CRISPR-based gene editing techniques may offer curative potential for sickle cell disease (SCD) when used to target KrÃ¼ppel-like factor 1 (KLF1) mutations, according to an oral abstract presented by Kevin Gillinder, PhD, of Monash University in Melbourne, Australia, during the 2020 ASH Annual Meeting.
"For years, geneticists have been looking at the idea of reversing the hemoglobin switch – the developmental process in which the genes contributing to the hemoglobin tetramer in a spatiotemporal manner," Dr. Gillinder explained. "The final switch from fetal hemoglobin to adult hemoglobin occurs after birth, however in patients carrying the sickle mutation, this leads to a swapping out of the perfectly functional gamma globin in exchange for the defective beta globin carrying the sickle mutation."
If this process could be reversed, it would represent an effective cure for SCD, he added.
In this study, Dr. Gillinder and researchers used CRISPR-based gene-editing techniques to achieve that goal by recreating heterozygosity for loss of function mutations in KLF1, which encodes a transcription factor that plays a vital role in red cell biology. Knocking out KLF1 expression, they hypothesized, could ultimately lead to increases in gamma-globin expression that may benefit patients with inherited blood disorders.
Investigators designed two single-guide RNAs (sgRNAs) with corresponding homology-directed repair templates that targeted and ablated the function of KLF1's second exon. Dr. Gillinder noted that he and his research team optimized transfection protocols and evaluated the on-target specificity of the sgRNAs, which resulted in achievement of a greater than 90% efficacy in all assayed cell types.
Editing of KLF1 may likely provide sufficient therapeutic benefit for patients with SCD, but this method will need to be explored in larger studies.
Through RNA sequencing, the researchers found that two cellular adhesion molecules (BCAM and ICAM-4) that are associated with vaso-occlusive episodes in SCD, were downregulated in a KLF1 gene dosage-dependent manner. "Their reduced expression may provide additional benefit in treating SCD," they noted.
However, they also discovered that complete loss of KLF1 is not tolerated by the HUDEP-2 cells, described as a conditionally immortalized erythroid cell line harboring three KLF1 copies. After 10 days in culture, only about 20% of the clones actually expanded, Dr. Gillinder reported. The investigators achieved 26 clones with wild-type, 13 with a single allele knocked out, and two double knockouts, but were unable to achieve any clones that had a triple knockout.
Cells with loss of KLF1 also appeared to grow and differentiate normally, although at a reduced rate compared with cells' parental lines, he added.
The investigators performed directed differentiation using an erythroid differentiation medium following transfection of the two CRISPR guides and assessed HbF levels. HbF levels ranged between 40% and 60%, and using high performance liquid chromatography, the researchers observed that nearly 80% of the hemoglobin produced in these cells was HbF.
Together, these results suggest that editing of KLF1 may likely provide sufficient therapeutic benefit for patients with SCD, but this gene-editing method will need to be explored in larger studies.
The authors report no relevant conflicts of interest.
Gillinder KR, Reed CL, Malelang S, et al. Gene editing of KLF1 to cure sickle cell disease. Abstract #87. Presented at the 2020 American Society of Hematology Annual Meeting, December 5, 2020.