GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells

Abstract

Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high-affinity binding to the asialoglycoprotein receptor (ASGPR) expressed at the surface of hepatocytes. Nevertheless, GalNAc conjugation does not prevent accumulation of AONs in the kidney cortex, and GalNAc-conjugated AONs might cause kidney toxicities, for example, under conditions of ASGPR saturation. Here, we investigated the nephrotoxicity potential of GalNAc-conjugated AONs by in vitro profiling of AON libraries in renal proximal tubule epithelial cells (PTECs) and in vivo testing of selected candidates. Whereas GalNAc-conjugated AONs appeared generally innocuous to PTECs, some caused mild-to-moderate nephrotoxicity in rats. Interestingly, the in vivo kidney liabilities could be recapitulated in vitro by treating PTECs with the unconjugated (or naked) parental AONs. An in vitro mechanistic study revealed that GalNAc conjugation attenuated AON-induced renal cell toxicity despite intracellular accumulation similar to that of naked AONs and independent of target knockdown. Overall, our in vitro findings reveal ASGPR-independent properties of GalNAc AONs that confer a favorable safety profile at the cellular level, which may variably translate in vivo due to catabolic transformation of circulating AONs.

Highly Potent GalNAc-Conjugated Tiny LNA Anti-miRNA-122 Antisense Oligonucleotides

“…No hepatotoxicity or kidney injury associated with the injection of tL-5G3 was found in the serum liver transaminase and creatinine analysis (Figure 1f), while at a dose of 10 mg/kg, tinyLNA showed a trend of liver and kidney injury (Figure 1b), whose toxic potential was also partially observed in previous studies [15]. The improved knockdown of miRNA-122 in the liver can be ascribed to an efficient shift of its biodistribution towards the liver, which can also lead to a reduction in its accumulation in the kidneys, resulting in a reduction of renal toxicity [22,26]. Another known benefit of GalNAc conjugation is its ability to mitigate off-target-associated hepatotoxicity [27].…”

Mesyl phosphoramidate backbone modified antisense oligonucleotides targeting miR-21 with enhanced in vivo therapeutic potency

“…In this direction, the use of N-acetylgalactosamine (GalNAc) moiety, providing interaction with asialoglycoprotein receptors, conjugated to antisense oligonucleotide was shown to greatly expand the therapeutic opportunities of ASOs (34)(35)(36). Conjugation of the GalNAc ligand to gapmer PS ASOs attenuates toxicity and promotes penetration into cells by a productive endocytic pathway that leads to a 30-fold increase in therapeutic potency and at least a 40-fold decrease in the dosage of PS ASOs (35,37).…”

Antibody Conjugates-Recent Advances and Future Innovations

“…Similar themes of heavy chemical modification have been utilized in ASOs. For example, one report principally noted that GalNAc conjugation ameliorated some nephrotoxicity signals in PCSK9-targeting ASOs, with data suggesting such conjugation could be a path towards addressing oligo-induced nephrotoxicity concerns more generally [115]. Beyond GalNAc, other oligonucleotide conjugation strategies have been found effective pre-clinically in targeting specific cellular populations or promoting systemic availability, as with conjugation of a GLP1R agonist [116] or conjugation to various lipids such as cholesterol or docosahexaenoic acid (DHA, Figure 5b) [117], respectively.…”

“…Antibodies 2020, 9, x FOR PEER REVIEW 10 of 27 oligo-induced nephrotoxicity concerns more generally [115]. Beyond GalNAc, other oligonucleotide conjugation strategies have been found effective pre-clinically in targeting specific cellular populations or promoting systemic availability, as with conjugation of a GLP1R agonist [116] or conjugation to various lipids such as cholesterol or docosahexaenoic acid (DHA, Figure 5b) [117], respectively.…”