Lakhasly

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). To mimic these
beneficial deletions with Streptococcus pyogenes (Sp)Cas9
nuclease, we designed a single gRNA that cuts both HBA1 and
HBA2 alleles removing 1 HBA2 gene per chromosome. 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). 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).
Importantly, our HBA2 deletion strategy reduced but did not abolish a-globin production, as observed in a-globin KO control cells
obtained with a gRNA targeting the coding sequence within the first
exon of HBA1 and HBA2 genes (Figure 1D).
We also measured the relative abundance of the different hemoglobin
forms by HPLC analysis. In the absence of b-globin, a portion of the
a-globin pool complexes with b-like globins, such as g- and d-globins
(to form HbF and HbA2, respectively), whereas the excess
precipitates in insoluble aggregates (a-precipitates). Remarkably,
RNP deletion of HBA2 genes significantly reduced a-precipitates
without affecting hemoglobin synthesis (Figure 1E-F). This is in
sharp contrast with a-globin KO cells, where the predominant
hemoglobin observed was the toxic HbBart (g-globin tetramers),
typical of severe forms of a-thalassemia (Figure 1E). This result
clearly indicates that only a controlled reduction of a-globin results
in a beneficial effect.

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. Alpha-thalassemia is associated with lower MCV and reduced hemolysis but does not impact HbF levels or complications. Certain rare alleles are linked to higher HbF levels and total hemoglobin, but this does not necessarily result in reduced SCD complications. Patients with specific polymorphisms may experience more pain crises and hospitalizations, despite having higher HbF levels. Co-inheriting multiple HbF-boosting alleles may have an additive effect on HbF levels.
The HBG2-polymorphism rs7482144 defines a group of patients with high risk of painful crises: Patients on hydroxyurea with the HBG2-polymorphism rs7482144 had higher levels of HbF and total hemoglobin compared to patients not on hydroxyurea.