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Stem cell gene therapy using the MFGS-gp91phoxretroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient’s refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOMlocus was identified in blast cells. The vector integration into MECOMwas detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

Chronic inflammation in adipose tissue contributes to obesity-related insulin resistance. The 3-phosphoinositide-dependent protein kinase 1 (Pdk1)/forkhead transcription factor (Foxo1) pathway is important in regulating glucose and energy homeostasis, but little is known about this pathway in adipose tissue macrophages (ATMs). To investigate this, we generated transgenic mice that carried macrophage/granulocyte-specific mutations, including a Pdk1 knockout ([LysMPdk1.sup.-/-]), a Pdk1 knockout with transactivation-defective Foxo1 (Δ256[LysMPdk1.sup.-/-]), a constitutively active nuclear (CN) Foxo1 (CN[Foxo1.sup.LysM]), or a transactivation-defective Foxo1 (Δ256[Foxo1.sup.LysM]). We analyzed glucose metabolism and gene expression in ATM populations isolated with fluorescence-activated cell sorting. The [LysMPdk1.sup.-/-] mice exhibited elevated M1 macrophages in adipose tissue and insulin resistance. Overexpression of transactivation-defective Foxo1 rescued these phenotypes. CN[Foxo1.sup.LysM] promoted transcription of the C-C motif chemokine receptor 2 (Ccr2) in ATMs and increased M1 macrophages in adipose tissue. On a high-fat diet, CN[Foxo1.sup.LysM] mice exhibited insulin resistance. Pdk1 deletion or Foxo1 activation in bone marrow-derived macrophages abolished insulin and interleukin-4 induction of genes involved in alternative macrophage activation. Thus, Pdk1 regulated macrophage infiltration by inhibiting Foxo1-induced Ccr2 expression. This shows that the macrophage Pdk1/Foxo1 pathway is important in regulating insulin sensitivity in vivo. Diabetes 61:1935-1948, 2012, Obesity is a predisposing factor for the development of type 2 diabetes, hypertension, hyperlipidemia, and atherosclerosis (1). Chronic activation of intracellular proinflammatory pathways in adipose tissue contributes to obesity-related insulin [...]

ABSTRACTMacroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of “autophagy profiles” revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations:ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-β: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

Haploinsufficiency of A20 (HA20) is caused by loss-of-function TNFAIP3variants. Phenotypic and genetic features of HA20 remain uncertain; therefore, the clinical distinction between HA20 and Behçet’s disease (BD) requires clarification. We have collected 12 Japanese BD-like families. Probands of these families were analyzed by whole exome sequencing (WES) and subsequent Sanger sequencing. Clinical features were compared between 54 HA20 patients (including previously reported and new cases) and 520 Japanese BD patients. We identified c.1434C>A:p.(Cys478*) in one family and a 236 kb deletion at 6q23.3 containing TNFAIP3in another family. Four HA20 patients in the two families presented with childhood-onset recurrent oral and genital ulcers and were initially diagnosed and treated as BD. Consistent with the clinical features of HA20, recurrent, refractory fever attacks (three of four patients), and digestive ulcers (two of the four patients) were observed. A comparison of clinical features between HA20 patients and cohorts of BD patients revealed several critical features specific to HA20. These were early-onset, familial occurrence, recurrent fever attacks, gastrointestinal involvement, and infrequent ocular involvement. We identified a novel nonsense variant and deletion of the entire TNFAIP3gene in two unrelated Japanese HA20 families. Genetic screening of TNFAIP3should be considered for familial BD-like patients with early-onset recurrent fevers.

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell‐based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor‐initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1‐eGSH). Chr1‐eGSH permitted robust transgene expression, but a 2 Mb‐distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase(HSV‐TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1‐eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV‐TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. Stem Cells Translational Medicine2019;8:627&638 Tumorigenesis and the risks associated with genome editing are major concerns of human induced pluripotent stem cells‐based therapies. In this report, we list the features of human genome safe harbor candidates and demonstrate that topological analysis facilitates prediction of the influence of genome editing. We also suggest that the herpes simplex virus thymidine kinase suicide gene system alone would not be an adequate safeguard.

Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.

Hematopoietic cell transplantation has proven beneficial for various intractable diseases, but it remains unclear how hematopoietic stem/progenitor cells (HSPCs) home to the bone marrow (BM) microenvironment, initiate hematopoietic reconstitution, and maintain life‐long hematopoiesis. The use of newly elucidated molecular determinants for overall HSPC engraftment should benefit patients. Here, we report that modification of C‐X‐C chemokine receptor type 4 (Cxcr4) signaling in murine HSPCs does not significantly affect initial homing/lodging events, but leads to alteration in subsequent BM repopulation kinetics, with observations confirmed by both gain‐ and loss‐of‐function approaches. By using C‐terminal truncated Cxcr4 as a gain‐of‐function effector, we demonstrated that signal augmentation likely led to favorable in vivo repopulation of primitive cell populations in BM. These improved features were correlated with enhanced seeding efficiencies in stromal cell cocultures and altered ligand‐mediated phosphorylation kinetics of extracellular signal‐regulated kinases observed in Cxcr4 signal‐augmented HSPCs in vitro. Unexpectedly, however, sustained signal enhancement even with wild‐type Cxcr4 overexpression resulted in impaired peripheral blood (PB) reconstitution, most likely by preventing release of donor hematopoietic cells from the marrow environment. We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing donor HSPCs with enhanced repopulation potential following transplantation, whilst preserving the ability to release HSPC progeny into PB for improved transplantation outcomes. StemCells2014;32:1929–1942

Although macrophage activation syndrome (MAS) develops in some patients with chronic granulomatous disease (CGD), all of the reported cases have been associated with pathogenic microbial infections. We report a 2-year-old boy with CGD-associated colitis who suffered from MAS without any clinical signs of a microbial infection. He was treated with 1 course of methylprednisolone pulse therapy and the clinical symptoms improved; however, the colitis was difficult to control even with immunosuppressive drugs, and he eventually required hematopoietic stem cell transplantation 1 year after the onset of MAS. It is likely that MAS develops in patients with CGD colitis independent of microbial infections.

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 & MIB2-6); these BiPSCs have the same VDJ rearrangement of IgHas the original B lymphocytes and differentiate into CD34+/CD38-hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgHand CCND1and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1lentiCRISPRv2 lentivirus, which targets the human IgHEµ region and 13kb upstream of the CCND1coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1and either an allele in which VDJ rearrangement of IgHhad been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ & AX) and MIB2-6 t(11;14) (BC & BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/-hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgHloxP-Neo-loxP knock-in vector and IgHlentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgHwere transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1loxP-FRT3-Neo-FRT3 knock-in vector and CCND1lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgHand loxP-FRT3-Neo-FRT3 in CCND1were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgHand CCND1-loxP-FRT3 in CCND1were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. αIIbβ3 has not been implicated in these conditions. We identified a novel, conserved heterozygous ITGA2BR995W mutation in 4 unrelated families. The surface expression of platelet αIIbβ3 was decreased to 50% to 70% of control. There was spontaneous PAC-1 and fibrinogen binding to resting platelets without CD62p expression. The activation state of αIIbβ3 in 293T cells was higher for αIIb-W995 than for β3-H723 but was weaker than for β3-N562. FAK was spontaneously phosphorylated in αIIb-W995/β3-transfected 293T cells. These results indicate that αIIb-W995/β3 has a constitutive, activated conformation but does not induce platelet activation. αIIb-W995/β3-transfected CHO cells developed membrane ruffling and abnormal cytoplasmic protrusions. The increased size and decreased number of proplatelet tips in αIIb-W995/β3-transduced mouse fetal liver-derived megakaryocytes indicate defective proplatelet formation. We propose that activating mutations in ITGA2Band ITGB3represent the etiology of a subset of congenital macrothrombocytopenias.

