Intracerebral lentiviral ABCD1 gene therapy in an early disease onset ALD mouse model.

X-linked adrenoleukodystrophy (ALD) is a genetic disorder of the ABCD1 gene. We aimed to treat ALD via direct intracerebral injection of lentiviral ABCD1 (LV.ABCD1). Lentiviral vectors (LVs) were injected into the brain of wild type mice to access toxicities and biodistribution. Confocal microscopy illustrated supraphysiological ABCD1 expression surrounding the injection sites, and LVs were also detected in the opposite site of the unilaterally injected brain. In multi-site bilateral injections (4, 6, 8, and 9 sites), LV.ABCD1 transduced most brain regions including the cerebellum. Investigation of neuronal loss, astrogliosis and microglia activation did not detect abnormality. For efficacy evaluation, a novel ALD knockout (KO) mouse model was established by deleting exons 3 to 9 of the ABCD1 gene based on CRISPR/Cas9 gene editing. The KO mice showed behavioral deficit in open-field test (OFT) and reduced locomotor activities in rotarod test at 6 and 7 months of age, respectively. We treated 3-month-old KO mice with bilateral LV.ABCD1 injections into the external capsule and thalamus. ABCD1 expression was detected 15 days later, and the impaired motor ability was gradually alleviated. Our studies established an early onset ALD model and illustrated neurological improvement after LV.ABCD1 intracerebral injection without immunopathological toxicity.

Safety and antitumor activity of GD2-Specific 4SCAR-T cells in patients with glioblastoma.

BACKGROUND: This study aimed to validate whether infusion of GD2-specific fourth-generation safety-designed chimeric antigen receptor (4SCAR)-T cells is safe and whether CAR-T cells exert anti-glioblastoma (GBM) activity. METHODS: A total of eight patients with GD2-positive GBM were enrolled and infused with autologous GD2-specific 4SCAR-T cells, either through intravenous administration alone or intravenous combined with intracavitary administration. RESULTS: 4SCAR-T cells expanded for 1-3 weeks and persisted at a low frequency in peripheral blood. Of the eight evaluable patients, four showed a partial response for 3 to 24 months, three had progressive disease for 6 to 23 months, and one had stable disease for 4 months after infusion. For the entire cohort, the median overall survival was 10 months from the infusion. GD2 antigen loss and infiltrated T cells were observed in the tumor resected after infusion. CONCLUSION: Both single and combined infusions of GD2-specific 4SCAR-T cells in targeting GBM were safe and well tolerated, with no severe adverse events. In addition, GD2-specific 4SCAR-T cells partially mediate antigen loss and activate immune responses in the tumor microenvironment. Validation of our findings in a larger prospective trial is warranted. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03170141 . Registered 30 May 2017.

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A.

BACKGROUND: Hemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities. METHODS: To investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters. RESULTS: To examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice. CONCLUSIONS: The LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A.

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.

Systemic Injection of RPE65-Programmed Bone Marrow-Derived Cells Prevents Progression of Chronic Retinal Degeneration.

Bone marrow stem and progenitor cells can differentiate into a range of non-hematopoietic cell types, including retinal pigment epithelium (RPE)-like cells. In this study, we programmed bone marrow-derived cells (BMDCs) ex vivo by inserting a stable RPE65 transgene using a lentiviral vector. We tested the efficacy of systemically administered RPE65-programmed BMDCs to prevent visual loss in the superoxide dismutase 2 knockdown (Sod2 KD) mouse model of age-related macular degeneration. Here, we present evidence that these RPE65-programmed BMDCs are recruited to the subretinal space, where they repopulate the RPE layer, preserve the photoreceptor layer, retain the thickness of the neural retina, reduce lipofuscin granule formation, and suppress microgliosis. Importantly, electroretinography and optokinetic response tests confirmed that visual function was significantly improved. Mice treated with non-modified BMDCs or BMDCs pre-programmed with LacZ did not exhibit significant improvement in visual deficit. RPE65-BMDC administration was most effective in early disease, when visual function and retinal morphology returned to near normal, and less effective in late-stage disease. This experimental paradigm offers a minimally invasive cellular therapy that can be given systemically overcoming the need for invasive ocular surgery and offering the potential to arrest progression in early AMD and other RPE-based diseases.

Immortalization of human normal and NF1 neurofibroma Schwann cells.

