Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.This publication reports that stem cell gene therapy using a codon-optimized gene encoding acid α-glucosidase (GAA) cures the mouse model of Pompe disease, a lysosomal storage disorder

Malignant Transformation Involving CXXC4 Mutations Identified in a Leukemic Progression Model of Severe Congenital Neutropenia

Olofsen et al. show that acquisition of a mutation in Cxxc4 results in increased CXXC4 protein levels, reduced TET2 protein, increased inflammatory signaling, and leukemic progression of a CSF3R/RUNX1 mutant mouse model of severe congenital neutropenia (SCN).Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN

Peroxiredoxin-controlled G-CSF signalling at the endoplasmic reticulum-early endosome interface

Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes

Suppressor of cytokine signaling 3 controls lysosomal routing of G-CSF receptor

The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output