Entity

Time filter

Source Type

Memphis, TN, United States

Greene M.R.,St. Jude Childrens Hospital | Lockey T.,Childrens GMP LLC | Mehta P.K.,Hartwell Center | Kim Y.-S.,St. Jude Childrens Hospital | And 3 more authors.
Human Gene Therapy Methods | Year: 2012

Self-inactivating (SIN)-lentiviral vectors have safety and efficacy features that are well suited for transduction of hematopoietic stem cells (HSCs), but generation of vector at clinical scale has been challenging. Approximately 280 liters of an X-Linked Severe Combined Immunodeficiency Disorder (SCID-X1) SIN-lentiviral vector in two productions from a stable cell line were concentrated to final titers of 4.5 and 7.2×108 tu/ml. These two clinical preparations and three additional development-scale preparations were evaluated in human CD34+ hematopoietic cells in vitro using colony forming cell (CFU-C) assay and in vivo using the NOD/Lt-scid/IL2Rγnull (NSG) mouse xenotransplant model. A 40-hour transduction protocol using a single vector exposure conferred a mean NSG repopulating cell transduction of 0.23 vector genomes/human genome with a mean myeloid vector copy number of 3.2 vector genomes/human genome. No adverse effects on engraftment were observed from vector treatment. Direct comparison between our SIN-lentiviral vector using a 40-hour protocol and an MFGγc γ-retroviral vector using a five-day protocol demonstrated equivalent NSG repopulating cell transduction efficiency. Clonality survey by linear amplification-mediated polymerase chain reaction (LAM-PCR) with Illumina sequencing revealed common clones in sorted myeloid and lymphoid populations from engrafted mice demonstrating multipotent cell transduction. These vector preparations will be used in two clinical trials for SCID-X1. © 2012, Mary Ann Liebert, Inc. Source


Pruitt H.C.,University of Alabama at Birmingham | Devine D.J.,Mitchell Institute | Devine D.J.,Childrens GMP LLC | Samant R.S.,University of Alabama at Birmingham
International Journal of Cancer | Year: 2016

N-myc & STAT Interactor, NMI, is a protein that has mostly been studied for its physical interactions with transcription factors that play critical roles in tumor growth, progression and metastasis. NMI is an inducible protein, thus its intracellular levels and location can vary dramatically, influencing a diverse array of cellular functions in a context-dependent manner. The physical interactions of NMI with its binding partners have been linked to many aspects of tumor biology including DNA damage response, cell death, epithelial-to-mesenchymal transition and stemness. Thus, discovering more details about the function(s) of NMI could reveal key insights into how transcription factors like c-Myc, STATs and BRCA1 are contextually regulated. Although a normal, physiological function of NMI has not yet been discovered, it has potential roles in pathologies ranging from viral infection to cancer. This review provides a timely perspective of the unfolding roles of NMI with specific focus on cancer progression and metastasis. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC. Source


Wang H.,St Jude Childrens Research Hospital | Wang H.,University of Tennessee Health Science Center | Wang H.,University of California at San Francisco | Holst J.,St Jude Childrens Research Hospital | And 16 more authors.
EMBO Journal | Year: 2010

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3 proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. © 2010 European Molecular Biology Organization | All Rights Reserved. Source


Hu H.,St Jude Childrens Research Hospital | Gomero E.,St Jude Childrens Research Hospital | Bonten E.,St Jude Childrens Research Hospital | Gray J.T.,St Jude Childrens Research Hospital | And 6 more authors.
Molecular Therapy | Year: 2012

Galactosialidosis (GS) is a lysosomal storage disease linked to deficiency of the protective protein/cathepsin A (PPCA). Similarly to GS patients, Ppca-null mice develop a systemic disease of the reticuloendothelial system, affecting most visceral organs and the nervous system. Symptoms include severe nephropathy, visceromegaly, infertility, progressive ataxia, and shortened life span. Here, we have conducted a preclinical, dose-finding study on a large cohort of GS mice injected intravenously at 1 month of age with increasing doses of a GMP-grade rAAV2/8 vector, expressing PPCA under the control of a liver-specific promoter. Treated mice, monitored for 16 weeks post-treatment, had normal physical appearance and behavior without discernable side effects. Despite the restricted expression of the transgene in the liver, immunohistochemical and biochemical analyses of other systemic organs, serum, and urine showed a dose-dependent, widespread correction of the disease phenotype, suggestive of a protein-mediated mechanism of cross-correction. A notable finding was that rAAV-treated GS mice showed high expression of PPCA in the reproductive organs, which resulted in reversal of their infertility. Together these results support the use of this rAAV-PPCA vector as a viable and safe method of gene delivery for the treatment of systemic disease in non-neuropathic GS patients. © The American Society of Gene & Cell Therapy. Source


Walker A.L.,St Jude Childrens Research Hospital | Steward S.,Childrens GMP LLC | Howard T.A.,Baylor College of Medicine | Mortier N.,Baylor College of Medicine | And 3 more authors.
Blood | Year: 2011

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea- mediated HbF induction by examining methylation patterns within the Gγ -globin promoter and miRNA expression within primary CD71 + erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated Gγ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNAexpression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials. gov (NCT00305175). © 2011 by The American Society of Hematology. Source

Discover hidden collaborations