Neurofibromas, which are benign Schwann cell tumors, are the hallmark feature in the autosomal dominant condition neurofibromatosis 1 (NF1) and are associated with biallelic loss of NF1 gene function. There is a need for effective therapies for neurofibromas, particularly the larger, plexiform neurofibromas. Tissue culture is an important tool for research. However, it is difficult to derive enriched human Schwann cell cultures, and most enter replicative senescence after 6-10 passages, impeding cell-based research in NF1. Through exogenous expression of human telomerase reverse transcriptase and murine cyclin-dependent kinase (mCdk4), normal (NF1 wild-type), neurofibroma-derived Schwann cells heterozygous for NF1 mutation, and neurofibroma-derived Schwann cells homozygous for NF1 mutation were immortalized, including some matched samples from the same NF1 patient. Initial experiments employed retroviral vectors, while subsequent work utilized lentiviral vectors carrying these genes because of improved efficiency. Expression of both transgenes was required for immortalization. Molecular and immunohistochemical analysis indicated that these cell lines are of Schwann cell lineage and have a range of phenotypes, many of which are consistent with their primary cultures. This is the first report of immortalization and detailed characterization of multiple human NF1 normal nerve and neurofibroma-derived Schwann cell lines, which will be highly useful research tools to study NF1 and other Schwann tumor biology and conditions.

The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation.

Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes.

STAT3 signaling contributes to the high effector activities of interleukin-15-derived dendritic cells.

Dendritic cells (DCs) are important innate and adaptive immune effectors, and have a key role in antigen presentation and T-cell activation. Different lineages of DCs can be developed from hematopoietic progenitors following cytokine signaling, and the various lineages of DCs display distinct morphology, phenotype and functions. There has been limited information on differential cytokine-mediated molecular signaling in DCs. Analyses of surface molecules by flow cytometry and quantitative RNA profiling revealed differences between DCs derived from interleukin-4 (IL-4) versus IL-15 signaling, yet both lineages of DCs exhibited similar levels of surface molecules key to immune activation. Functional assays confirmed that IL-15-derived DCs elicited greater antigen-specific, primary and secondary CD8 and CD4 T-cell responses than did IL-4-derived DCs. Importantly, IL-15 DCs secreted substantial amounts of proinflammatory cytokines, including IL-6, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNFα), which helped polarize a strong T-cell response. Assessment of signaling pathways revealed that IL-15 DCs exhibited a lower levels of activated signal transducer and activator of transcription 5 (STAT5), STAT6 and extracellular signal-regulated kinase 1/2 than IL-4 DCs, but after lipopolysaccharide (LPS)/TNFα treatment, the STAT3 and p38 mitogen-activated protein kinase (MAPK) activities were significantly enhanced in the IL-15 DCs. Surprisingly, contrary to the canonical IL-15-mediated STAT5 signaling pathway in lymphoid cells, IL-15 did not mediate a strong STAT5 or STAT3 activation in DCs. Further analysis using specific inhibitors to STAT3 and p38 MAPK pathways revealed that the STAT3 signaling, but not p38 MAPK signaling, contributed to IFN-γ production in DCs. Therefore, while IL-15 does not promote the STAT signaling in DCs, the increased STAT3 activity after LPS/TNFα treatment of the IL-15 DCs has a key role in their high IFN-γ effector activities.

Defining a new role of GW182 in maintaining miRNA stability.

GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA-mediated gene silencing. Using lentiviral shRNA-induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA-mimic half-lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA-mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3'-5' exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute-bound miRNA.

Pre-B cell leukemia homeobox 1 is associated with lupus susceptibility in mice and humans.

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-ß-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

Synergistic effects of interleukin-7 and pre-T cell receptor signaling in human T cell development.

The role of IL-7 in pre-T cell receptor (TCR) signaling during human T cell development is poorly understood. To study this, we engineered Molt3, a T cell progenitor T-acute lymphoblastic leukemia cell line, using lentiviral IL-7 receptor α (IL-7Rα) to serve as a model system. IL-7 promoted pre-TCR activation in IL-7Rα(hi) Molt3 as illustrated by CD25 up-regulation after anti-CD3 stimulation. Anti-CD3 treatment activated Akt and Erk1/2 signaling pathways as proven using specific inhibitors, and IL-7 further enhanced both signaling pathways. The close association of IL-7Rα with CD3ζ in the pre-TCR complex was illustrated through live imaging confocal fluorescence microscopy. These results demonstrate a direct and cooperative role of IL-7 in pre-TCR signaling.

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25- effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.

The engagement of CTLA-4 on primary melanoma cell lines induces antibody-dependent cellular cytotoxicity and TNF-α production.

