Clinical data suggest that a reduction of a-globin to 75% to 25% of
its physiological levels is safe and beneficial to patients with
b-thalassemia.3,35 The most common natural mutations affecting
a-globin synthesis are gene deletions that remove a single HBA
gene from 1 or both chromosomes generating -a/aa or -a/-a
genotypes (-a3.7 and -a4.2deletions36) (Figure 1A).Effects of genetic modifiers can be either mediated by HbF or independent of HbF: Genetic modifiers affecting sickle cell disease (SCD) may be linked to increased fetal hemoglobin (HbF) expression.To establish a correlation between
a-globin expression and number of HBA genes, we generated
multiple cell clones with mono- or biallelic HBA2 deletions (-a/aa
and -a/-a, respectively, n 5 3 per genotype) and we showed
a significant amelioration of the a/b-like globin imbalance upon
deletion of HBA2, with the -a/-a clones being indistinguishable
from wild-type HUDEP-2 cells (Figure 1D; supplemental Figure 1D).To
minimize the possibility of generating a a-globin KO, the sgRNA
was designed to target the 59UTR (HBA15) of HBA1 and HBA2
(Figure 1A), where the presence of InDels resulting from doublestrand
breaks (DSBs) does not affect a-globin production.37 As
a b0-thalassemia cell model, we used immortalized HUDEP-2,
which can differentiate and express adult hemoglobin (supplemental
Figure 1A-B), and we knocked out b-globin genes (HUDEP-2 b0)
(supplemental Figure 1C).We transfected both wild-type HUDEP-2 and HUDEP-2 b0 with
RNP targeting HBA and we achieved efficient editing (83.1% 6
12.1 and 77.3% 6 18.2, respectively, n 5 3; Figure 1B) and
genomic deletion of HBA2 gene (0.9 6 0.2 and 0.9 6 0.1 HBA2
copy per cell, n 5 3; Figure 1C), which resulted in a decrease of
a-globin messenger RNA (mRNA) expression upon erythroid
differentiation (Figure 1D).