Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of ß-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling ßAS3-globin transgene is potentially curative for all ß-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for ßAS3-globin addition and evaluates strategies for an increased ß-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-ßAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior ß-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent ßAS3-globin expression. LVs were initially compared for their ability to achieve high ß-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that ß-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased ß-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with ßAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-ßAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-ßAS3 for the treatment of severe ß-hemoglobinopathies.

Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.

Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.This article has an associated First Person interview with the first author of the paper.

Mouse Stbd1 is N-myristoylated and affects ER-mitochondria association and mitochondrial morphology.

Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N-myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria.

Impaired proliferative potential of bone marrow mesenchymal stromal cells in patients with myelodysplastic syndromes is associated with abnormal WNT signaling pathway.

It has been shown that bone marrow mesenchymal stromal cells (MSCs) from patients with myelodysplastic syndromes (MDSs) display defective proliferative potential. We have probed the impaired replicative capacity of culture-expanded MSCs in MDS patients (n=30) compared with healthy subjects (n=32) by studying senescence characteristics and gene expression associated with WNT/transforming growth factor-ß1 (TGFB1) signaling pathways. We have also explored the consequences of the impaired patient MSC proliferative potential by investigating their differentiation potential and the capacity to support normal CD34(+) cell growth under coculture conditions. Patient MSCs displayed decreased gene expression of the senescence-associated cyclin-dependent kinase inhibitors CDKN1A, CDKN2A, and CDKN2B, along with PARG1, whereas the mean telomere length was upregulated in patient MSCs. MDS-derived MSCs exhibited impaired capacity to support normal CD34(+) myeloid and erythroid colony formation. No significant changes were observed between patients and controls in gene expression related to TGFB1 pathway. Patient MSCs displayed upregulated non-canonical WNT expression, combined with downregulated canonical WNT expression and upregulated canonical WNT inhibitors. MDS-derived MSCs displayed defective osteogenic and adipogenic lineage priming under non-differentiating culture conditions. Pharmacological activation of canonical WNT signaling in patient MDSs led to an increase in cell proliferation and upregulation in the expression of early osteogenesis-related genes. This study indicates abnormal WNT signaling in MSCs of MDS patients and supports the concept of a primary MSC defect that might have a contributory effect in MDS natural history.