BACKGROUND: CTLA-4 (Cytotoxic T lymphocyte antigen-4) is traditionally known as a negative regulator of T cell activation. The blocking of CTLA-4 using human monoclonal antibodies, such as Ipilimumab, is currently used to relieve CTLA-4-mediated inhibition of anti-tumor immune response in metastatic melanoma. Herein, we have analyzed CTLA-4 expression and Ipilimumab reactivity on melanoma cell lines and tumor tissues from cutaneous melanoma patients. Then, we investigated whether Ipilimumab can trigger innate immunity in terms of antibody dependent cellular cytotoxicity (ADCC) or Tumor Necrosis Factor (TNF)-α release. Finally, a xenograft murine model was set up to determine in vivo the effects of Ipilimumab and NK cells on melanoma. METHODS: CTLA-4 expression and Ipilimumab reactivity were analyzed on 17 melanoma cell lines (14 primary and 3 long-term cell lines) by cytofluorimetry and on 33 melanoma tissues by immunohistochemistry. CTLA-4 transcripts were analyzed by quantitative RT-PCR. Soluble CTLA-4 and TNF-α were tested by ELISA. Peripheral blood mononuclear cells (PBMC), NK and γδT cells were tested in ADCC assay with Ipilimumab and melanoma cell lines. TNF-α release was analyzed in NK-melanoma cell co-cultures in the presence of ipilimumab. In vivo experiments of xenotransplantation were carried out in NOD/SCID mice. Results were analyzed using unpaired Student's t-test. RESULTS: All melanoma cell lines expressed mRNA and cytoplasmic CTLA-4 but surface reactivity with Ipilimumab was quite heterogeneous. Accordingly, about 2/3 of melanoma specimens expressed CTLA-4 at different level of intensity.Ipilimumab triggered, via FcγReceptorIIIA (CD16), ex vivo NK cells as well as PBMC, IL-2 activated NK and γδT cells to ADCC of CTLA-4+ melanoma cells. No ADCC was detected upon interaction with CTLA-4- FO-1 melanoma cell line. TNF-α was released upon interaction of NK cells with CTLA-4+ melanoma cell lines. Remarkably, Ipilimumab neither affected proliferation and viability nor triggered ADCC of CTLA-4+ T lymphocytes. In a chimeric murine xenograft model, the co-engraftment of Ipilimumab-treated melanoma cells with human allogeneic NK cells delayed and significantly reduced tumor growth, as compared to mice receiving control xenografts. CONCLUSIONS: Our studies demonstrate that Ipilimumab triggers effector lymphocytes to cytotoxicity and TNF-α release. These findings suggest that Ipilimumab, besides blocking CTLA-4, can directly activate the elimination of CTLA-4+ melanomas.

Lentiviral Gene Therapy of Chronic Granulomatous Disease: Functional Assessment of Universal and Tissue-Specific Promoters.

Chronic granulomatous disease (CGD) is a rare congenital immunodeficiency characterized by a defect in nicotinamide adenine dinucleotide phosphate oxidase required for phagocytosis. Hematopoietic stem cell (HSC) transplantation is currently the only curative treatment, but it is ladened with morbidities and mortality. Gene therapy is a promising treatment for CGD. However, if not properly designed, the gene therapy approach may not be successful. We engineered lentiviral vectors (LVs) carrying a universal promoter (EF1a) and two myeloid-specific promoters (miR223 and CD68) to drive the expression of green fluorescence protein (GFP) or CYBB, one of the key defective genes causing CGD. Tissue-specific LV expression was investigated in vitro and in a CGD mouse model. We compared GFP expression in both myeloid differentiated and undifferentiated HSCs. The CGD mice were transplanted with LV-modified mouse HSCs to investigate expression of CYBB and restoration of reactive oxygen species. The LV promoters were further compared under low and high-transgenic conditions to assess safety and therapeutic efficacy. A pneumonia disease model based on pathogenic Staphylococcus aureus challenge was established to assess the survival rate and body weight change. All three promoters demonstrated ectopic CYBB expression in vitro and in vivo. The EF1a promoter showed the highest expression of GFP or CYBB in transduced cells, including HSCs without cytotoxicity, whereas the LV-miR223 showed the highest transgene delivery efficiency with high myeloid specificity. Importantly, under low-transgenic condition, only the LV-EF1a-CYBB showed high antibacterial activity in vivo.

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation.

BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαß+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vß repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Molecular therapeutics of hemophilia A and B.