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. αIIbβ3 has not been implicated in these conditions. We identified a novel, conserved heterozygous ITGA2B R995W mutation in 4 unrelated families. The surface expression of platelet αIIbβ3 was decreased to 50% to 70% of control. There was spontaneous PAC-1 and fibrinogen binding to resting platelets without CD62p expression. The activation state of αIIbβ3 in 293T cells was higher for αIIb-W995 than for β3-H723 but was weaker than for β3-N562. FAK was spontaneously phosphorylated in αIIb-W995/β3-transfected 293T cells. These results indicate that αIIb-W995/β3 has a constitutive, activated conformation but does not induce platelet activation. αIIb-W995/β3-transfected CHO cells developed membrane ruffling and abnormal cytoplasmic protrusions. The increased size and decreased number of proplatelet tips in αIIb-W995/β3-transduced mouse fetal liver-derived megakaryocytes indicate defective proplatelet formation. We propose that activating mutations in ITGA2B and ITGB3 represent the etiology of a subset of congenital macrothrombocytopenias.

Gene transfer vectors based on retroviruses are commonly used in gene therapy applications because of their unique ability to integrate efficiently into host genomes. This ability also forms the basis of a transformation event that can be induced in transduced cells by transactivation of proto-oncogenes near the vector integration sites. Here, we report on the development of lymphoma in mice generated from embryonic stem cells transduced with an enhanced green fluorescent protein. The cells expressed B220, CD5, Mac1, and IgM on their surfaces and expressed transcription factors characteristic of B-cell lymphoma. Importantly, each mouse had a single copy of the provirus in its genome; the copy was integrated into the second intron of the dopamine receptor 3 (D3) gene, and high-level expression of D3 was detected only in the lymphoma cells. Ectopic expression of D3 in murine marrow cells resulted in preferential proliferation of cells at the pre–B-cell stage in response to a D3-specific agonist, but this proliferation was not observed in vivo. Cells cotransduced with D3 and Bcl-xL genes had a phenotype similar to that of lymphoma in vivo, suggesting that the leukemogenesis induced by retroviral integration required “second hit” mutations of additional genes.

Generation of induced pluripotent stem cells (iPSCs) generally uses fibroblastic cells, but other cell sources may prove useful in both research and clinical settings. Although proof of cellular origin requires genetic-marker identification in both target cells and established iPSCs, somatic cells other than mature lymphocytes mostly lack such markers. Here we show definitive proof of direct reprogramming of murine hematopoietic cells with no rearranged genes. Using iPSC factor transduction, we successfully derived iPSCs from bone marrow progenitor cells obtained from a mouse whose hematopoiesis was reconstituted from a single congenic hematopoietic stem cell. Established clones were demonstrated to be genetically identical to the transplanted single hematopoietic stem cell, thus proving their cellular origin. These hematopoietic cell–derived iPSCs showed typical characteristics of iPSCs, including the ability to contribute to chimerism in mice. These results will prompt further use of hematopoietic cells for iPSC generation while enabling definitive studies to test how cellular sources influence characteristics of descendant iPSCs.

