Determinants of specific RNA interference-mediated silencing of human β-globin alleles differing by a single nucleotide polymorphism

A single nucleotide polymorphism (SNP) in the sickle β-globin gene (βS) 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 βS siRNA with position 10 of the guide strand designed to align with the targeted βS SNP specifically silences βS gene expression without affecting the expression of the γ-globin or normal β-globin (βA) 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 βS silencing was demonstrated by using a luciferase reporter and full-length βS 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 βE, silencing was not limited to the mutant gene but also targeted the normal βA 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 βS-globin gene, as compared with βE, 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.