOXA7 (accession number NM_006896), HOXB3 (accession number NM_002146), or HOXB6 (accession number NM_018952). Other non-restricted examples of the HOX gene include the activity-dependent neuroprotective homeobox (ADNP; accession number NM_001282531), the homeobox protein aristaless-like 4 (ALX4; accession number NM_021926), the homeobox protein DBX1 (accession number NM_001029865), the dual homeobox 4 (DUX4; NM_001127386), and the homeobox protein EMX1 (accession number NM_0010404). 04), GBX2 (accession number NM_001301687), homeobox 1 expressed in ES cells (HESX1; accession number NM_003865), NANOG (accession number NM_001297698), PAX3 (accession number NM_000438), retinal and anterior neural folding homeobox (RAX; accession number NM_013435) or zinc finger E-box binding homeobox 1 (ZEB1; accession number NM_001128128). In the specific implementation, the homeobox gene is HOXA9.

[0107] B. Hematopoietic stem cell programming factors

[0108] Certain aspects of this disclosure provide constructs encoding hematopoietic stem cell programming factors for long-term engraftment potential. Long-term engraftable hematopoietic stem cells can be generated directly from multilineage hematopoietic progenitor cells of this disclosure by increasing the levels of hematopoietic stem cell programming genes in the cells (particularly those listed in Table 1). The inventors also contemplate including all isotypes and variants of the genes listed in this section within this disclosure, and provide non-limiting examples of accession numbers for certain isotypes or variants.

[0109] Table 1 provides a list of genes used to program multi-lineage hematopoietic precursors into hematopoietic stem cells capable of long-term engraftment. All gene sequences and related information provided by the listed gene IDs and accession numbers are incorporated herein by reference from the filing date of this application.

[0110] Table 1: Hematopoietic stem cell programming genes for long-term implantation potential.

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] In some implementations, the hematopoietic stem cell programming gene is any of the genes included in Table 1, including genes involved in the specialization of hematopoietic cells, genes involved in the maintenance and / or proliferation of hematopoietic cells, and genes expressed in hematopoietic cells.

[0117] In some embodiments, one or more hematopoietic stem cell programming genes are combined to program hematopoietic stem cells that can be implanted long-term. In some embodiments, three or more, such as 4, 5, 6, 7, 8, 9, 10, 15, up to 20, or any range thereof, of hematopoietic stem cell programming genes are combined to program hematopoietic stem cells that can be implanted long-term.

[0118] Hematopoietic stem cell programming genes can be fused with sequences known in the art for expanding hematopoietic progenitor cells (US Patent Publication No. US20080299095, incorporated herein by reference). An exemplary sequence is NUP98 or its homologous domain.

[0119] IV. Delivery of hematopoietic programming genes

[0120] In some implementations, vectors are constructed for delivering nucleic acids encoding programming factors to express those factors in pluripotent stem cells. Details of the components and delivery methods of these vectors are disclosed below.

[0121] On the other hand, the following systems and methods can also be used to deliver reporter gene expression cassettes to identify desired cell types, such as hematopoietic progenitor cells. Specifically, reporter gene expression can be driven using regulatory elements specific to hematopoietic stem cells or hematopoietic progenitors. Thus, reporter molecules can be used to characterize, select, or enrich hematopoietic stem cells or progenitors derived from programmed hematopoietic stem cells or progenitors.

[0122] A. Nucleic acid delivery system

[0123] Those skilled in the art will be able to construct vectors using standard recombination techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996, both incorporated herein by reference). Vectors include, but are not limited to, plasmids, serosomes, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC), such as retroviral vectors (e.g., vectors derived from Moloney mouse leukemia virus (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., vectors derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenovirus (AAV) vectors including those with replication capability, replication defects, and gutless forms, simian virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey mouse sarcoma virus vectors, mouse mammary tumor virus vectors, and Rous sarcoma virus vectors.

[0124] 1. Viral vector

[0125] Viral vectors may be provided in certain aspects of this disclosure. In generating recombinant viral vectors, non-essential genes are typically replaced with genes or coding sequences of heterologous (or non-natural) proteins. A viral vector is an expression construct that utilizes a viral sequence to introduce nucleic acids and, possibly, proteins into a cell. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome, and to stably and efficiently express viral genes, makes them attractive candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). The following describes non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain aspects of this disclosure.

[0126] Retroviruses are promising as gene delivery vectors due to their ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specific cell lines (Miller, 1992).

[0127] To construct retroviral vectors, nucleic acids are inserted into the viral genome to replace certain viral sequences, producing replication-defective viruses. To generate viral particles, packaging cell lines containing the gag, pol, and env genes (but without LTRs and packaging components) are constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA along with the retroviral LTR and packaging sequence is introduced into a specific cell line (e.g., via calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require host cell division (Paskind et al., 1975).

[0128] Lentivirals are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol, and env. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136).

[0129] Recombinant lentiviral vectors can infect non-dividing cells and can be used for in vivo and in vitro gene transfer and nucleic acid sequence expression. For example, U.S. Patent 5,994,136 describes recombinant lentiviruses capable of infecting non-dividing cells, wherein suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, and rev and tat, which is incorporated herein by reference.

[0130] 2. Free carrier

[0131] The use of plasmid- or liposome-based extrachromosomal (i.e., free) vectors is also provided in certain aspects of this disclosure. Such free vectors may include, for example, oriP-based vectors and / or vectors encoding EBNA-1 derivatives. These vectors allow large fragments of DNA to be introduced into cells and maintained extrachromosomally, replicated once per cell cycle, efficiently distributed to daughter cells, and substantially without evoking an immune response.

[0132] In particular, EBNA-1 (the only viral protein required for replication of oriP-based expression vectors) does not elicit a cellular immune response because efficient mechanisms have been developed to bypass the processing required to present its antigen to MHC class I molecules (Levitskaya et al., 1997). Furthermore, EBNA-1 can transact to enhance clonal gene expression, inducing up to 100-fold expression in some cell lines (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the manufacture of such oriP-based expression vectors is inexpensive.

[0133] Other extrachromosomal vectors include other vectors based on lymphotrophic herpesviruses. Lymphotrophic herpesviruses are plasmid-based herpesviruses that replicate in lymphoblasts (such as human B lymphoblasts) and become part of their natural life cycle. Herpes simplex virus (HSV) is not a “lymphotrophic” herpesvirus. Exemplary lymphotrophic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); squirrel monkey herpesvirus (HSV), and Marek's disease virus (MDV). Other sources of exosome-based vectors, such as yeast ARS, adenovirus, SV40, or BPV, are also considered.

[0134] Those skilled in the art will be able to construct vectors using standard recombination techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0135] The vector may also contain other components or functional elements that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to target cells. Such other components include, for example, components that affect binding to or targeting cells (including components that mediate cell type or tissue-specific binding); components that affect cellular uptake of the vector nucleic acid; components that affect the intracellular localization of polynucleotides after uptake (e.g., reagents that mediate nuclear localization); and components that affect polynucleotide expression.

[0136] These components may also include markers, such as detectable and / or selectable markers that can be used to detect or select cells that have taken up and are expressing nucleic acids delivered by the vector. These components may be provided as a natural feature of the vector (e.g., using certain viral vectors with components or functional elements that mediate binding and uptake), or the vector may be modified to provide such functional elements. Various such vectors are known in the art and are generally available. When the vector is held in a host cell, it can stably replicate as an autonomous structure during mitosis, be incorporated into the host cell's genome, or remain in the host cell's nucleus or cytoplasm.

[0137] 3. Transposon-based systems

[0138] In some respects, the delivery of programming factors can be achieved using transposon-transposase systems. For example, transposon-transposase systems can be the well-known Sleeping Beauty, Frog Prince transposon-transposase systems (for a description of the latter, see, for example, EP1507865) or the TTAA-specific transposon PiggyBac system.

[0139] Transposons are DNA sequences that can move to different locations within the genome of a single cell, a process known as transposition. During this process, they can cause mutations and alter the amount of DNA in the genome. Transposons have also been called jumping genes and are examples of mobile genetic elements.

[0140] Various types of mobile genetic elements exist and can be grouped based on their transposition mechanisms. Class I mobile genetic elements, or retrotransposons, are first transcribed into RNA, then reverse transcribed into DNA by reverse transcriptase, and then inserted into another location in the genome to replicate themselves. Class II mobile genetic elements move directly from one location to another, using transposases to "cut and paste" them in the genome.

