RESUMO
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.
Assuntos
Ativação Transcricional , Talassemia beta/genética , Globinas delta/genética , Animais , Modelos Animais de Doenças , Eritrócitos/citologia , Eritrócitos/metabolismo , Eritropoese/genética , Expressão Gênica , Ordem dos Genes , Genes Reporter , Loci Gênicos , Humanos , Ferro/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Regiões Promotoras Genéticas , Talassemia beta/terapia , Globinas delta/química , Globinas delta/metabolismoRESUMO
Introduction: T cell reactivity against pancreatic autoantigens is considered one of the main contributors to the destruction of insulin-producing cells in type 1 diabetes (T1D). Over the years, peptide epitopes derived from these autoantigens have been described in NOD mice and in both HLA class II transgenic mice and humans. However, which ones are involved in the early onset or in the progressive phases of the disease is still unclear. Methods: In this work we have investigated, in early-onset T1D pediatric patients and HLA-matched controls from Sardinia, the potential of preproinsulin (PPI) and glutamate decarboxylase 65 (GAD65)-derived peptides to induce spontaneous T cell proliferation responses of peripheral blood mononuclear cells (PBMCs). Results: Significant T cell responses against PPI1-18, PPI7-19 and PPI31-49, the first two belonging to the leader sequence of PPI, and GAD65271-285 and GAD65431-450, were found in HLA-DR4, -DQ8 and -DR3, -DQ2 T1D children. Conclusions: These data show that cryptic epitopes from the leader sequence of the PPI and GAD65271-285 and GAD65431-450 peptides might be among the critical antigenic epitopes eliciting the primary autoreactive responses in the early phases of the disease. These results may have implications in the design of immunogenic PPI and GAD65 peptides for peptide-based immunotherapy.
Assuntos
Diabetes Mellitus Tipo 1 , Criança , Humanos , Autoantígenos , Epitopos , Leucócitos Mononucleares , Camundongos Endogâmicos NOD , Peptídeos , Sinais Direcionadores de Proteínas , Camundongos , AnimaisRESUMO
A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.