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgHand chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem cells (BiPSCs: BiPSC13 and MIB2-6); these BiPSCs have the same VDJ rearrangement of IgHas the original B lymphocytes and induce the expression of activation-induced cytidine deaminase (AID) by the Tet-off system (Sci Rep, 2017). Subsequently, we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgHEμ region of either the VDJ rearranged allele or non-rearranged allele of IgHand the 5′-upsteam region of the CCND1(two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14) carrying the DJ-rearranged allele of IgHthat reciprocally translocated with CCND1(G28). All these BiPSCs differentiated into hematopoietic progenitor cells (HPCs) (Sci Res, 2021). Next, we created AID fused with the hormone-binding domain of the estrogen receptor T2 mutant (AID-ER) to analyze the function of AID. The ER sequence of pCAG-ERT2CreERT2 (Addgene # 13777) was amplified by PCR and cloned into the downstream part of the TREtight promoter of pRetroX-Tight-Hyg as an open reading frame using an In-Fusion HD cloning kit. The ER sequence was also cloned into the same vector as a negative control for the analysis of AID function. These vectors were packaged into a retrovirus using gp293 and pMD2.G, and the viruses were infected into HEK293 cells expressing Tet-off transcription factor and fluorescence-deficient EGFP, in which the artificially-introduced stop codon was at the 106 thcodon of the EGFPgene. The expression of AID-ER fusion protein was induced by removal of doxycycline three days before analysis. AID activity was induced by the addition of 4-hydroxytamoxifen, an agonist of ER mutants, one day before analysis. Therefore, AID activity was monitored by detecting the frequency of EGFP-positive cells, i.e., the recovery of fluorescence, by flow-cytometry. Mutation of the artificial stop codon (from TAG to TAC; from stop codon to Tyrosine) was confirmed by sequencing the EGFPDNA amplified by PCR from the sorted EGFP-positive cells. Subsequently, we examined the introduction of mutated KRASand NRASgenes into G28, because the most frequently occurring mutations in patients with MM are in KRASand NRAS. Mutated KRAS(G12A) and NRAS(G13D) were amplified by RT-PCR from poly (A) mRNA of MM.1S and KMM1 of MM cell lines, respectively. After confirming the sequence of these mutants, the mutant cDNA of KRASor NRASwas cloned into pRetroX-Tight-Hyg in combination with AID-ER. These two vectors were packaged into retroviruses, following which the viruses were infected into BiPSC13 with t(11;14) expressing Tet-off transcription factors, respectively. These BiPSCs also differentiated into HPCs. However, unlike cord blood, these HPCs did not differentiate into B lymphocytes by co-culture with BM stromal cell (MS-5). Therefore, further ingenuity is required to differentiate the BiPSC-derived HPCs into B lymphocytes.

Recent studies of enhanced hippocampal neurogenesis by antidepressants suggest enhancement of neurogenesis is a potentially effective therapy in neurodegenerative diseases. In this study, we evaluated nigral neurogenesis in animals and autopsy brains including patients with Parkinson's disease (PD). First, proliferating cells in substantia nigra were labeled with retroviral transduction of green fluorescent protein, which is an efficient method to label neuronal stem cells. Subsequent differentiation of labeled cells was followed; many transduced cells became microglia, but no differentiation into tyrosine hydroxylase–positive neurons was detected at 4 weeks after injection, in both intact rodents and those treated with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. Second, polysialic acid (PSA)–like immunoreactivity, indicative of newly differentiated neurons, was detected in the substantia nigra of rodent, primate, and human midbrains. A large number of PSA‐positive cells were detected in the substantia nigra pars reticulata of some patients with PD. In rats and a macaque monkey, the dopamine‐depleted hemispheres showed more PSA staining than the intact side. A small number of tyrosine hydroxylase–positive cells were PSA‐positive. Our results suggest enhanced neural reconstruction in PD, which may be important in the design of new therapies against the progression of PD. Ann Neurol 2005

