Abstract
A single nucleotide polymorphism (SNP) in the sickle β-globin gene (β
S
) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal γ-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A β
S
siRNA with position 10 of the guide strand designed to align with the targeted β
S
SNP specifically silences β
S
gene expression without affecting the expression of the γ-globin or normal β-globin (β
A
) genes. Silencing is increased by altering the 5′ end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific β
S
silencing was demonstrated by using a luciferase reporter and full-length β
S
cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous β-globin gene promoter and the locus control region enhancers. When this strategy was used to target β
E
, silencing was not limited to the mutant gene but also targeted the normal β
A
gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the β
S
-globin gene, as compared with β
E
, as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.
