Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice.

BACKGROUND: Gastrointestinal complications are the main cause of death in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Available treatments often restore biochemical homeostasis, but fail to cure gastrointestinal symptoms. METHODS: We evaluated the small intestine neuromuscular pathology of an untreated MNGIE patient and two recipients of hematopoietic stem cells, focusing on enteric neurons and glia. Additionally, we evaluated the intestinal neuromuscular pathology in a mouse model of MNGIE treated with hematopoietic stem cell gene therapy. Quantification of muscle wall thickness and ganglion cell density was performed blind to the genotype with ImageJ. Significance of differences between groups was determined by two-tailed Mann-Whitney U test (P < 0.05). RESULTS: Our data confirm that MNGIE presents with muscle atrophy and loss of Cajal cells and CD117/c-kit immunoreactivity in the small intestine. We also show that hematopoietic stem cell transplantation does not benefit human intestinal pathology at least on short-term. CONCLUSIONS: We suggest that hematopoietic stem cell transplantation may be insufficient to restore intestinal neuropathology, especially at later stages of MNGIE. As interstitial Cajal cells and their networks play a key role in development of gastrointestinal dysmotility, alternative therapeutic approaches taking absence of these cells into account could be required.

SMAD signaling regulates CXCL12 expression in the bone marrow niche, affecting homing and mobilization of hematopoietic progenitors.

We recently demonstrated that ex vivo activation of SMAD-independent bone morphogenetic protein 4 (BMP4) signaling in hematopoietic stem/progenitor cells (HSPCs) influences their homing into the bone marrow (BM). Here, we assessed whether alterations in BMP signaling in vivo affects adult hematopoiesis by affecting the BM niche. We demonstrate that systemic inhibition of SMAD-dependent BMP signaling by infusion of the BMP antagonist noggin (NGN) significantly increased CXCL12 levels in BM plasma leading to enhanced homing and engraftment of transplanted HSPCs. Conversely, the infusion of BMP7 but not BMP4, resulted in decreased HSPC homing. Using ST2 cells as an in vitro model of BM niche, we found that incubation with neutralizing anti-BMP4 antibodies, NGN, or dorsomorphin (DM) as well as knockdown of Smad1/5 and Bmp4, all enhanced CXCL12 production. Chromatin immunoprecipitation identified the SMAD-binding element in the CXCL12 promoter to which SMAD4 binds. When deleted, increased CXCL12 promoter activity was observed, and NGN or DM no longer affected Cxcl12 expression. Interestingly, BMP7 infusion resulted in mobilization of only short-term HSCs, likely because BMP7 affected CXCL12 expression only in osteoblasts but not in other niche components. Hence, we describe SMAD-dependent BMP signaling as a novel regulator of CXCL12 production in the BM niche, influencing HSPC homing, engraftment, and mobilization.

LGR5-Positive Supporting Cells Survive Ototoxic Trauma in the Adult Mouse Cochlea.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters' cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

Gastrointestinal Dysmotility in MNGIE: from thymidine phosphorylase enzyme deficiency to altered interstitial cells of Cajal.

BACKGROUND: MNGIE is a rare and fatal disease in which absence of the enzyme thymidine phosphorylase induces systemic accumulation of thymidine and deoxyuridine and secondary mitochondrial DNA alterations. Gastrointestinal (GI) symptoms are frequently reported in MNGIE patients, however, they are not resolved with the current treatment interventions. Recently, our understanding of the GI pathology has increased, which rationalizes the pursuit of more targeted therapeutic strategies. In particular, interstitial cells of Cajal (ICC) play key roles in GI physiology and are involved in the pathogenesis of the GI dysmotility. However, understanding of the triggers of ICC deficits in MNGIE is lacking. Herein, we review the current knowledge about the pathology of GI dysmotility in MNGIE, discuss potential mechanisms in relation to ICC loss/dysfunction, remark on the limited contribution of the current treatments, and propose intervention strategies to overcome ICC deficits. Finally, we address the advances and new research avenues offered by organoids and tissue engineering technologies, and propose schemes to implement to further our understanding of the GI pathology and utility in regenerative and personalized medicine in MNGIE. CONCLUSION: Interstitial cells of Cajal play key roles in the physiology of the gastrointestinal motility. Evaluation of their status in the GI dysmotility related to MNGIE would be valuable for diagnosis of MNGIE. Understanding the underlying pathological and molecular mechanisms affecting ICC is an asset for the development of targeted prevention and treatment strategies for the GI dysmotility related to MNGIE.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp-/-Upp1-/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp-/-Upp1-/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

Mitochondrial Neurogastrointestinal Encephalomyopathy Caused by Thymidine Phosphorylase Enzyme Deficiency: From Pathogenesis to Emerging Therapeutic Options.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive metabolic disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients, clinical features include mental regression, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also causes imbalances in mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may play a direct or indirect role in the mtDNA depletion/deletion abnormalities, although the exact underlying mechanism remains unknown. The available therapeutic approaches include dialysis and enzyme replacement therapy, both can only transiently reverse the biochemical imbalance. Allogeneic hematopoietic stem cell transplantation is shown to be able to restore normal enzyme activity and improve clinical manifestations in MNGIE patients. However, transplant related complications and disease progression result in a high mortality rate. New therapeutic approaches, such as adeno-associated viral vector and hematopoietic stem cell gene therapy have been tested in Tymp-/-Upp1-/- mice, a murine model for MNGIE. This review provides background information on disease manifestations of MNGIE with a focus on current management and treatment options. It also outlines the pre-clinical approaches toward future treatment of the disease.

Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.