[0141] In specific implementations, the constructs provided in this disclosure (e.g., multi-lineage constructs) use the PiggyBac expression system. PiggyBac (PB) DNA transposons move via a "cut-and-paste" mechanism, where a transposase (PB transposase) encoded by the transposon itself is excised and reintegrated at other sites within the genome. The PB transposase specifically recognizes PB inverted terminal repeat (ITR) sequences flanking the transposon; it binds to these sequences and catalyzes the excision of the transposon. The PB then integrates into TTAA sites throughout the genome in a relatively random manner. To generate gene-trapping mutations (or to produce transgenic animals), the transposase is trans-supplied on a plasmid and co-transfected with a plasmid containing a donor transposon, which is a recombinant transposon containing a gene trap with flanking transposase (ITR) binding sites. The transposase catalyzes the excision of the transposon from the plasmid and its subsequent integration into the genome. Integration within the coding region captures the elements required for gene trap expression. PB has several desirable properties: (1) it preferentially inserts into genes (50% to 67% of inserted genes hit); (2) it does not have local skipping (extensive genome coverage); (3) it is insensitive to overproduction inhibition (elevated transposase levels lead to reduced transposition); and (4) it is cleanly excised from the donor site, unlike Sleeping Beauty which leaves a “footprint”.

[0142] 4. Homologous recombination

[0143] In some respects, nucleic acid molecules can be introduced into cells in specific ways for genome engineering, such as through homologous recombination. As mentioned above, some methods for expressing genes in cells involve the use of viral vectors or transgenes that integrate randomly into the genome. However, these methods have the drawback of integration occurring at sites where the expression of the integrated nucleic acid cannot be effectively mediated or where it leads to the destruction of the natural gene. The problems associated with random integration can be partially overcome by homologous recombination into specific loci in the target genome (e.g., the Rosa26 locus).

[0144] Homologous recombination (HR), also known as general recombination, is a genetic recombination process used in all life forms in which nucleotide sequences are exchanged between two similar or identical DNA strands. Since the mid-1980s, this technique has been the standard method for genome engineering in mammalian cells. The process involves several steps of physical disruption and eventual recombination of DNA. This process is widely used to repair potentially fatal double-strand breaks in DNA. Furthermore, homologous recombination produces new combinations of DNA sequences during meiosis, the process by which eukaryotes produce germ cells such as sperm and eggs. These new DNA combinations represent genetic variation in offspring, allowing populations to adapt to changing environmental conditions over time. Homologous recombination is also used for horizontal gene transfer to exchange genetic material between different strains and bacterial and viral species. Homologous recombination is also used in molecular biology as a technique to introduce genetic changes into target organisms.

[0145] Homologous recombination can be used as a targeted genomic modification. The efficiency of standard HR in mammalian cells is only 10⁻⁶ to 10⁻⁹ in treated cells (Capecchi, 1990). Broad-spectrum nucleases or homing endonucleases such as I-SceI have been used to increase the efficiency of HR. Both native broad-spectrum nucleases and engineered broad-spectrum nucleases with modified target specificity have been used to improve HR efficiency (Pingoud and Silva, 2007; Chevalier et al., 2002).

[0146] One approach to improving HR efficiency is to modify chimeric endonucleases with programmable DNA-specific domains (Silva et al., 2011). Zinc finger nucleases (ZFNs) are an example of such chimeric molecules, in which the zinc finger DNA-binding domain is fused with the catalytic domain of an IIS-type restriction endonuclease such as FokI (as described in Durai et al., 2005).

[0147] Another class of such specific molecules includes transcription activator-like effector (TALE) DNA-binding domains fused to the catalytic domain of IIS-type restriction endonucleases such as FokI (Miller et al., 2011; PCT / IB2010 / 000154). TALENs can be engineered for site-specific genomic modifications at virtually any given site of interest (Cermak et al., 2011; Christian et al., 2010; Li et al., 2011; Miller et al., 2011; Weber et al., 2011; Zhang et al., 2011). Site-specific DNA-binding domains are expressed as fusion proteins with DNA-cutting enzymes such as Fok I. The DNA-binding domain is the scaffold of repeating amino acids; linking each repeat sequence are two variable amino acids that bind to a single nucleotide in the DNA. For example, Asn-Asn binds guanosine, Asn-Ile binds adenosine, Asn-Gly binds thymine, and His-Asp binds cytosine. These two amino acids are referred to as repeating variable diresidues, or RVDs. There are many different RVDs that can be engineered into TAL effector / Fok1 protein constructs to create specific TALENs. The RNA encoding the recombinant TALEN can then be purified and transfected into cells for site-specific genomic modification. Once the TALEN introduces a double-strand DNA break, the DNA can be modified via non-homologous end joining (NHEJ) or homology-directed repair (HDR). This allows for DNA mutagenesis, deletion, or addition depending on the presence of other sequences during DNA repair.

[0148] B. Control elements

[0149] The expression cassette contained in the vector used in this disclosure preferably contains (in the 5' to 3' orientation) a eukaryotic transcription promoter effectively linked to a protein-coding sequence, a splicing signal for the insertion sequence, and a transcription termination / polyadenylation sequence.

[0150] 1. Promoter / Enhancer

[0151] The expression constructs provided in this article contain promoters that drive the expression of programmed genes. Promoters typically contain sequences that locate the start site for RNA synthesis. The best example of this is the TATA box, but in some promoters lacking a TATA box (e.g., the promoters of mammalian terminal deoxynucleotidyl transferase genes and SV40 late genes), discrete elements covering the start site themselves help to fix the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these promoters are located in a 30–110 bp region upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. To make the coding sequence “controlled” by the promoter, the 5’ end of the transcription start site of the transcription reading frame is positioned “downstream” (i.e., 3’) of the selected promoter. The “upstream” promoter stimulates DNA transcription and promotes the expression of the encoded RNA.

[0152] The spacing between promoter elements is typically flexible, allowing the promoter to maintain function when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline. Depending on the promoter, individual elements can work together or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers," which are cis-regulatory sequences involved in the transcriptional activation of nucleic acid sequences.

[0153] Promoters can be promoters naturally associated with a nucleic acid sequence, such as promoters obtained by isolating a 5' non-coding sequence located upstream of a coding region and / or exon. Such promoters can be referred to as "endogenous." Similarly, enhancers can be enhancers naturally associated with a nucleic acid sequence located downstream or upstream of that sequence. Alternatively, certain advantages can be obtained by placing the coding nucleic acid fragment under the control of a recombinant or heterologous promoter, which is a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers are also enhancers that are not normally associated with a nucleic acid sequence in their natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, and promoters or enhancers that are not "naturally present," i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, the promoters most commonly used for recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthetically generating nucleic acid sequences for promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR) can be used. TMThe sequences generated, together with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each incorporated herein by reference), are also intended to guide the transcription and / or expression of sequences in nonnuclear organelles such as mitochondria, chloroplasts, etc.

[0154] Of course, it is important to use promoters and / or enhancers that effectively guide the expression of DNA fragments in the selected organelles, cell types, tissues, organs, or organisms for expression. Those skilled in the art of molecular biology are generally familiar with the applications of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoters used can be constitutive, tissue-specific, inducible, and / or, under suitable conditions, advantageous for guiding high-level expression of the introduced DNA fragment, for example, in the large-scale production of recombinant proteins and / or peptides. Promoters can be heterologous or endogenous.

[0155] Additionally, any promoter / enhancer combination (e.g., based on the eukaryotic promoter database EPDB via the World Wide Web at epd.isb-sib.ch / ) can also be used to drive expression. Using T3, T7, or SP6 cytoplasmic expression systems is another possible implementation. If appropriate bacterial polymerases are provided, eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, either as part of a delivery complex or as an additional gene expression construct.

[0156] Non-limiting examples of promoters include early or late viral promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus (RSV) early promoter; eukaryotic promoters, for example, the β-actin promoter (Ng, 1989; Quitsche et al., 1989), the GADPH promoter (Alexander et al., 1988; Ercolani et al., 1988), the metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and linked response element promoters, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the response element promoter near the minimal TATA box (tre). Alternatively, human growth hormone promoter sequences (e.g., the minimal human growth hormone promoter described in Genbank, accession number X05244, nucleotides 283-341) or mouse mammary tumor promoters (available from ATCC, catalog number ATCC 45007) can be used.

[0157] As a method for identifying derived hematopoietic cells and precursors, tissue-specific transgenic expression, particularly reporter gene expression in hematopoietic cells and precursors derived from programmed hematopoietic cells, is likely desirable. To improve specificity and activity, the use of cis-regulatory elements has been considered. For example, hematopoietic cell-specific promoters can be used. Many such hematopoietic cell-specific promoters are known in the art, such as the promoters of hematopoietic genes provided in Table 1.

[0158] In some aspects, the method of the present invention also relates to enhancer sequences, i.e., nucleic acid sequences that increase promoter activity and have cis-action regardless of their orientation, even at relatively long distances (up to several thousand bases from the target promoter). However, enhancer function is not necessarily limited to such long distances, as they can also function close to a given promoter.

[0159] Numerous hematopoietic cell promoter and enhancer sequences have been identified and can be used in the methods of this invention. See, for example, U.S. Patent 5,556,954; U.S. Patent Application 20020055144; and U.S. Patent Application 20090148425.

[0160] In a specific sense, the promoter is an inducible promoter. The activity of an inducible promoter can be induced by the presence or absence of biotic or abiotic factors. Inducible promoters are powerful tools in genetic engineering because the expression of genes operably linked to them can be turned on or off at a specific stage of an organism's development or in a particular tissue. For example, Tet-On and Tet-Off inducible gene expression systems based on the fundamental regulatory components of the *E. coli* tetracycline resistance operon can be used. Once established in the starting cells, the inducer doxycycline (Dox, a tetracycline derivative) can be used to control the expression system in a dose-dependent manner, allowing for precise modulation of the expression level of the programmed gene. In an exemplary embodiment, the inducible promoter is the rtTET inducible tight promoter (pTight). Therefore, the pTight promoter can be used to induce the expression of multi-lineage programmed genes such as ETV2, GATA2, and HOXA9 for a sufficient period to allow PSCs to be programmed as hematopoietic progenitor cells, and subsequently, this expression can be turned off. The pTight promoter can also be a bidirectional promoter.

[0161] 2. Initiation signals and linkage expression

[0162] Specific start signals can also be used in the expression constructs provided in the method of this invention to efficiently translate coding sequences. These signals include the ATG start codon or adjacent sequences. Exogenous translation control signals, including the ATG start codon, may need to be provided. Those skilled in the art will be able to readily determine this and provide the necessary signals. It is well known that the start codon must "frame-match" the reading frame of the desired coding sequence to ensure the translation of the entire insert. Exogenous translation control signals and start codons can be natural or synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements.

[0163] In some implementations, internal ribosome entry site (IRES) elements are used to generate multigene or polycistronic information. IRES elements are able to bypass ribosome scanning models of 5'-methylation-dependent Cap translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES in mammalian information (Macejak and Sarnow, 1991) have described IRES components from two members of the piconeravirus family (poliomyelitis and encephalomyocarditis) (Pelletier and Sonenberg, 1988). IRES elements can be linked to heterologous open reading frames (ORFs). Multiple ORFs can be transcribed together, each frame separated by an IRES, thus forming polycistronic information. With IRES elements, ribosomes can utilize each ORF for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, each incorporated herein by reference).

[0164] Additionally, certain 2A sequence elements can be used in the constructs provided in this disclosure to generate linked or co-expressed programmed genes. For example, a cleavage sequence can be used to co-express a gene by connecting open reading frames to form a single cistron. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or “2A-like” sequences (e.g., Thoseea asigna virus 2A; T2A) (Minskaia and Ryan, 2013). In a specific embodiment, the F2A cleavage peptide is used to link gene expression in a multilineage construct.

[0165] 3. Copy the starting point

[0166] To propagate the vector in host cells, it may contain one or more replication initiation sites (commonly referred to as "ori"), for example, nucleic acid sequences corresponding to the oriP of the aforementioned EBV or genetically engineered oriP with similar or enhanced function in programming, which are the specific nucleic acid sequences that initiate replication. Alternatively, replication origins or autonomous replication sequences (ARS) of other extrachromosomal replicating viruses as described above may be used.

[0167] 4. Selection and Filterable Tags

[0168] In some embodiments, cells containing nucleic acid constructs of this disclosure can be identified in vitro or in vivo by including a marker in the expression vector. This marker confers identifiable changes to the cells, thereby facilitating the identification of cells containing the expression vector. Typically, a selection marker is a marker that confers the property of allowing selection. A positive selection marker is one in which the presence of the marker allows selection, while a negative selection marker is one in which the presence of the marker prevents selection. An example of a positive selection marker is a drug resistance marker.

[0169] Drug-selective markers are commonly used to aid in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin, and histidine are useful selectable markers. Besides markers that confer resistance allowing condition-based differentiation of transformant phenotypes, other types of markers are envisioned, including colorimetric-based selectable markers such as GFP. Alternatively, selectable enzymes, such as herpes simplex virus thymidine kinase (TK) or chloramphenicol acetyltransferase (CAT), can be used as negative selectable markers. Those skilled in the art also know how to use immunological markers, which may be combined with FACS analysis. The marker used is not considered critical, as long as it can be co-expressed with the nucleic acid encoding the gene product. Other examples of selectable and selectable markers are well known to those skilled in the art.

[0170] C. Nucleic acid delivery

[0171] As described herein, the introduction of nucleic acids (e.g., DNA or RNA) into pluripotent stem cells to be programmed into hematopoietic progenitor cells using the methods of the present invention can be achieved using any suitable method for nucleic acid delivery to transform the cells, or as is known to those skilled in the art. These methods include, but are not limited to, direct delivery of DNA, such as by in vitro transfection (Wilson et al., 1989; Nabel et al., 1989), by injection (US Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; US Patent No. 5,789,215, incorporated herein by reference); by electroporation (US Patent No. 5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986; Potter et al., 1984); and by calcium phosphate precipitation (Graham and Van Der). Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-glucan and polyethylene glycol (Gopal, 1985); by direct acoustic loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988); by particle bombardment (PCT applications WO 94 / 09699 and 95 / 06128; US patents 5,610,042; 5,322,783). U.S. Patents 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each incorporated herein by reference; by stirring with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacterium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); by drying / inhibition-mediated DNA uptake (Potrykus et al., 1985); and any combination of these methods. By applying techniques such as these, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0172] 1. Liposome-mediated transfection

[0173] In one implementation, nucleic acids can be introduced into pluripotent stem cells via liposome-mediated transfection. In this method, the nucleic acids are encapsulated in lipid complexes, such as liposomes. Liposomes are vesicle structures characterized by a phospholipid bilayer and an internal aqueous medium. Multilayered liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement prior to the formation of the encapsulated structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also considered. The amount of liposomes used can vary depending on the properties of the liposomes and the cells used; for example, 1 to 10 x 10^6 liposomes per liposome can be considered. 6 Each cell contains approximately 5 to 20 μg of vector DNA.

[0174] In vitro liposome-mediated delivery of nucleic acids and expression of exogenous DNA has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of exogenous DNA in cultured chicken embryos, HeLa cells, and hepatocellular carcinoma cells has also been demonstrated (Wong et al., 1980).

[0175] In some embodiments, liposomes may be complexed with hemagglutinin virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote the entry of liposome-encapsulated DNA into the cell (Kaneda et al., 1989). In other embodiments, liposomes may be combined with or used in conjunction with nuclear non-histone chromosomal protein (HMG-1) (Kato et al., 1991). In some embodiments, liposomes may be complexed with or used in conjunction with both HVJ and HMG-1. In other embodiments, the delivery vector may comprise a ligand and liposomes.

[0176] 2. Electroporation

[0177] In some implementations, nucleic acids are introduced into organelles, cells, tissues, or organisms via electroporation. Electroporation involves exposing a suspension of cells and DNA to a high-voltage discharge. Recipient cells can be more easily transformed by mechanical damage. The amount of carrier used can also vary depending on the nature of the cells used; for example, 1 to 10 x 10^6 carriers per cell can be considered. 6 Each cell contains approximately 5 to 20 μg of vector DNA.

