Multi-Organ Morphological Findings in a Humanized Murine Model of Sickle Cell Trait.

Sickle cell disease (SCD) is caused by the homozygous beta-globin gene mutation that can lead to ischemic multi-organ damage and consequently reduce life expectancy. On the other hand, sickle cell trait (SCT), the heterozygous beta-globin gene mutation, is still considered a benign condition. Although the mechanisms are not well understood, clinical evidence has recently shown that specific pathological symptoms can also be recognized in SCT carriers. So far, there are still scant data regarding the morphological modifications referable to possible multi-organ damage in the SCT condition. Therefore, after genotypic and hematological characterization, by conventional light microscopy and transmission electron microscopy (TEM), we investigated the presence of tissue alterations in 13 heterozygous Townes mice, one of the best-known animal models that, up to now, was used only for the study of the homozygous condition. We found that endothelial alterations, as among which the thickening of vessel basal lamina, are ubiquitous in the lung, liver, kidney, and spleen of SCT carrier mice. The lung shows the most significant alterations, with a distortion of the general tissue architecture, while the heart is the least affected. Collectively, our findings contribute novel data to the histopathological modifications at microscopic and ultrastructural levels, underlying the heterozygous beta-globin gene mutation, and indicate the translational suitability of the Townes model to characterize the features of multiple organ involvement in the SCT carriers.

Delta-globin gene expression improves sickle cell disease in a humanised mouse model.

Sickle cell disease (SCD) is a widespread genetic disease associated with severe disability and multi-organ damage, resulting in a reduced life expectancy. None of the existing clinical treatments provide a solution for all patients. Gene therapy and fetal haemoglobin (HbF) reactivation through genetic approaches have obtained promising, but early, results in patients. Furthermore, the search for active molecules to increase HbF is still ongoing. The delta-globin gene produces the delta-globin of haemoglobin A2 (HbA2). Although expressed at a low level, HbA2 is fully functional and could be a valid anti-sickling agent in SCD. To evaluate the therapeutic potential of a strategy aimed to over-express the delta-globin gene in vivo, we crossed transgenic mice carrying a single copy of the delta-globin gene, genetically modified to be expressed at a higher level (activated), with a humanised mouse model of SCD. The activated delta-globin gene gives rise to a consistent production of HbA2, effectively improving the SCD phenotype. For the first time in vivo, these results demonstrate the therapeutic potential of delta-globin, which could lead to novel approaches to the cure of SCD.

In vivo activation of the human δ-globin gene: the therapeutic potential in ß-thalassemic mice.

ß-thalassemia and sickle cell disease are widespread fatal genetic diseases. None of the existing clinical treatments provides a solution for all patients. Two main strategies for treatment are currently being investigated: (i) gene transfer of a normal ß-globin gene; (ii) reactivation of the endogenous γ-globin gene. To date, neither approach has led to a satisfactory, commonly accepted standard of care. The δ-globin gene produces the δ-globin of hemoglobin A2. Although expressed at a low level, hemoglobin A2 is fully functional and could be a valid substitute of hemoglobin A in ß-thalassemia, as well as an anti-sickling agent in sickle cell disease. Previous in vitro results suggested the feasibility of transcriptional activation of the human δ-globin gene promoter by inserting a Kruppel-like factor 1 binding site. We evaluated the activation of the Kruppel-like factor 1 containing δ-globin gene in vivo in transgenic mice. To evaluate the therapeutic potential we crossed the transgenic mice carrying a single copy activated δ-globin gene with a mouse model of ß-thalassemia intermedia. We show that the human δ-globin gene can be activated in vivo in a stage- and tissue-specific fashion simply by the insertion of a Kruppel-like factor 1 binding site into the promoter. In addition the activated δ-globin gene gives rise to a robust increase of the hemoglobin level in ß-thalassemic mice, effectively improving the thalassemia phenotype. These results demonstrate, for the first time, the therapeutic potential of the δ-globin gene for treating severe hemoglobin disorders which could lead to novel approaches, not involving gene addition or reactivation, to the cure of ß-hemoglobinopathies.

miR-365-3p mediates BCL11A and SOX6 erythroid-specific coregulation: A new player in HbF activation.