Naturally occurring CD25+CD4+ regulatory T cells are engaged in the maintenance of immunological self-tolerance and down-regulation of various immune responses. Recent studies with mice showed that Foxp3, which encodes the transcription factor Scurfin, is a master regulatory gene for the development and function of CD25+CD4+ regulatory T cells. Here we examined the role of FOXP3 in human CD25+CD4+ regulatory T cells. The FOXP3 gene and its protein product were preferentially expressed in peripheral CD25+CD4+ T cells, in particular CD25+CD45RO+CD4+ T cells in normal individuals and, interestingly, in some human T cell leukemia virus type 1-infected T cell lines, which constitutively express CD25. TCR stimulation of CD25−CD45RO−CD4+ naive T cells failed to elicit FOXP3 expression at the gene or protein level. Ex vivo retroviral gene transfer of FOXP3, on the other hand, converted peripheral CD25−CD45RO−CD4+ naive T cells into a regulatory T cell phenotype similar to CD25+CD4+ regulatory T cells. For example, FOXP3-transduced T cells exhibited impaired proliferation and production of cytokines including IL-2 and IL-10 upon TCR stimulation, up-regulated the expression of regulatory T cell-associated molecules such as CD25 and CTL-associated antigen-4 and suppressed in vitro proliferation of other T cells in a cell–cell contact-dependent manner. Thus, human FOXP3 is a crucial regulatory gene for the development and function of CD25+CD4+ regulatory T cells, and can be used as their reliable marker. Furthermore, regulatory T cells de novo produced from normal naive T cells by FOXP3 transduction can be instrumental for treatment of autoimmune/inflammatory diseases and negative control of various immune responses.

Upon antigen recognition by the T cell receptor, lymphocyte function–associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12–independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1–mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells.

Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo. The aim of this study was to accomplish xenotransplantation of human HSPCs into pig.

In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains.Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression ofGfi-1B in human CD34+ hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression ofGfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non–zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.

GATA transcription factors are major regulators of hematopoietic and immune system. Among GATA factors, GATA-1, GATA-2, and GATA-3 play crucial roles in the development of erythroid cells, hematopoietic stem, and progenitor cells, and T helper type 2 (Th2) cells, respectively. A high level of GATA-1 and GATA-2 expression has been observed in eosinophils, but their roles in eosinophil development remain uncertain both in vitro and in vivo. Here we show that enforced expression of GATA-1 in human primary myeloid progenitor cells completely switches myeloid cell fate into eosinophils. Expression of GATA-1 exclusively promotes development and terminal maturation of eosinophils. Functional domain analyses revealed that the COOH-terminal finger is essential for this capacity while the other domains are dispensable. Importantly, GATA-1–deficient mice failed to develop eosinophil progenitors in the fetal liver. On the other hand, GATA-2 also showed instructive capacity comparable to GATA-1 in vitro and efficiently compensated for GATA-1 deficiency in terms of eosinophil development in vivo, indicating that proper accumulation of GATA factors is critical for eosinophil development. Taken together, our findings establish essential and instructive roles of GATA factors in eosinophil development. GATA-1 and GATA-2 could be novel molecular targets for therapeutic approaches to allergic inflammation.

Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer–retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte–macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)α, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFα-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFα, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized.

Clinical gene therapy trials for adenosine deaminase (ADA) deficiency have shown limited success of corrective gene transfer into autologous T lymphocytes and CD34+ cells. In these trials, the levels of gene transduction and expression in hematopoietic cells have been assessed by DNA- or RNA-based assays and measurement of ADA enzyme activity. Although informative, these methods are rarely applied to clonal analysis. The results of these assays therefore provide best estimates of transduction efficiency and gene expression in bulk populations based on the assumption that gene transfer and expression are uniformly distributed among transduced cells. As a useful additional tool for evaluation of ADA gene expression, we have developed a flow cytometry (fluorescence-activated cell sorting, FACS) assay capable of estimating the levels of intracellular ADA on a single-cell basis. We validated this technique with T cell lines and peripheral blood mononuclear cells (PBMCs) from ADA-deficient patients that showed severely reduced levels of ADA expression (ADA-dull) by FACS and Western blot analyses. After retrovirus-mediated ADA gene transfer, these cells showed clearly distinguishable populations exhibiting ADA expression (ADA-bright), thus allowing estimation of transduction efficiency. By mixing ADA-deficient and normal cells and using enzymatic amplification, we determined that our staining procedure could detect as little as 5% ADA-bright cells. This technique, therefore, will be useful to quickly assess the expression of ADA in hematopoietic cells of severe combined immunodeficient patients and represents an important tool for the follow-up of patients treated in clinical gene transfer protocols.