[0178] Electroporation transfection of eukaryotic cells has been quite successful. Mouse pre-B lymphocytes have been transfected with the human κ-immunoglobulin gene (Potter et al., 1984), and rat hepatocytes have been transfected in the same manner with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986).

[0179] V. Methods for producing hematopoietic precursor cells

[0180] A. Multilineage hematopoietic progenitor cells

[0181] This disclosure provides a method for generating multilineage hematopoietic progenitor cells from pluripotent stem cells (PSCs). PSCs (e.g., ESCs or iPSCs) are genetically modified to express the hematopoietic progenitor programming genes described herein, which forward-program the PSCs into multilineage hematopoietic progenitor cells. Specifically, the multilineage hematopoietic progenitors have the potential to differentiate into myeloid and lymphoid lineage cells. Preferably, the hematopoietic progenitor programming genes include ETS genes, hematopoietic development genes, and homeobox genes. Exemplary hematopoietic progenitor programming genes include EVT2 or ERG, GATA2, and HOXA9.

[0182] Additional hematopoietic progenitor programming genes, such as HOXA10, can enhance the efficiency of forward programming. In some aspects, the hematopoietic programming gene is fused with sequences known in the art for expanding hematopoietic progenitor cells (US Patent Publication No. US20080299095, incorporated herein by reference). An exemplary sequence is NUP98 or its homologous domain. In one exemplary method, the hematopoietic progenitor programming gene includes EVT2 or ERG, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10.

[0183] Hematopoietic precursor programming genes can be encoded by one or more expression constructs. Preferably, the gene is encoded by a single expression construct. Therefore, the expression of hematopoietic precursor programming genes can be controlled by a single promoter. The expression of hematopoietic programming genes can be operatively linked, for example, via IRES or 2A sequence elements.

[0184] Preferably, the three hematopoietic precursor programming genes are expressed only for a period of time sufficient to positively program PSCs into hematopoietic precursor cells. Therefore, the hematopoietic precursor programming genes can be under the control of an inducible promoter. Thus, the expression of the hematopoietic precursor programming genes in PSCs can be induced for a sufficient period to positively program up to multiple lineages of hematopoietic precursor cells. This time period can be from approximately 1 day to approximately 20 days, such as approximately 3, 4, 5, 6, 7, 8, 9, or 10 days. Alternatively, the hematopoietic precursor programming genes can be introduced into PSCs via a free vector. Therefore, the hematopoietic precursor programming genes can be expressed transiently in PSCs.

[0185] Then, multi-lineage hematopoietic progenitor cells can be further cultured to generate lymphoid and myeloid lineage cells and further programmed into implantable hematopoietic stem cells.

[0186] B. Hematopoietic cells for long-term implantation

[0187] Multilineage hematopoietic progenitor cells can be further programmed into hematopoietic stem cells capable of long-term engraftment. Preferably, PSCs or hematopoietic progenitor cells are transfected with one or more additional expression constructs encoding one or more hematopoietic stem cell programming genes described herein (e.g., Table 1), the expression of which enables stable engraftment of multilineage hematopoietic progenitors in vivo. One or more additional expression constructs may be introduced into PSCs simultaneously with the multilineage constructs or after PSCs have been positively programmed into immature hematopoietic progenitor cells.

[0188] Hematopoietic stem cell programming genes for long-term implantation can be encoded by one or more expression constructs. Preferably, multiple genes are encoded by expression constructs. Thus, the expression of one or more hematopoietic stem cell programming genes (i.e., long-term implantation genes) can be under the control of a single promoter. The expression of long-term implantation genes can be operatively linked, for example, via IRES or 2A sequence elements.

[0189] In some respects, hematopoietic stem cell programming genes for long-term implantation are expressed in multilineage hematopoietic progenitor cells and not in PSCs. Therefore, the hematopoietic stem cell programming genes can be under the control of promoters that are substantially silent in PSCs. In one exemplary approach, the hematopoietic stem cell programming genes are under the control of a cytomegalovirus (CMV) promoter. Alternatively, the hematopoietic stem cell programming genes can be under the control of an inducible promoter. Thus, the expression of the hematopoietic stem cell programming genes can be induced after PSCs have been positively programmed into multilineage hematopoietic progenitor cells. In yet another alternative, constructs encoding the hematopoietic stem cell programming genes can be transfected into immature hematopoietic progenitor cells after they have been positively programmed from PSCs.

[0190] C. Cell Culture

[0191] Multi-lineage hematopoietic progenitor cells or hematopoietic stem cells capable of long-term engraftment can be cultured under hematopoietic stem cell culture conditions known in the art. Specifically, hematopoietic progenitor cells can also be cultured under conditions that derive specific hematopoietic lineages, such as myeloid or lymphoid lineages.

[0192] Typically, the cells of this disclosure are cultured in a culture medium that is a nutrient-rich buffer solution capable of sustaining cell growth. Culture media suitable for isolating, expanding, and differentiating pluripotent stem cells into hematopoietic progenitor cells and hematopoietic cells according to the methods described herein include, but are not limited to, high-glucose Dulbecco modified Eagle medium (DMEM), DMEM / F-15, Liebovitz L-15, RPMI 1640, Iscove modified Dulbecco medium (IMDM), and Opti-MEM SFM (Invitrogen Inc.). Minimal basal media containing supplements of human serum albumin, human ExCyte lipoprotein, transferrin, insulin, vitamins, essential and non-essential amino acids, sodium pyruvate, glutamine, and mitogens, such as Iscove modified Dulbecco medium (IMDM), are also suitable. As used herein, a mitogen refers to an agent that stimulates cell division. A drug can be a chemical substance, typically a protein in some form, that stimulates the cell to initiate cell division, thereby triggering mitosis. In one embodiment, serum-free culture media as described in U.S. Serial Nos. 08 / 464,599 and WO96 / 39487 and “complete culture media” as described in U.S. Patent No. 5,486,359 are intended to be used with the methods described herein. In some embodiments, the culture medium is supplemented with 10% fetal bovine serum (FBS), human autologous serum, human AB serum, or platelet-rich plasma supplemented with heparin (2 U / ml). Cell cultures may be maintained in a CO2 atmosphere (e.g., 5% to 12%) to maintain the pH of the culture medium, incubated at 37°C in a humid atmosphere, and passaged to maintain confluence below 85%.

[0193] Pluripotent stem cells intended to differentiate into hematopoietic cells and their precursors can be cultured in a medium sufficient to maintain pluripotency. The culture of induced pluripotent stem cells generated in certain aspects of this disclosure can utilize various media and techniques developed for culturing primate pluripotent stem cells, more particularly embryonic stem cells, as described in U.S. Patent Application 20070238170 and U.S. Patent Application 20030211603. For example, similar to human embryonic stem cells, induced pluripotent stem cells can be maintained in 80% DMEM / F12 (Gibco #11330032 or #11320082), 20% KnockOut serum substitute, 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and bFGF (4-100 ng / mL) (PCT Application WO99 / 20741). Alternatively, human ES cells and iPS cells can be maintained in a chemically defined serum-free medium such as mTeSR1.

[0194] Hematopoietic cells and their precursors can be generated by culturing pluripotent stem cells or other non-hematopoietic cells in a culture medium under conditions that increase intracellular levels of hematopoietic programming factors sufficiently to promote cell programming into hematopoietic progenitor cells. The culture medium may also contain one or more hematopoietic cell differentiation and maturation agents, such as various growth factors. These agents can help induce cells to exhibit a more mature phenotype—or preferentially promote the survival of mature cells—or a combination of both effects. Hematopoietic progenitor cells and hematopoietic cell differentiation and maturation agents may include soluble growth factors (peptide hormones, cytokines, ligand-receptor complexes, and other compounds) capable of promoting the growth of hematopoietic lineage cells. Non-limiting examples of these reagents include, but are not limited to, hematopoietic or endothelial growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), thrombopoietin (TPO), FLT-3 (FLT3L), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-9 (IL-9), or granulocyte colony-stimulating factor (G-CSF) or their isotypes or variants.

[0195] VI. Characteristics of hematopoietic progenitor cells and hematopoietic stem cells

[0196] The hematopoietic progenitor cells and hematopoietic stem cells of this disclosure can be characterized according to a number of phenotypic criteria. Criteria include, but are not limited to, the detection or quantification of expressed cellular markers, functional activity, and characterization of morphological features and intercellular signaling. In other aspects, the cells to be programmed may contain a reporter gene expression cassette containing tissue- or cell-specific transcriptional regulatory elements, such as hematopoietic cell-specific promoters, for hematopoietic cell recognition.

[0197] The hematopoietic progenitor cells covered in certain aspects of this disclosure possess morphological characteristics characteristic of natural hematopoietic progenitor cells. These characteristics are readily understood by those skilled in the art when assessing these conditions and include the detection of cell clusters that produce round, non-adherent cells. Additionally, the hematopoietic progenitor cells have a round shape and a low cytoplasm-to-nucleus ratio.

[0198] The cells of the present invention can also be characterized by whether they express certain characteristic markers of hematopoietic lineage cells. Non-limiting examples of cellular markers used to distinguish hematopoietic stem cells from hematopoietic precursors include: CD43, CD33, CD34, CD45, CD235a, CD38, CD90, CD133, CD105, CD117 (c-kit; SCF receptor), CD74, and CD41a. For example, immature hematopoietic precursors capable of differentiating into myeloid and lymphoid lineages can be distinguished by being positive for CD43 and CD34. To identify cells that have differentiated from pluripotent initiating cells (e.g., ESCs or iPSCs), it may be useful to identify cells that do not express certain markers present on pluripotent stem cells or somatic cells (such as TRA-1-60, TRA-1-81, CD166, or CD140b).

[0199] The expression level of this marker can be assessed by comparison with other cells. Positive controls for hematopoietic progenitor cells or hematopoietic cell markers include adult hematopoietic cells or hematopoietic stem cells of the species of interest, and established hematopoietic cell lines. Note that permanent cell lines or long-term hematopoietic cell cultures may be metabolically altered and may not express certain characteristics of primary hematopoietic cells and hematopoietic progenitor cells. Negative controls include cells from individual lineages, such as adult fibroblast lines, adult mesenchymal stem cells, or retinal pigment epithelial cells (RPE). As the examples below illustrate, undifferentiated stem cells are positive for some of the markers listed above, but negative for certain markers of hematopoietic cells and hematopoietic progenitor cells.

[0200] The hematopoietic-specific proteins and oligosaccharide determinants listed in this disclosure can be detected using any suitable immunological technique, such as flow cytometry for cell surface labeling, immunohistochemistry for intracellular or surface labeling (e.g., fixed cells or tissue sections), Western blot analysis and enzyme-linked immunosorbent assay (ELISA) of cell extracts, and cell extracts or products secreted into culture media. If, in a standard immunocytochemical or flow cytometry assay, optionally after cell fixation, and optionally using labeled secondary antibodies or other conjugates (e.g., biotin-avidin conjugates) to amplify the labeling, a significantly detectable amount of antibody will bind to the antigen, then the cell expressing the antigen is referred to as “antibody detectable.”

[0201] Expression of specific (e.g., hematopoietic progenitor cell-specific) markers can also be detected at the mRNA level by Northern blotting analysis, dot blot hybridization analysis, or by reverse transcription polymerase chain reaction (RT-PCR) using sequence-specific primers in standard amplification methods (US Patent No. 5,843,780). Sequence data for the specific markers listed in this disclosure are available from public databases such as GenBank. Expression at the mRNA level is considered "detectable" according to one of the assays described in this disclosure if the performance of the assay performed on a cell sample according to a standard procedure of a typical plus-control experiment results in a clearly identifiable hybridization or amplification product within a standard time window. Unless otherwise required, expression of a specific marker is indicated if the corresponding mRNA is detectable by RT-PCR. Expression of a specific marker detected at the protein or mRNA level is considered positive if the level is at least 2-fold higher and preferably more than 10 or 50-fold higher than the level in control cells such as undifferentiated pluripotent stem cells, fibroblasts, or other unrelated cell types.

[0202] Cells can also be characterized by whether they exhibit characteristic functional activities of hematopoietic lineage cells. For example, hematopoietic progenitor cells have the capacity for self-renewal and can generate more than one type of hematopoietic cell. In specific embodiments, the obtained hematopoietic progenitor cells can efficiently generate lymphoid cells (e.g., T cells, B cells, and NK cells), erythromegakaryocytic cells (e.g., erythrocytes and coagulation cells), and myeloid cells (e.g., granulocytes and monocytes). In other embodiments, hematopoietic stem cells can be engrafted long-term in mammals. For example, long-term engraftment in mouse models can be achieved by, for example, the presence of artificial blood cells (e.g., CD45) in peripheral blood and / or bone marrow at 6, 12, 18, 20, or 25 weeks post-engraftment. + Characterized by HLA class I+ cells.

[0203] Hematopoietic progenitor cells and hematopoietic stem cells provided by the method according to the invention may possess many characteristics of the cellular stage they are intended to represent. The more of these characteristics present in a particular cell, the more it is characterized as a cell of a hematopoietic lineage. Cells having at least 2, 3, 5, 7, or 9 of these characteristics are increasingly preferred. Regarding a particular cell population that may be present in a culture vessel or in an administered formulation, consistency among cells expressing these characteristics is generally advantageous. In this case, a population in which at least about 40%, 60%, 80%, 90%, 95%, or 98% of the cells have the desired characteristics is increasingly preferred.

[0204] VII. Uses of hematopoietic progenitor cells and hematopoietic stem cells

[0205] Hematopoietic progenitor cells and hematopoietic stem cells provided by methods and compositions of certain aspects of this disclosure can be used in a wide range of applications. These include, but are not limited to, in vivo transplantation or implantation of hematopoietic cells and progenitor cells; screening for cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, pharmaceutical compounds, etc.; elucidating the mechanisms of blood disorders and injuries; studying the mechanisms of action of drugs and / or growth factors; diagnosing and monitoring cancer in patients; gene therapy; and the production of bioactive products, to name just a few.

[0206] A. Screening of test compounds

[0207] The programming-derived hematopoietic progenitor cells and hematopoietic stem cells of this disclosure can be used to screen for factors (such as solvents, small molecule drugs, peptides, and polynucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of the hematopoietic cells provided herein.

[0208] The specific screening applications of this disclosure relate to the testing of drug compounds in drug research. Readers typically refer to the standard textbook "In vitro Methods in Pharmaceutical Research," Academic Press, 1997, and U.S. Patent No. 5,030,015. In some respects, cells programmed into hematopoietic lineages serve as test cells in standard drug screening and toxicity assays, as previously done with hematopoietic cells and precursors in short-term cultures. The evaluation of the activity of candidate drug compounds typically involves combining hematopoietic cells or precursors, provided in certain aspects, with the candidate compound, identifying any changes in morphology, marker phenotype, or metabolic activity of the cells attributable to the compound (compared to untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed changes. Screening can be performed because the compound is designed to have a pharmacological effect on hematopoietic cells or precursors, or because a compound designed to have other effects may have an unexpected effect on hematopoietic cells or precursors. Two or more drugs may be used in combination (by simultaneous or sequential combination with cells) to detect potential drug-drug interaction effects.

[0209] In some applications, compounds can be screened for their toxicity to hematopoietic stem cells or hematopoietic progenitor cells.

[0210] B. Hematopoietic cell therapy

[0211] This disclosure also provides information on the potential use of the hematopoietic stem cells and hematopoietic progenitor cells provided herein to restore the function of subjects who may require such treatment due to blood disorders, conditions, or injuries. For example, hematopoietic cells and hematopoietic progenitor cells derived using the methods disclosed herein can be used to treat hematological disorders and conditions such as hemoglobinopathies, anemia, etc. Additionally, hematopoietic stem cells and their precursors can be used to supply blood or blood cells (e.g., red blood cells, platelets, and neutrophils) to subjects in need (e.g., subjects requiring blood transfusions or subjects with blood disorders). Such cells can be used to treat hematopoietic cell defects caused by cell-suppressive therapies (e.g., chemotherapy).