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as ß-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal ß-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, we identified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating ß-hemoglobinopathies.

Delta-Globin Gene Expression Is Enhanced in vivo by Interferon Type I.

Beta hemoglobinopathies are widely spread monogenic lethal diseases. Delta-globin gene activation has been proposed as a possible approach for curing these pathologies. The therapeutic potential of delta-globin, the non-alpha component of Hemoglobin A2 (α2δ2; HbA2), has been demonstrated in a mouse model of beta thalassemia, while its anti-sickling effect, comparable to that of gamma globin, was established some time ago. Here we show that the delta-globin mRNA level is considerably increased in a Deoxyribonuclease II-alpha knockout mouse model in which type 1 interferon (interferon beta, IFNb) is activated. IFNb activation in the fetal liver improves the delta-globin mRNA level, while the beta-globin mRNA level is significantly reduced. In addition, we show that HbA2 is significantly increased in patients with multiple sclerosis under type 1 interferon treatment. Our results represent a proof of principle that delta-globin expression can be enhanced through the use of molecules. This observation is potentially interesting in view of a pharmacological approach able to increase the HbA2 level.

Thyroid hormone (T3) and TRbeta agonist GC-1 inhibit/reverse nonalcoholic fatty liver in rats.

Nonalcoholic fatty liver disease is the most common noninfectious liver disease in clinical practice, and there is an increasing need for new therapeutic approaches for the treatment of this liver disease. Here, we examined the effect of the thyroid hormone triiodothyronine (T3) and the agonist of the thyroid hormone receptor beta isoform (TRbeta), GC-1, on fatty liver and steatohepatitis induced in rodents by a choline-methionine deficient (CMD) diet. Male Fischer 344 rats fed a CMD diet for 1 wk developed a marked fatty liver and mild hepatitis. Concurrent administration of T3 resulted in a complete prevention of the fatty change associated with increased fatty acid mitochondrial and peroxisomal beta-oxidation. To investigate whether T3 could also reverse fully established fatty liver, rats were fed a CMD diet for 10 wk and then cofed T3 for 1 wk. Coadministration of T3 resulted in a complete regression of liver steatosis associated with a decrease of lipid peroxidation, cyclooxygenase-2 expression, and activation of phospho-STAT3 and phospho-SAPK/JNK. Finally, additional experiments showed that GC-1, which has no significant side effects on heart rate, prevented and reverted CMD-induced fat accumulation, and ameliorated steatohepatitis. These results indicate that TR agonists have the potential to inhibit or reverse hepatic steatosis induced by a nutritional model.

Potential utility of xenobiotic mitogens in the context of liver regeneration in the elderly and living-related transplantation.

Although liver regeneration occurring after partial hepatectomy (PH) is greatly reduced in aged mice, liver hyperplasia induced by xenobiotic mitogens was found to be age independent. Here, we investigated the potential utility of mitogens in stimulating liver regeneration in old mice subjected to two-third PH. Although virtually no hepatocytes entered S phase 48 h after PH, pretreatment (2 h prior to surgery) with 1,4-bis(2-(3,5-dichloropyridyloxy)benzene (TCPOBOP), a ligand of constitutive androstane receptor, induced an increase of bromodeoxyuridine incorporation and enhanced the expression of cyclin D1, cyclin A and proliferating cell nuclear antigen . Next, we investigated the potential utility of mitogens in the context of donor conditioning prior to living-related transplantation. Three days after TCPOBOP administration to intact young mice, an almost doubling of the liver mass and DNA content occurred; the regenerative response to two-third resection of the TCPOBOP-induced hyperplastic liver was similar to that of mice subjected to PH alone, suggesting that an increased liver mass at the time of surgery does not inhibit the regenerative capacity. The present results suggest that mitogen-induced hyperplasia is a promising tool in conditions characterized by reduced regenerative capacity, such as in the elderly, or when a rapid increase of liver mass is required, such as in living-related transplantation.

Deficiency in interferon type 1 receptor improves definitive erythropoiesis in Klf1 null mice.

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-ß (IFN-ß) is activated in the fetal liver's CMEI of Klf1 null mice. The inhibitory effect of IFN-ß on erythropoiesis is known and, therefore, we speculated that IFN-ß could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-ß activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation.