LIN28A-Mediated Expression Of Fetal Hemoglobin Ameliorates Erythrocyte Sickling

Highly-conserved LIN28 proteins regulate certain developmentally-timed events in multicellular organisms by decreasing the levels of let-7 miRNAs. It was recently reported that increased expression of LIN28 proteins or decreased expression of the target let-7 miRNAs in human erythroblasts cultured from healthy adult humans causes increased fetal hemoglobin expression. Here LIN28A expression in sickle cell donors’ cells was explored for its potential to regulate fetal and sickle hemoglobin, and to affect the morphological sickling of the mature erythrocytes.

After obtaining consent and assent, CD34(+) cells from five pediatric research subjects with HbSS genotype (ages 9-16 years old) were harvested from discarded whole blood following partial manual exchange transfusions. Transgenic expression of LIN28A was accomplished using lentiviral transduction of human CD34(+) sickle cells cultivated ex vivo in serum-free medium for a total of 21 days. On culture day 14, LIN28A over-expression (LIN28A-OE) was confirmed by Q-RT-PCR (control: 8.6E+00 ± 8.1E+00 copies/ng, LIN28A-OE: 2.3E+05 ± 2.1E+05 copies/ng) and Western blot analyses. Erythroblast differentiation and terminal maturation were not affected by LIN28A-OE. Enucleation, as assessed by thiazole orange (TO) staining, was equivalent between the LIN28A-OE cells and control transductions (LIN28A-OE enucleation 40.8 ± 17.0% compared to control 49.9 ± 23.4%, p=0.19). LIN28A-OE strongly suppressed all members of the let-7 family of miRNAs, with average reductions from 66% to 96% for let-7a, let-7b, let-7c, let-7d, let-7e, let-7f-2, let-7g and let-7i. LIN28A-OE caused reduced expression of BCL11A, a known repressor of gamma-globin gene expression. Gamma-, beta (sickle)- and alpha-globin mRNA levels were also investigated by Q-RT-PCR. Gamma-globin mRNA expression levels were significantly increased in LIN28A-OE samples (control: 2.0E+06 ± 7.0E+05 copies/ng, LIN28A-OE: 2.0E+07 ± 6.0E+06 copies/ng, p=0.006), and beta (sickle)-globin mRNA significantly decreased in LIN28A-OE samples (control: 2.0E+07 ± 5.2E+06 copies/ng, LIN28A-OE: 1.6E+07 ± 6.3E+06 copies/ng, p=0.024). Differences in alpha-globin mRNA expression were not statistically significant. Hemoglobin chromatography (HPLC) demonstrated that LIN28A-OE significantly increased the proportion of fetal hemoglobin (HbF control: 10.8 ± 7.1%, LIN28A-OE: 40.1 ± 14.0%; p=0.003) that was balanced by a significant decrease in the proportion of sickle hemoglobin. HbA was not detected.

For investigation of the sickling phenotype, enucleated [TO(-)] sickle erythrocytes from LIN28A-OE and control transductions of two subjects’ cells were sorted at the end of the culture period into duplicate tissue culture wells. The sorted erythrocytes were incubated in hypoxia (2% oxygen) for 16 hours, and imaged using inverted microscopy within three minutes after removal from the hypoxia incubator. Four random microscopic field images from each well were acquired. Blinded observers then scored the images from the control and LIN28A-OE transductions according to non-sickled versus sickled morphologies. Cultured erythrocytes from the control transductions demonstrated 86.3 ± 9.5% with sickled morphologies. By comparison, a significant reduction in sickling morphology was observed in the LIN28A-OE cells (56.2 ± 23.1% sickled morphologies; p=0.000009).

These results demonstrate that transgenic expression of LIN28A during ex vivo erythropoiesis causes increased gamma-globin gene and protein expression balanced with decreased beta (sickle)-globin at levels that are sufficient to ameliorate hypoxia-related sickling of mature erythrocytes.