INTRODUCTION: Hemophilia A (HA) or B (HB) is an X-linked recessive disorder caused by a defect in the factor VIII (FVIII) or factor IX (FIX) gene which leads to the dysfunction of blood coagulation. Protein replacement therapy (PRT) uses recombinant proteins and plasma-derived products, which incurs high cost and inconvenience requiring routine intravenous infusions and life-time treatment. Understanding of detailed molecular mechanisms on FVIII gene function could provide innovative solutions to amend this disorder. In recent decades, gene therapeutics have advanced rapidly and a one-time cure solution has been proposed. AREAS COVERED: This review summarizes current understanding of molecular pathways involved in blood coagulation, with emphasis on FVIII's functional role. The existing knowledge and challenges on FVIII gene expression, from transcription, translation, post-translational modification including glycosylation to protein processing and secretion, and co-factor interactions are deciphered and potential molecular interventions discussed. EXPERT OPINION: This article reviews the potential treatment targets for HA and HB, including antibodies, small molecules and gene therapeutics, based on molecular mechanisms of FVIII biosynthesis, and further, assessing the pros and cons of these various treatment strategies. Understanding detailed FVIII protein synthesis and secretory pathways could provide exciting opportunities in identifying novel therapeutics to ameliorate hemophilia state.

GD2-specific chimeric antigen receptor-modified T cells for the treatment of refractory and/or recurrent neuroblastoma in pediatric patients.

PURPOSE: This study aimed to evaluate the safety and efficacy of chimeric antigen receptor (CAR) disialoganglioside 2 (GD2)-specific (4SCAR-GD2) T cells for treatment of refractory and/or recurrent neuroblastoma (NB) in pediatric patients. EXPERIMENTAL DESIGN: A phase I clinical study using 4SCAR-GD2 T cells for the treatment of NB in pediatric patients was conducted. This study was registered at www. CLINICALTRIALS: gov (NCT02765243). A lentiviral CAR with the signaling domains of CD28/4-1BB/CD3ζ-iCasp9 was transduced into activated T cells. The response to 4SCAR-GD2 T-cell treatment, and 4SCAR-GD2 T-cell expansion and persistence in patients were evaluated. Toxicities were determined based on the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v4.03. RESULTS: Twelve patients were enrolled and finally ten patients were included in this clinical trial which started from January 1, 2016, to August 1, 2017. These patients had progressive disease (PD) before CAR T-cell infusion. After 4SCAR-GD2 T-cell treatment, 6 (6/10) had stable disease (SD) at 6 months, and 4 (4/10) remained SD at 1 year and alive after 3-4 years of follow-up. Six patients died due to disease progression by the end of July 1, 2020. The median overall survival (OS) time was 25 months (95% CI, 0.00-59.43), and the median progression-free survival (PFS) time was 8 months (95% CI, 0.25-15.75). Grade 3 or 4 hematological toxicities were the common adverse events frequently occurred after fludarabine and cyclophosphamide (Flu/cy) chemotherapy. Grade 1-2 toxicities such as cytokine release syndrome (CRS) and neuropathic pain were common, but were transient and mild. CONCLUSIONS: The 4SCAR-GD2 T-cell therapy demonstrated antitumor effect and manageable toxicities, indicating its potential to benefit children with refractory and/or recurrent NB.

Ex vivo transduction and transplantation of bone marrow cells for liver gene delivery of alpha1-antitrypsin.

Adult stem cell-based gene therapy holds several unique advantages including avoidance of germline or other undesirable cell transductions. We have previously shown that liver progenitor (oval) cells can be used as a platform for liver gene delivery of human alpha1-antitrypsin (hAAT). However, this cell source cannot be used in humans for autologous transplantation. In the present study, we tested the feasibility of bone marrow (BM) cell-based liver gene delivery of hAAT. In vitro studies showed that BM cells can be transduced by lentiviral vector (Lenti-CB-hAAT) and recombinant adeno-associated viral vectors (rAAV1-CB-hAAT, and rAAV8-CB-hAAT). Transplantation studies showed that transplanted BM cells homed into liver, differentiated into hepatocytes and expressed hAAT in the liver. Importantly, we showed that transplantation of rAAV8-CB-hAAT vector-transduced BM cells resulted in sustained levels of hAAT in the systemic circulation of recipient mice. These results demonstrated that rAAV vector-mediated BM cell-based liver gene therapy is feasible for the treatment of AAT deficiency and implies a novel therapy for the treatment of liver diseases.