Despite efforts toward improvements in retrovirus-mediated gene transfer, stable high-level expression of a therapeutic gene in human hematopoietic stem cells remains a great challenge. We have evaluated the efficiency of different viral long terminal repeats (LTRs) in long-term expression of a transgene in vivo, using severe combined immunodeficiency (SCID)-repopulating cell assays. Vectors used were variants of the simplified retroviral vector GCsap with the different LTRs of Moloney murine leukemia virus (MLV), myeloproliferative sarcoma virus (MPSV), and murine stem cell virus (MSCV). The enhanced green fluorescent protein (EGFP) gene was used as a marker to assess levels of transduction efficiency. CD34+ cells isolated from human cord blood were transduced by exposure to virus-containing supernatants on fibronectin fragments and in the presence of stem cell factor, interleukin 6, Flt-3 ligand, and thrombopoietin, and then transplanted into nonobese diabetic/SCID mice. Engraftment of human cells highly expressing EGFP, with differentiation along multiple cell lineages, was demonstrated for up to 18 weeks posttransplant, although the three different vectors showed different transduction frequencies (MLV, <0.1-33.2%; MPSV, <0.1-22.8%; MSCV, 0.3-51.7%). Of importance is that high-level transduction frequencies in human progenitor cells were also confirmed by colony-forming cell assays using bone marrow from transplanted mice, in which EGFP-expressing, highly proliferative potential colonies were observed by fluorescence microscopy. In these mice the vector carrying the MSCV LTR generated more EGFP-expressing human cells than did either of the other two constructs, indicating that GCsap carrying the MSCV LTR may be an efficient tool for stem cell gene therapy.

In the past decade, the advent of gene therapy has been acclaimed as a revolutionary medical intervention, embraced with great enthusiasm. However, recent disappointing results of the considerable clinical trials have also clearly demonstrated that such an initial expectation was an overestimation of gene therapy. There are only a few successful cases despite the 3000 patients who have been treated with various forms of gene therapy. Gene therapy for severe combined immunodeficiency (SCID) caused by adenosine deaminase (ADA) deficiency is one of the few such cases where results have been promising. In particular, peripheral T-lymphocytes-directed gene therapy provides further immunological improvements for patients with ADA-SCID receiving the PEG-ADA treatment whereas gene therapy targeting haematopoietic stem cell has so far proved insufficient for clinical benefits. This report will review crucial problems elucidated in the past five clinical trials for ADA-SCID and gives an outline of the next generation of stem cell gene therapy in Japan.

A total of 2728 (96) immunocompromised Japanese children received ≥4 doses of palivizumab. No respiratory syncytial virus-associated hospitalizations occurred. Mean palivizumab trough concentrations were 59.0 and 91.8 gmL 30 days after the 1st and 4th doses, respectively. Of 28 subjects, 27 (96) experienced ≥1 adverse event and 7 (25) experienced ≥1 serious adverse event, none of which was considered related to palivizumab.

ABSTRACTA number of different viruses have been adapted as gene transfer vectors, including retroviruses, adenoviruses, adenoassociated viruses (AAVs), herpes simplex virus, SV40 viruses, and alphaviruses (both Semliki Forest and Sindbis viruses). One of the major rate-limiting and time-consuming steps in the characterization of these vectors is the process of determining the viral vector titers. In addition, there is no universal method that can be used to rapidly estimate the titer and the utility of viral vector preparations. We demonstrate here that supernatant from diverse classes of viral victors, with either RNA or DNA genomes, can be rapidly evaluated by a simple virus dot-blot hybridization without prior extraction of nucleic acids. This system can provide a reliable screen for physical titer of viral vector supernatants in 1 day.

Ten patients with adenosine deaminase deficiency (ADA-) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA- severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA-SCID.

ABSTRACTA series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA-) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA-severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.