[0212] To determine the suitability of the hematopoietic stem cells and precursors provided herein for therapeutic applications, the cells can first be tested in suitable animal models. At one level, the ability of the cells to survive in vivo and maintain their phenotype is assessed. The programmed cells provided herein are administered to immunodeficient animals (e.g., NOG mice or chemically or irradiated immunodeficient animals) at locations suitable for further observation (e.g., under the renal sac, in the spleen, in the liver lobules, or in the bone marrow). Tissues are harvested after days to weeks or longer, and the presence of the initiating cell type, such as pluripotent stem cells, is assessed. This can be done by providing the administered cells with detectable markers (e.g., green fluorescent protein or β-galactosidase); or by measuring constitutive markers specific to the administered human cells. When testing the programmed cells provided herein in rodent models, the presence and phenotype of the administered cells can be assessed by immunohistochemistry or ELISA using human-specific antibodies or by RT-PCR analysis using primers and hybridization conditions that induce amplification specific to human polynucleotide sequences. Suitable markers for assessing gene expression at the mRNA or protein level are provided elsewhere in this disclosure.

[0213] The hematopoietic stem cells and hematopoietic precursors provided by the methods of this disclosure can be tested in a variety of animal models to treat blood disorders and injuries. For example, sickle cell anemia mouse models or T / B cell-deficient Rag-2 knockout mice may be particularly useful animal models for testing the hematopoietic cells and hematopoietic precursors disclosed herein.

[0214] Hematopoietic stem cells and hematopoietic precursor cells that exhibit the desired functional characteristics or efficacy in animal models, as provided in certain aspects of this disclosure, may also be suitable for direct administration to human subjects in need. For hemostatic purposes, the cells may be administered at any site with sufficient access. Hematopoietic cells or their precursors may also be delivered at sites of injury or disease.

[0215] The cells described in this article can be used to treat any subject in need. Possible human conditions suitable for this treatment include various anemias and hemoglobinopathies, as well as diseases characterized by a decrease in the number of hematopoietic cells (e.g., myeloid dysplasia syndromes, myeloid fibrosis, neutropenia, agranulocytosis, Gransmann's platelet dysfunction, thrombocytopenia, and acquired immunodeficiency syndromes). For human treatment, the dosage is typically around 10... 9 Up to 10 12 Between cells, and usually at about 5 × 10 9 Up to 5×10 10 Between individual cells, the treatment modulates the subject's weight, the nature and severity of the disease, and the replicative capacity of the administered cells. Ultimately, the responsibility for determining the treatment modality and appropriate dosage rests with the managing clinician.

[0216] C. Allocation of funds for commercial, therapeutic, and research purposes

[0217] For manufacturing, distribution and use purposes, the hematopoietic progenitor cells and hematopoietic stem cells of this disclosure are generally supplied in the form of cell cultures or suspensions in isotonic excipients or culture media, optionally frozen to facilitate transport or storage.

[0218] This disclosure also includes various reagent systems comprising cell collections or combinations present at any point during the manufacturing, dispensing, or use process. Cell collections include any combination of two or more cell populations described in this disclosure, such as, but not limited to, programmed-derived cells (hematopoietic lineage cells, their precursors and subtypes), combinations with undifferentiated stem cells, somatic-derived hematopoietic cells, or other differentiated cell types. Cell populations in this collection may sometimes share the same genome or its genetic modifications. Each cell type may be packaged together at the same or different times under the control of the same or different entities with a shared commercial relationship, or packaged in different containers within the same entity, or packaged in different locations. V. Example

[0219] The following embodiments are included to illustrate preferred embodiments of the invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention, and therefore can be considered as constituting preferred modes of practice. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the disclosed specific embodiments and similar or analogous results can still be obtained without departing from the spirit and scope of the invention.

[0220] Example 1 - Linkage expression of ETV2 / ERG, GATA2, and HOXA9 effectively programmed human PSCs into immature CD34 cells. + hematopoietic progenitor cells

[0221] This study aimed to generate hematopoietic precursors with multi-lineage potential, including myeloid and lymphoid potential. Programming efficiency was tested for various configurations of the programmed genes to achieve multi-lineage potential (Table 2). The coding region of the transgene was cloned into the PiggyBac expression vector under the control of the rtTET-inducible Tight promoter (pTight). ETV2 / ERG and GATA2 (E+G) were cloned into separate expression vectors, respectively resistant to blastomycin and hematoxylin and cyproterone acetate. Co-expression of ETV2 / ERG and GATA2 in transfected cells was achieved using combined blastomycin and hematoxylin and cyproterone acetate selection. Alternatively, to ensure balanced expression in transfected cells, ETV2 / ERG and GATA2 (EG) were linked via an F2A-cleaving peptide in a pTight-controlled expression cassette. Linkage co-expression of ETV2 / ERG and GATA2(EG) was found to significantly improve programming efficiency and appeared to bypass the intermediate endothelial cell stage seen when ETV2 / ERG and GATA2(E+G) were expressed on separate vectors (Figure 1).

[0222] Table 2: Configuration of Programming Genes

[0223]

[0224] Next, HOXA9 was linked to the EG expression cassette (EGH) using a bi-pTight promoter to determine whether HOXA9 could improve programming efficiency to generate hematopoietic precursors with multi-lineage potential, including myeloid and lymphoid potential. E+G, EG, and EGH-inducible gene configurations were tested for doxycycline (DOX)-inducible gene expression using human PSCs modified to constitutively express the rtTET protein. The PiggyBac transgenic vector was introduced into human PSCs expressing rtTET using electroporation along with a vector expressing hPBase. Cells with stable PiggyBac transposon integration were selected in culture using 100 μg / ml blastcin and / or genimycin. For transgenic-induced hematopoietic programming, transfected PSCs were dissociated with 0.5 mM EDTA for approximately 5–10 minutes, resuspended in PSC medium (e.g., TeSR or E8), and incubated at 5–10 × 10⁻⁶ rpm in PSC medium supplemented with 5 μM ROCK inhibitor myosin inhibitor. 4Cells / well were spread onto a matrix gel-coated 6-well plate. The following day, transgenic expression and hematopoietic induction were initiated by replacing the PSC medium with induction medium supplemented with 3 ml / well of 0.25 μg / ml doxycycline (Table 3). The induction medium was changed every other day, and the culture was harvested on day 8 of induction using Accutase (Innovative Cell Technologies) cell dissociation solution.

[0225] Table 3: Induction Culture Medium

[0226]

[0227]

[0228] The harvested cells were counted and analyzed by flow cytometry to measure CD34. + CD43 - Endothelial cells, CD43 + Total hematopoietic cells and CD43 + CD34 + Immature hematopoietic progenitor cells. Dot plots and images show that in an ETV2 and ERG-based gene configuration, induction with either the E+G gene alone results in the production of mixed CD34. + CD43 - Endothelial cells and CD43 + Hematopoietic cell populations, and linked EG genes effectively (>80%) program human PSCs to CD43. + Hematopoietic cell population. More importantly, although immature CD43 cells were present in E+G and EG-induced cultures. + CD34 + The ratio of progenitor cells is similar (total CD43) + The proportion of cells (30-40%) indicates that hematopoietic cells have similar differentiation rates, but the ETV2 / ERG-GAT2-HOXA9 (EGH) gene configuration effectively induces and maintains an immature population of hematopoietic progenitor cells, such as through more than 90% of CD43 cells. + CD34 in cells + The expression shown ( Figure 1B Absolute cell counts in DOX-induced cultures over 8 days indicated that, in an ETV2 and ERG-based gene configuration, CD43 was induced via linkage to the inducible gene (EG). + The total number of hematopoietic cells increased significantly (e.g., more than 5-fold), and the EGH gene configuration containing HOXA9 specifically induced immature CD34 cells. + CD43 + More than twice the number of progenitor cells ( Figure 1C ).

