New therapeutic strategies for sickle-cell disease using CRISPR/Cas9 to produce hereditary persistence of non-deletional fetal hemoglobin of the types: Brazilian (-195 C>G), "black" (-175 T>C) and Greek (-117 G>A)
Sickle-cell disease is a serious, chronic disorder and undoubtedly represents a public health problem in Brazil. Patients need to be accompanied for lifelong in specialized centers with a multidisciplinary team to control symptoms and prevent sickle cell crisis. The only treatment option for sickle cell anemia so far is related hematopoietic stem cell transplantation (BMT), indicated in severe cases (such as stroke in childhood) and that have a compatible donor in the family. Thereby, gene therapy represents a possible alternative that in the future may lead to new possibilities of cure or better control of these diseases. Our laboratory recently proposed a new therapeutic strategies using the CRISPR/Cas9 System to increase fetal hemoglobin (HbF) through the insertion of the -195C>G mutation in the promoter region of the HBG1 gene, which corresponds to hereditary persistence of non-deletional fetal hemoglobin (ndPHHF) of the Brazilian type. The effectiveness of this methodology has been successfully tested in the generation of an immortalized erythroid cell model. To continue this project, we will seek to improve the technique of application in erythroid progenitor cells (Sca1 + ckit+ lin-) of model animals for sickle-cell anemia. Thus, the aim of this project is to continue and expand this therapeutic strategy developed for sickle-cell anemia producing the Brazilian type ndPHHF (-195 C>G) and to test new mutations of the Black type (-175 T>C) and Greek type ndPHHF (-117 G>A). All these alterations are located in the promoter region of the HBG1 gene and are responsible for an increase in fetal hemoglobin levels more expressive than that observed in the Brazilian type ndPHHF. The proposed strategy to achieve this goal will be the use of the high-fidelity CRISPR/Cas9 system (CRISPR/Cas9-HF1).
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