ABSTRACTSimplified retroviral vectors that lack dominant selectable markers are being used with increasing frequency. These simplified vectors may offer a number of advantages over selectable marker-containing constructs, including potentially higher titers and less immunogenicity. However, the use of these vectors has been limited by the cumbersome experimental approaches in establishing and characterizing useful producer cell clones. To address this issue, a simple and reliable assay was developed to identify retroviral producer cell lines with or without dominant selectable markers. Producer cells were first generated by standard transfection/transduction and clones isolated by limiting dilution. Supernatant from each clone was then screened by RNA dot blot to identify the best producer clone candidates. The semiquantitative nature of the RNA dot blot assay was validated using a retroviral vector containing neomycin phosphotransferase (neo). Titers obtained by conventional G418-resistant colony forming units/ml (G418Rcfu/ml) assays strongly correlated with the values by RNA dot blot procedure. RNA dot blot results also correlated well with titers estimated by Southern analysis of HeLa cells transduced with supernatant from each clone. The RNA dot blot technique is a rapid (2 days) and reliable method to screen retroviral producer cells, thereby facilitating the generation and characterization of simplified retroviral producer cell clones.

The cellular origin of multiple myeloma (MM) has not ben identified. Based on the results of transplantation experiments using bone marrow samples from MM patients in immunodeficient mice, so-called myeloma stem cells have been inferred to be present in CD19-/CD38++/CD138+or CD138-plasma cells populations; however, it is possible that the results indicated the presence of plasma cell populations with cell proliferation ability rather than the cellular origin of myeloma cells. On the other hand, based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation play a critical role in early tumorigenesis of MM. We hypothesize that the reprograming of mature B cells, in which IgHgene rearrangements have maintained, are the origin of MM. We propose that mature B cells gain potential by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To identify myeloma-initiating cells, we established normal B cell-derived induced pluripotent stem cells (BiPSCs; BiPSC13 and MIB2-6). These BiPSCs maintain VDJ rearrangement of the IgHgene, and they can be induced by the tet-off system to express activation-induced cytidine deaminase (AID; BiPSC13-AID and MIB2-6-AID) and differentiate into hematopoietic progenitor cells (HPCs) (Scientific Rep, 2017). Subsequently, we used the CRISPR/Cas9 system (Oncology Letters, 2019) to establish two BiPSCs with chromosomal translocation t(11;14); the cleavage site were located in the IgHEμ region of the VDJ non-rearranged allele of the IgHgene and the CCND15'-upsteam region of the CCND1gene (BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53was deleted using the CRISPR/Cas9 system (Ota A, J Cell Sci, 2017) in BiPSC13 with t(11;14) and BiPSC13-AID with t(11;14). These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43-HPCs in co-culture with stromal cell, AGM-S3, and their ability to subsequently differentiate into granulocytes, macrophages, and erythroblasts was confirmed by colony-formation assay. Until now, the co-culture of BiPSC13, MIB2-6, and MIB2-6 with t(11;14) with AGM-S3 followed by co-culture with stromal cell, MS-5, showed the appearance of CD34-/CD45+and CD34-/CD10+cell population. Also of interest, by RT-PCR, the expression levels of E2F, EBF, GATA3 and CD10 were higher in CD34+HPCs differentiated by the co-culture of BiPSC13 and MIB2-6 with AGM-S3 when compared to the levels in cord blood-derived CD34+cells. Overall, the results suggest that these BiPSCs have the ability to differentiate into HPCs, and that they may further differentiate into B cells. If these BiPSCs could be differentiated into mature B cells, they may be useful for the elucidation and study of myeloma-initiating cells derived from mature B cells.

