5613327 KNOCK-DOWN OF AUTOPHAGY-RELATED GENE 5 (ATG5) EXPRESSION IN THALASSEMIC CD34+ CELLS LEADS TO AMELIORATION OF ERYTHROPOIESIS

Abstract

Background: β-thalassemia is characterized by ineffective erythropoiesis due to increased apoptosis of the thalassemic erythroid precursors. Recent studies documented enhanced initiation of autophagy in β-thalassemia erythroblasts, which may contribute to the elevated levels of apoptosis.1,2 Therefore, genetic manipulation of key genes of autophagy employing shRNA technology, could improve the pathophysiology of the disease. Aim: To this end, our study aimed to investigate the role of ATG5 in autophagy and/or apoptosis in β-thalassemia, by downregulating its expression via shRNA technology, and potentially exploit this effect for the amelioration of the disease pathophysiology. Materials and Methods: Lentiviral vectors (LVs), harboring three distinct shRNAs (sh11840, sh11841 and sh11842) targeting discrete regions of endogenous ATG5 mRNA, were used to transduce K562 cells at a multiplicity of infection (MOI) 25. A lentiviral vector carrying a shRNA against no human gene (shNT; No Target), was used as a control. Flow cytometry and confocal microscopy using an ATG5-specific antibody were employed to determine ATG5 expression at the protein level, while qPCR assessed ATG5 knock-down at the mRNA level. The sh11842 LV exhibited the highest ATG5 knock-down and was selected for in vitro assessment in thalassemic cells, following initial transduction of CD34+ cells at MOI 25, for a differentiation time period to include the early autophagy activation at basophilic stage. Autophagic flux in untransduced, shNT-transduced and shATG5-transduced CD34+ cells, was assessed using confocal microscopy to determine the level of the LC3/LAMP co-localization, markers of autophagosomes and lysosomes, while flow cytometry was employed to determine erythroid differentiation and apoptosis in the differentiated cells. Results: Transduction of K562 cells with the sh11842 LV led to the highest ATG5 knock-down, reaching 93% and 38% at mRNA and protein level, respectively (n=4). Transduction of thalassemic CD34+ cells with the sh11842 LV at day 6 of differentiation showed advanced erythropoiesis compared to shNT-transduced cells. Specifically, the percentage of basophilic erythroblasts was significantly reduced following sh11842-transduction as compared to shNT-transduction, (43.5 ± 4.5% vs 59.6 ± 8.1%, p=0.0127, n=4, respectively), leading to a transient increase of 28.9 ± 5.9% in polychromatic erythroblasts, compared to 16.3 ± 3.7% (p=0.0118, n=4) in shNT-transduced cells. As a result of this accelerated erythropoiesis, the percentage of orthochromatic erythroblasts increased to 5.5 ± 2.4%, compared to 2.8 ± 1.7% observed in shNT-transduced cells (p=0.199, n=4). This was followed by a significant reduction of LC3/LAMP co-localization in sh11842-transduced cells, reaching a Pearson’s correlation coefficient of 0.103 ± 0.29%, compared to 0.165 ± 0.4% seen in shNT-transduced and 0.372 in untransduced cells (p<0.0001). Furthermore, no differences in apoptosis were observed between sh11842- and shNT- transduced cells (59.0 ± 18.2% vs 50.3 ± 8.6%, p=0.6618 n=3). Pilot studies indicate that transduction with sh11842 LV may lead to increased mitochondrial membrane potential, as demonstrated by increased Mitotracker® staining, suggesting a potential reduction in mitophagy, as well. Summary - Conclusion: Knock-down of ATG5 expression leads to improvement of the thalassemic erythropoiesis in vitro, highlighting its potential synergistic role in the amelioration of thalassemic pathophysiology.