LAUSR

Sickle cell disease (SCD) is a serious condition, chronic and undoubtedly represents a public health problem worldwide. SCD is caused by a point mutation in codon 6 of the β globin gene resulting in the production of a structurally abnormal hemoglobin, hemoglobin S. Although the cause of the disease has been known for more than fifty years, therapeutic options are still quite limited. High levels of fetal hemoglobin (HbF) in the blood are associated with a better clinical outcome in SCD patients. In some individuals, the expression of γ-globin gene persists into adulthood in elevated levels, which is called hereditary persistence of fetal hemoglobin (HPFH). A single nucleotide mutation from C to G at position -195 of the HBG1 gene promoter, called non deletional HPFH Brazilian type (nd-HPFH-B), augments the levels of HbF in patients in 7%- 30%. Nd-HPFH-B has been described by our group, but the mechanism and how this single mutation rises HbF levels differently in red blood cells is still unknown. Genome editing using CRISPR/Cas9 in HUDEP-2 cell, an erythroid precursor line, has been developed through homologous direct repair from a small single DNA strand containing the guanine in -195 position at HBG1 gene promotor. All the other genes, including the second HBG1 allele were unaltered. This point mutation has been carried out by CRISPR/Cas9 high fidelity system, capable of performing a specific break in the DNA target sequence, that improves homologous recombination rate of the donor sequence containing the -195 CT mutation in HBG1 promoter using CRISPR/Cas9 genome editing. The HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1uM ssODN -195, using CD34+ human cell kit and program E-001 in AMAXA Nucleofector 4D- device (Lonza). Seven days after nucleofection, the transformed cells were submitted to clonal selection for 25 days. The genomic DNA from 48 clones were submitted by Sanger Sequencing. The sequencing analysis showed highest Crispr/Cas9 efficiency in genomic DNA cut (77.08%; 37/48) and satisfactory ssODN -195 homologous recombination (10.4%; 5/48). Five nd-HPFH-B HUDEP-2 clones and three other clones without the mutation, but with indels after Cas9 DNA cut (controls), were expanded in culture and the HbF levels were measure with anti-HbF antibody by flow cytometry in two biological replicates. HbF levels in nd-HPFH-B HUDEP-2 clones were 6.02%±1.4, 8.25% ± 0.28, 10.18% ± 3.71, 11.95% ± 0.49, 26,3% ± 4,6 while in controls were 1.69% ± 0.26, 1.66% ± 0.26, 0.59% ± 0.06. Two nd-HPFH-B clones were differentiated into erythrocyte in vitro, and fetal hemoglobin levels persisted at high levels seen previously. In addition, α-globin, β-globin and γ-globin mRNA levels were evaluated in three nd-HPFH-B HUDEP-2 clones and two control clones. The mRNA HBG1/HBG1+HBB percentage in nd-HPFH-B were 96.16% ± 4.10 against 22.63% ± 9.64 in controls. The monoallelic single nucleotide mutation -195 C>G is capable to increase the fetal hemoglobin levels up to 30% in nd-HPFH-B HUDEP-2, and our results shows that this is a potential experimental in vitro model to be used in future studies. No relevant conflicts of interest to declare.