Abstract
Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2′O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage.
Highlights
Single-stranded antisense oligonucleotides are a promising therapeutic platform to treat a variety of genetic diseases, cancer, and viral infections
Antisense oligonucleotide therapeutics accumulate dose dependently in renal proximal tubular cells, and renal tubular effects are commonly associated with oligonucleotide treatment in both animal studies and in the clinic.[1,3,4,7]
Our data support the hypothesis that 2OMePS oligonucleotides compete with receptor-mediated endocytosis in the proximal tubule epithelium and reduce the uptake of endogenous ligands such as A1M, resulting in reversible low molecular weight proteinuria in patients and animals
Summary
Single-stranded antisense oligonucleotides are a promising therapeutic platform to treat a variety of genetic diseases, cancer, and viral infections. Accumulation of antisense oligonucleotides in the kidney was demonstrated in animals by the presence of basophilic granules in the cytoplasm of proximal tubule epithelial cells.[4,5,6] These granules are considered to reflect physiological clearance of oligonucleotides and, in the absence of associated degenerative changes, are not considered of toxicological importance.[7,8] To increase resistance to endogenous nuclease breakdown and/or target engagement, the backbone structures of antisense oligonucleotides have been chemically modified using sulfur or hydroxyl groups, resulting in phosphorothioate (PS) and 2ʹ-O-methyl (2OMe) oligonucleotides, respectively.[9,10] Dose- and concentration-dependent effects on renal tubule cells were described for the class of 20-O-methoxyethylribose PS oligonucleotides. These principles likely apply to 2OMePS antisense oligonucleotides as they share physicochemical properties.[7,11]
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