Hidefumi Hiramatsu

Identification of Hematopoietic Stem Cell–Specific miRNAs Enables Gene Therapy of Globoid Cell Leukodystrophy

Hematopoietic stem cell–specific microRNAs allow regulation of therapeutic transgene expression and enable effective gene therapy of globoid cell leukodystrophy. Scratching the Surface of the Holy Grail In Monty Python and the Holy Grail, when King Arthur cuts off one of the arms of the Black Knight, he claims it is only a scratch. Similarly, gene therapy—the insertion of genes into cells to reverse a condition or repair a biological process—has been heralded as a Holy Grail for the treatment of genetic diseases for nearly 40 years. Yet, the complications of gene therapy, including immune responses to the viral vector and cancers that result from insertional mutagenesis, are more comparable to a severed arm than a surface wound. However, researchers with the resiliency of the Black Knight have presided over recent successes, most notably in metastatic melanoma and immune cells, and have reignited the quest for gene therapy solutions to otherwise untreatable diseases. Gentner et al. build on these successes by identifying new microRNAs that can restrict gene therapy vectors to particular immune cell types and thus be used to safely treat globoid cell leukodystrophy (also known as Krabbe disease). Globoid cell leukodystrophy is a rare metabolic disorder caused by a mutation in a lysosomal enzyme called galactocerebrosidase (GALC). In patients who carry the mutation in both copies of the GALC gene, unmetabolized lipids accumulate in myelin-secreting glial cells, rendering them unable to produce the myelin sheath that normally wraps and protects nerves. This aberration results in severe and often fatal degeneration of motor skills. Bone marrow transplantation has been shown to benefit these patients if the disease is caught early enough. Genetic manipulation of the hematopoietic stem and progenitor cells (HSPCs) found in bone marrow may improve this therapy; however, high-level GALC expression in HSPCs, but not in more differentiated immune cells, is toxic. To address this issue, Gentner et al. identified miRNAs—short RNA sequences that often silence gene expression—that were specifically expressed in HSPCs but not in more differentiated cells. They then used these miRNAs in a GALC/HSPC gene therapy system to suppress GALC function in HSPCs upon transfer into a mouse model of globoid cell leukodystrophy. As these cells matured, amounts of HSPC-specific miRNA decreased and GALC expression increased. This approach protected the HSPCs from GALC toxicity, but allowed for successful gene therapy of the disease. In addition, these hematopoietic stem cell–specific miRNAs could be used as simple markers with which to isolate HSPCs for study and transplantation. This work thus provides a basis for improvements in HSPC-mediated gene therapy and may offer globoid cell leukodystrophy patients a new therapeutic option that resembles a scratch more than a chop. Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)–based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.

Attenuation of miR-126 Activity Expands HSC In Vivo without Exhaustion

Summary Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia

Background In a single‐center phase 1–2a study, the anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B‐cell acute lymphoblastic leukemia (ALL). Methods We conducted a phase 2, single‐cohort, 25‐center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B‐cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. Results For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event‐free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. Conclusions In this global study of CAR T‐cell therapy, a single infusion of tisagenlecleucel provided durable remission with long‐term persistence in pediatric and young adult patients with relapsed or refractory B‐cell ALL, with transient high‐grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849.)

Selective infection of CD4+ effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγnull mice

To investigate the events leading to the depletion of CD4(+) T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34(+) cells-transplanted NOD/SCID/IL-2Rgamma(null) mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4(+) thymocytes and both CD45RA(+) naïve and CD45RA(-) memory CD4(+) T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA(-) memory CD4(+) T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T(EM)) rather than central memory T lymphocytes. In addition, the majority of p24(+) cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T(EM) in cycling phase and further suggest that the predominant infection in T(EM) would lead to the depletion of memory CD4(+) T lymphocytes in CCR5-tropic HIV-1-infected mice.

Living-Donor Liver Transplantation for Hepatoblastoma

Hepatoblastoma is the most common malignant liver tumor in children. Recently, liver transplantation has been indicated for unresectable hepatoblastoma. We retrospectively reviewed 14 children with a diagnosis of hepatoblastoma who had undergone living‐donor liver transplantation (LDLT) at Kyoto University Hospital. During the period from June 1990 to December 2004, 607 children underwent LDLT. Of these interventions, 2.3% were performed for hepatoblastoma. Based on radiological findings, the pre‐treatment extent of disease (PRETEXT) grouping was used for pre‐treatment staging of the tumor. There were grade III in seven patients and grade IV in seven patients. Thirteen patients received chemotherapy, and seven underwent hepatectomy 11 times. Immunosuppressive treatment consisted of tacrolimus monotherapy in 11 patients. Actuarial 1‐ and 5‐year graft and patient survival rates were 78.6% and 65.5%. The poor prognostic factors were macroscopic venous invasion and extrahepatic involvement with 1‐year and 5‐year survival rates of 33.0% and 0%. Pediatric patients without these factors showed an acceptable 5‐year survival rate of 90.9%. LDLT provides a valuable alternative with excellent results in children with hepatoblastoma because it allows optimal timing of the liver transplantation, given the absence of delay between the completion of chemotherapy and planned liver transplantation.

Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion

Several studies have shown that hepatocytes can be generated from hematopoietic stem cells, but this event is believed to be rare and to require hepatic damage. To investigate this phenomenon in human cells, we used a NOD/SCID/γcnull (NOG) mouse model that can achieve a tremendously high level of chimerism when transplanted with human hematopoietic cells. Even without hepatotoxic treatment other than irradiation, human albumin and α‐1‐antitrypsin‐positive cells were invariably detected in the livers of NOG mice after i.v. transplantation of human cord blood CD34+ cells. Human albumin was detected in the murine sera, indicating functional maturation of the human hepatocytes. Flow cytometric analysis of recipient liver cells in single‐cell suspension demonstrated that human albumin‐positive cells were also positive for both murine and human MHC and were negative for human CD45. PCR analysis of recipient livers revealed the expression of a wide variety of human hepatocyte‐ or cholangiocyte‐specific mRNAs. These results show that human CD34+ cells fuse with hepatocytes of NOG mice without liver injury, lose their hematopoietic phenotype, and begin hepatocyte‐specific gene transcription. These phenomena were not observed when CD34− cells were transplanted. Thus, our model revealed a previously unidentified pathway of human hematopoietic stem/progenitor cell differentiation.—Fujino, H., Hiramatsu, H., Tsuchiya, A., Niwa, A., Noma, H., Shiota, M., Umeda, K., Yoshimoto, M., Ito, M., Heike, T., Nakahata, T. Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/γcnull mice through cell fusion. FASEB J. 21, 3499–3510 (2007)

Genetic correction of HAX1 in induced pluripotent stem cells from a patient with severe congenital neutropenia improves defective granulopoiesis

HAX1 was identified as the gene responsible for the autosomal recessive type of severe congenital neutropenia. However, the connection between mutations in the HAX1 gene and defective granulopoiesis in this disease has remained unclear, mainly due to the lack of a useful experimental model for this disease. In this study, we generated induced pluripotent stem cell lines from a patient presenting for severe congenital neutropenia with HAX1 gene deficiency, and analyzed their in vitro neutrophil differentiation potential by using a novel serum- and feeder-free directed differentiation culture system. Cytostaining and flow cytometric analyses of myeloid cells differentiated from patient-derived induced pluripotent stem cells showed arrest at the myeloid progenitor stage and apoptotic predisposition, both of which replicated abnormal granulopoiesis. Moreover, lentiviral transduction of the HAX1 cDNA into patient-derived induced pluripotent stem cells reversed disease-related abnormal granulopoiesis. This in vitro neutrophil differentiation system, which uses patient-derived induced pluripotent stem cells for disease investigation, may serve as a novel experimental model and a platform for high-throughput screening of drugs for various congenital neutrophil disorders in the future.

Identification of Hepatic Niche Harboring Human Acute Lymphoblastic Leukemic Cells via the SDF-1/CXCR4 Axis

In acute lymphoblastic leukemia (ALL) patients, the bone marrow niche is widely known to be an important element of treatment response and relapse. Furthermore, a characteristic liver pathology observed in ALL patients implies that the hepatic microenvironment provides an extramedullary niche for leukemic cells. However, it remains unclear whether the liver actually provides a specific niche. The mechanism underlying this pathology is also poorly understood. Here, to answer these questions, we reconstituted the histopathology of leukemic liver by using patients-derived primary ALL cells into NOD/SCID/Yc null mice. The liver pathology in this model was similar to that observed in the patients. By using this model, we clearly demonstrated that bile duct epithelial cells form a hepatic niche that supports infiltration and proliferation of ALL cells in the liver. Furthermore, we showed that functions of the niche are maintained by the SDF-1/CXCR4 axis, proposing a novel therapeutic approach targeting the extramedullary niche by inhibition of the SDF-1/CXCR4 axis. In conclusion, we demonstrated that the liver dissemination of leukemia is not due to nonselective infiltration, but rather systematic invasion and proliferation of leukemic cells in hepatic niche. Although the contribution of SDF-1/CXCR4 axis is reported in some cancer cells or leukemic niches such as bone marrow, we demonstrated that this axis works even in the extramedullary niche of leukemic cells. Our findings form the basis for therapeutic approaches that target the extramedullary niche by inhibiting the SDF-1/CXCR4 axis.

Successful treatment of a patient with Klinefelter's syndrome complicated by mediastinal germ cell tumor and AML(M7)

Successful treatment of a patient with Klinefelters syndrome complicated by mediastinal germ cell tumor and AML(M7)

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor α-positive and -negative human hematopoietic progenitors

Interleukin (IL)‐6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL‐6 receptor‐α subunit (IL‐6Rα)+ but also IL‐6Rα− hematopoietic progenitors via soluble IL‐6R. The Notch ligand Delta‐1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL‐6 are influenced by Delta‐1. When CD34+CD38− hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL‐3, Delta‐1, in combination with the IL‐6R/IL‐6 fusion protein FP6, increased the generation of glycophorin A+ erythroid cells but counteracted the effects of IL‐6 and FP6 on the generation of CD14+ monocytic and CD15+ granulocytic cells. Although freshly isolated CD34+CD38− cells expressed no or only low levels of IL‐6Rα, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta‐1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL‐6Rα− erythroid progenitors. In contrast, Delta‐1 antagonized the effects of IL‐6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14−CD1a+ dendritic cells, from the IL‐6Rα+ myeloid progenitors. These results indicate that Delta‐1 interacts differentially with gp130 activation in IL‐6Rα− erythroid and IL‐6Rα+ myeloid progenitors. The present data suggest a divergent interaction between Delta‐1 and gp130 activation in human hematopoiesis. (Cancer Sci 2007; 98: 1597–1603)