Hanamura: Mundi: Honoraria; Yamada Yohojo: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Nihon Shinyaku: Honoraria, Research Funding; Fukuyu Hospital: Research Funding; Fujimoto: Research Funding; Taiho: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Chugai: Research Funding; Eisai: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Eli Lilly: Research Funding; Asai Clinic: Research Funding; AbbVie: Honoraria; MSD: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Zenyaku: Research Funding; Sanofi: Research Funding; Shionogi: Honoraria, Research Funding; Kyowa Kirin: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Astellas: Research Funding; Pfizer: Honoraria, Research Funding.

Hanamura: CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Company, Limited: Research Funding; Fujimoto Pharmaceutical Corporation: Research Funding; Takeda Pharmaceutical Company Limited.: Other: Lecture fee; Bristol-Myers Squibb: Other: Lecture fee, Research Funding; Celgene: Other: Lecture fee.

Induced pluripotent stem cells (iPSCs) are generated by the genetic reprogramming of somatic cells. In addition to regenerative medicine, these cells have been used in studies on the pathogenesis of inherited genetic diseases or neoplasms. If B cell-derived iPSCs (BiPSCs) could be developed from mature B cells or plasma cells, the genomic rearrangement of the B cell receptor of the BiPSCs would take over that of the original B cell. BiPSCs were recently established from peripheral blood B cells by the simultaneous introduction of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) with CCAAT/enhancer-binding protein-alpha using a Sendai virus vector (Bueno C, et al. Leukemia 2016). In this study, we were also able to generate BiPSCs, albeit using different method. The BiPSCs had immunoglobulin heavy chain (IgH) gene rearrangement but no class-switch in both alleles from normal B cells purified from lymph nodes and peripheral blood; therefore, we postulated that they were most likely established from naive B cells or IgM-expressing mature B cells. The main points of our method are consecutive transfection of a high concentration of Yamanaka factors into B cells using a retrovirus vector, which were pre-stimulated with interleukin (IL)-21 and CD40 ligand (CD40L), combined with consecutive centrifugations of the cells after their activation by IL-4, IL-2, and CD40L on a retronectin coated plate. We also established doxycycline-controlled (Tet-off system) activation-induced cytidine deaminase (AID)-induced BiPSCs (BiPSCs-A). AID is an enzyme that initiates somatic hypermutation (SHM) and class-switch recombination (CSR) in B cells. Furthermore, it has been noted recently that AID has similar effects on other than immunoglobulin genes. We hypothesized that myeloma cells are derived from reprogrammed mature B cells, in which reciprocal chromosome translocation occurs by double-strand DNA breaks induced by AID activation in the nonproductive (nonfunctional) allele of chromosome 14 and in another chromosome containing genes such as cyclin D1, Maf. To test our hypothesis, we used the comet assay to confirm that DNA cleavage was induced by AID in the BiPSCs-A. Next, we analyzed the ability of these BiPSCs to differentiate into hematopoietic progenitor cells (HPCs), and found that both parental BiPSCs and BiPSCs-A were capable of differentiating into HSCs, as determined by evaluating CD34 expression and the colony-formation of macrophages, granulocytes, and erythrocytes from CD34-positive cells. However, these cells were negative for CD38, CD43, and CD45 expression, indicating that they may be hematoendothelial cells as Maxim proposed previously (Vodyanik MA et al. Blood 2006). Based on our findings regarding the differentiation of BiPSCs-A into HPCs and their retention of the IgH gene rearrangement, it is possible that the induction of AID expression might induce chromosomal translocations in the process of differentiation of these BiPSCs into HPCs and further into mature or antibody-producing B cells in experiments using mouse. Because the BiPSCs had teratoma-forming ability, if a chromosome break were to occur in either an allele with IgH gene rearrangement (productive allele) or in another nonproductive IgH allele followed by reciprocal translocation with another chromosome and oncogene activation, BiPSCs reactivation and differentiation into B cells might be a cause of multiple myeloma in vivo. Thus, these BiPSCs might be useful for elucidating the origin of abnormal B cells in this disease.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Abstract 1888

No relevant conflicts of interest to declare.

Omine: Alexion: Consultancy, Research Funding.