[0229] To examine the functional properties of EG- and EGH-induced hematopoietic cells, the expansion and differentiation potential of DOX-induced cells were tested after 8 days in co-culture with MS5 stromal cells. The induced cells were cultured at 10... 4 Cells / well were seeded in 4 ml / well co-culture medium (Table 4) into 6-well plates containing a mitomycin C-treated MS5 cell monolayer. Cultures were maintained for 2 weeks, with half the medium replaced every 3 days. Non-adherent cells were collected and the cell monolayer was dissociated by sequential treatment with 1 mg / ml collagenase IV for 15 minutes followed by Accutase for 15 minutes. The non-adherent and dissociated adherent cell fractions were combined for flow cytometry analysis of absolute cell count and total CD43. + and immature CD34 + CD43 + The ratio of hematopoietic cells was analyzed, and colony-forming cells were analyzed using a MethodCult assay (StemCell Technologies). Compared to EG-induced cells (which showed very limited growth, mainly attributed to small floating cell clusters), EGH-induced cells exhibited strong expansion with significant and extensive cobblestone-like growth regions. Figure 1D This is a well-known characteristic of very primitive hematopoietic progenitor cells. Total CD43 + and immature CD43 + CD34 + Absolute cell counts indicated that total CD43 was present in expanded EG-induced cells. + The number of cells increased only about 5-fold and lacked immature CD34. + CD43 + Cells. Conversely, in EGH-induced cells, total CD43 + Hematopoietic cell amplification exceeding 30-fold, CD43 + CD34 + Immature cells expanded approximately 5-fold. Figure 1E Although colony-forming potential was severely depleted in EG-induced cells, with only a small number of myeloid colonies detected, multi-lineage colony-forming potential was detected in EGH-induced cells after 2 weeks of co-culture with MS5 matrix. Figure 1F ).

[0230] Table 4: Co-culture medium

[0231] Components concentration Iscove Modified Dulbecco Medium (IMDM) FBS (HyClone) 10％ Glutamax (Gibco) 1:100 Monothioglycerol 100μM FGF1 2ng / mL IGF1 50 ng / mL SCF 100ng / mL FLT3L 100ng / mL TPO 10 ng / mL IL3 (added only during cell plating) 10 ng / mL

[0232] Example 2 - Enhancement of the multi-lineage potential of immature hematopoietic precursors

[0233] Screening for other genes to improve the positive programming efficiency of PSCs to immature hematopoietic progenitor cells. Designing screening models to detect additional genes complementary to ETV2 / ERG-GATA2-HOXA9 (EGH) for improving immature CD43. + CD34 + and CD43 + CD34 + CD133 + Cell production.

[0234] Since pCMV has been shown to be substantially suppressed in undifferentiated human PSCs and actively expressed in differentiated cells such as HPCs, this study was conducted to determine whether this characteristic of the CMV promoter can be used in induced CD43... + Post-induced gene expression in cells. pCMV-EGFP and pTight-EG constructs were introduced into human PSCs expressing rtTET using the PiggyBac expression vector. CD43 expression was monitored after DOX-induced hematopoietic programming. + EGFP expression in cells. In undifferentiated / uninduced iPSCs, up to day 8 of DOX induction, including induced CD43... + Low EGFP expression was detected in approximately 20% of the cells. Conversely, after another 4 days (e.g., day 12), more than 50% of the induced CD43 cells showed low expression. + High EGFP expression was detected in cells ( Figure 2 Therefore, the pCMV promoter can be used for transgenic expression of other genes after induction.

[0235] To screen for additional genes, PSCs expressing rtTET were transfected into the PiggyBac expression vector with pTight-EGH (blast fungicide resistance) and an additional pTight- or pCMV-test gene (genimycin resistance). Co-expression of EGH and the test gene in transfected cells was achieved through selection using a combination of blast fungicide and genimycin. Human PSCs transfected with EGH and the test gene were induced with DOX for 8 days to produce CD43. + Cells were then further expanded in two consecutive 2-week MS5 co-cultures. Figure 3B For each test gene, the production of the total and primitive hematopoietic cell populations in the amplified culture was calculated and represented as part of the internal EGH control. Screening results for genes showing a positive role in EGH-induced primitive progenitor cell amplification were shown... Figure 3CIn this study, the addition of HOXA10, driven by the cytomegalovirus (CMV) promoter (which allows transgene expression after day 8), significantly improved cell proliferation and CD43 levels induced during two weeks of expansion culture on MS-5 matrix-fed cells. + CD34 + CD133 + Cell generation ( Figure 3A ).

[0236] Next, a screening model was designed to detect genes that improve lymphoid cell development from EGH-induced cells. Figure 4A For T / NK cells, cells induced on day 8 by transfection with EGH and the test gene combination were used at a rate of 5 x 10⁸ cells / year. 3 cells / cm 2 T / NK cell differentiation medium (e.g., StemSpan SFEM (Stem Cell Technologies) supplemented with ascorbic acid, magnesium phosphate (95 μM), Glutamax (1 / 100; Gibco), penicillin / streptomycin (1 / 100; Gibco), and cytokines SCF, FLT3L, TPO, and IL7 (50 ng / ml each)) was plated onto DLL4-Fc / fibronectin-coated plates (0.5 μg / cm³ each). 2 The culture was maintained under hypoxic conditions (e.g., 5% O2), with half of the medium replaced every 2 or 3 days. After 2 weeks, the cells were transferred to fresh DLL4-Fc / fibronectin-coated plates for another 2 weeks. CD3 counts of cells harvested after 4 weeks were analyzed by flow cytometry. + CD8 + T cells and CD3 - CD8 + NK cells. For B cells, cells induced on day 8 by transfection with EGH and the test gene combination were used at 10 3 cells / cm 2B cells were plated on mitomycin C-treated MS5 monolayers in B cell differentiation medium (e.g., IMDM supplemented with FBS (10%, HyClone), Glutamax (1 / 100, Gibco), penicillin / streptomycin (1 / 100; Gibco), monothioglycerol (100 μM) and cytokines-SCF, FLT3L, TPO (50 ng / ml each), IL7 (20 ng / ml) and IL3 (10 ng / ml, added only at cell plate formation); or, for example, DMEM-F12 supplemented with FBS (10%, HyClone), Glutamax (1 / 100, Gibco), penicillin / streptomycin (1 / 100; Gibco), ascorbic acid (95 μM) and cytokines-SCF and FLT3L (50 ng / ml each), IL7 (20 ng / ml, added only during the first 2 weeks of B cell culture) and IL3 (10 ng / ml, added only during the first 1 week of B cell culture). Cultures were maintained for 4 weeks, with half the volume of medium replaced every 2 or 3 days. Each time fresh medium was added, the suspended non-adherent cells were resuspended and removed using the new medium. CD45 was analyzed by flow cytometry. + CD19 + B cell analysis was performed on cells collected 4 weeks later. The combination of the EGH, pCMV-NA9HD (NUP98-HOXA9 homology domain fusion protein), and pCMV-NA10 (NUP98-HOXA10 fusion protein) genes was found to enable efficient differentiation into T, NK, and B cells. Figure 4B ).

[0237] Example 3 - Hematopoietic stem cell programming genes endow long-term engraftment potential

[0238] A screening model was designed to detect hematopoietic stem cell programming genes that can be combined with EGH expression vectors for long-term hematopoietic transplantation. CD34 cells transfected with EGH and different test gene combinations (e.g., up to 20 genes per combination) were purified from day 8 DOX-induced cultures using magnetically activated cell sorting (MACS). + cell( Figure 5A For immediate post-induction injection, administer 4 × 10⁻⁶ doses. 6 CD34 + Cells at 10 6 Cells / ml were seeded in rehabilitative cultures in HSC medium (e.g., StemSpan SFEM supplemented with SCF, FLT3L, and TPO (100 ng / ml each)) and recovered after 18–24 hours. NOD / SCID / IL2Rg was then added to the culture at 6–8 weeks of age. - / c-kit W41(NBSGW) mice were injected intravenously. The injected mice were then placed in cages containing a DOX-containing diet (i.e., 625 mg DOX / kg) to provide a 7-day supply of DOX for sustained in vivo transgene expression. To inject cells adapted to HSC cultures, 0.5 × 10⁻⁶ cells were injected. 6 CD34 + Cells at 0.2 × 10 6 Cells / ml were seeded in HSC medium onto DLL4-Fc / fibronectin-coated plates and cultured under hypoxic conditions (e.g., 5% O2) for 7 days, with half the culture volume replaced on day 4. After culture, the harvested cells were injected into cells previously injected with CD34 on day 8. + Cells were injected into the same mice fed a DOX-containing diet for 7 days. After injection, the mice were transferred to a normal DOX-free diet, and peripheral blood samples containing artificial blood cells (i.e., CD45) were tested at 6, 12, and 18 weeks. + Type 1 HLA + The presence of cells was detected, and bone marrow was tested at 20-24 weeks.

[0239] Artificial blood CD45 was detected in the peripheral blood and bone marrow of NBSGW mice 12 weeks after injection. + cell( Figure 5B Human CD45 was not detected in mice injected with EGH-induced cells. + Cells, and EGH, combined with 10 other programming genes (Table 1), produce detectable human CD45 in peripheral blood and bone marrow. + Cells. Further analysis was performed to detect CD43 / 45 in the bone marrow and peripheral blood of NBSGW mice 12 weeks post-injection. + These CD43 / 45 cells were not detected in mice injected with EGH-induced cells. + Cells, and the combination of EGH with other programmed genes resulted in the production of 4.64% CD43 / 45 in mouse bone marrow. + cell( Figure 5C ).

[0240] Next, H1-A16-TET ESCs were co-transfected with various combinations of the hematopoietic-inducible EGH gene (blastcin selection vector) and the test gene for HSC programming (G418 selection vector). The transfected ESCs were cultured for two generations in the presence of blastcin and G418 to select double transfectants (EGH + test gene combination), and then induced to CD34. + Cells were tested for implantation in NSGW mice, such as Figure 5AThe expression of each tested transgene in transplanted mouse bone marrow samples was analyzed by transgene-specific qPCR, normalized to the total transgene cell population detected by EGH-specific primers. This expression was then compared with injected CD34. + Initial transgene expression in cells was compared. Each implantation / injection expression ratio indicates the enrichment (positive value) or depletion (negative value) of transgenes after cell transplantation. Figure 6 ).

[0241] Forty candidate HSC programming genes selected from initial in vitro screening were tested in three independent transplantation experiments (using pCMV expression vectors). More than 12 weeks after cell injection, a total of 20 human transgenes were detected in the bone marrow of transplanted mice. Figure 6 As shown, with injected CD34 + Compared to other cells, the expression levels of some transgenes were significantly higher or lower in bone marrow, indicating in vivo selection for transplantable cells. However, regardless of expression levels, all detected human genes likely contribute to PSC-derived CD34 expression in the bone marrow environment. + Cell migration, survival, and persistence are known characteristics of transplantable hematopoietic stem cells / progenitor cells.

[0242] Therefore, by using a combined screening strategy in NBSGW mice (McIntosh et al., 2015), hematopoietic stem cell programming genes that confer long-term implantation of human PSC-derived cells in bone marrow and peripheral blood were identified.

[0243] ***

[0244] Based on this disclosure, all methods disclosed and claimed herein can be prepared and performed without excessive experimentation. While the compositions and methods of the invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations in the steps or order of the steps of the methods can be made without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for those described herein to obtain the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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Claims

1. An in vitro method for generating hematopoietic progenitor cells from pluripotent stem cells, comprising: (a) Providing pluripotent stem cells (PSCs) comprising an expression construct encoding at least one hematopoietic precursor programming gene, wherein the hematopoietic precursor programming gene comprises co-expressed ERG, GATA2 and HOXA9 genes, or comprises co-expressed ETS, GATA2 and HOXA9 genes. and (b) culturing the pluripotent stem cells, wherein the pluripotent stem cells are induced to express hematopoietic progenitor programming genes, thereby generating hematopoietic progenitor cells (HPCs); and (c) The HPC is further cultured, wherein the expression of the hematopoietic precursor programming gene is no longer induced, thereby generating a cell population containing at least 70% multilineage HPCs with CD34 and CD43 expression. The pluripotent stem cells mentioned above are selected from embryonic stem cells (ESCs) from non-human organisms, established human embryonic stem cell lines, or induced pluripotent stem cells (iPSCs).

2. The method of claim 1, wherein the multi-lineage HPC is capable of differentiating into myeloid and lymphoid lineages.

3. The method of claim 1, wherein the expression construct is a transposon- or free-body expression construct.

4. The method of claim 1, wherein the hematopoietic precursor programming gene is under the control of a single promoter.

5. The method of claim 4, wherein the single promoter is an inducible promoter.

6. The method of claim 5, wherein the inducible promoter is a tetracycline-inducible promoter.

7. The method of claim 1, wherein the culture in step (b) is for four to ten days.

8. The method of claim 1, wherein the pluripotent stem cells are established human pluripotent stem cells.

9. The method of claim 1, wherein the multi-lineage HPC expresses one or more hematopoietic precursor markers in addition to CD34 and CD43.

10. The method of claim 9, wherein the hematopoietic precursor marker is selected from CD33, CD34, CD45, CD235a and CD41a.

11. The method of claim 9, wherein one or more hematopoietic precursor markers are CD45.

12. The method of claim 1, wherein the multi-lineage HPC is an immature HPC.

13. The method of claim 12, wherein at least 90% of the multi-lineage HPCs are immature HPCs.

14. The method of claim 1, wherein the HPC in step (c) is cultured in the absence of stromal cells.

15. The method of claim 1, wherein the HPC in step (c) is cultured in a serum-free or defined culture medium.

16. The method of claim 14, wherein the multilineage HPC can differentiate into two or more cell types selected from: plasma cells, natural killer cells, macrophages, mast cells, megakaryocytes, erythrocytes, granulocytes, lymphocytes, monocytes, leukocytes, and platelets.

17. The method of claim 16, wherein the lymphocytes are B lymphocytes and / or T lymphocytes.

18. The method of claim 1, wherein the hematopoietic precursor programming gene is fused with the target sequence.

19. The method of claim 18, wherein the target sequence is NUP98 or its homologous domain.

20. The method of claim 1, wherein the hematopoietic precursor programming genes include ERG, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10.

21. The method of claim 1, wherein the hematopoietic precursor programming genes include ETV2, GATA2, HOXA9, NUP98-HOXA9, and NUP98-HOXA10.

22. The method of claim 1, wherein the PSC of step (a) further comprises at least one additional expression construct encoding one or more hematopoietic stem cell programming genes.

23. The method of claim 22, wherein the one or more hematopoietic stem cell programming genes are selected from BCL2, BEND4, BMI1, CIITA, EGR3, ETV6, EZH1, EZH2, FOXL1, HIF3A, HLF, HMGA2, HOXA10, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXB3, HOXB6, HSF5, KLF2, KLF4, MECOM, MEIS1, MIR29A, MIR29B1. MSI2, MYB, MYCN, NKX2-3, NR4A2, PEG3, PRDM12, PRDM16, RBAK, RUNX1, RUNX3, SETBP1, SOX17, SOX8, TFEC, ZBTB14, Z BTB20, ZMAT1, ZNF131, ZNF134, ZNF136, ZNF256, ZNF26, ZNF300, ZNF337, ZNF350, ZNF414, ZNF662, ZNF667 and ZNF682.

24. The method of claim 22, wherein the expression of one or more hematopoietic stem cell programming genes is constitutive in the HPC of step (b).

25. The method of claim 22, wherein the expression of one or more hematopoietic stem cell programming genes is substantially silenced in the pluripotent stem cells.

26. The method of claim 22, wherein the hematopoietic stem cell programming gene is fused with a target sequence.

27. The method of claim 26, wherein the target sequence is NUP98 or its homologous domain.

28. The method of claim 5, wherein the induced promoter is a bidirectional tight promoter.

29. The method of claim 1, wherein the ERG or ETV2 gene, the GATA2 gene and / or the HOXA9 gene are linked via a 2A cleavage peptide.

30. The method of claim 1, further comprising step (d) differentiating the hematopoietic progenitor cells into myeloid lineage cells or lymphoid lineage cells.

31. The method of claim 6, wherein the PSC in step (b) is cultured in the presence of 0.25 μg / ml doxycycline to induce gene expression.

32. An in vitro method for generating hematopoietic progenitor cells from pluripotent stem cells, comprising: (a) Providing pluripotent stem cells (PSCs) containing expression constructs encoding ERG, GATA2, and HOXA9 under the control of a single promoter; and (b) Pluripotent stem cells were cultured with co-expression of ERG, GATA2, and HOXA9 to generate hematopoietic progenitor cells (HPCs); and (c) The hematopoietic progenitor cells (HPCs) were cultured without co-expression of ERG, GATA2 and HOXA9 to produce a cell population comprising at least 70% multilineage HPCs with CD34 and CD43 expression. The pluripotent stem cells mentioned above are selected from embryonic stem cells (ESCs) from non-human organisms, established human embryonic stem cell lines, or induced pluripotent stem cells (iPSCs).

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