Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats

Abstract

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

“…Theoretically, MS-based detection of radioactive isotopes (as used in QWBA and MAR) is also possible but would require sufficient dedicated facilities. It was recently shown [84] that a non-endogenous phosphorothioate fragment of an antisense oligonucleotide could be used as a marker for biodistribution in matrix assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance-MS (MALDI-FT-ICR-MS) imaging. The use of MALDI ionization for direct on-tissue, spatially resolved quantification for therapeutics has brought exciting results for small molecule drugs [85] and even antibodies [86] , and it remains to be seen if this applicability can be extended fully to RNA therapeutics.…”

Recent Progress and Challenges in the Development of Antisense Therapies for Myotonic Dystrophy Type 1

“…Alternatively, the addition of an extra methylene unit in the bridge connecting the 2′ oxygen and the 4′ carbon led to the generation of 2′-O,4′-C-ethylene-bridged nucleic acid (ENA) with better nuclease resistance and RNA affinity properties than the LNA chemistry [ 34 ]. LNA ASOs have been reported to be hepatotoxic, limiting their potential for clinical use [ 33 , 35 , 36 , 37 ]. A compromise between the LNA and 2′-MOE chemistries has led to the generation of a 2′,4′-constrained ethyl (cEt) modification in which the 2′-O-ethyl substitute is linked to the 4′ position.…”

Kidney Effects by Alternative Classes of Medicines in Patients and Relationship to Effects in Nonclinical Toxicity Studies

“…53 A few ASOs and siRNAs have resulted in off-target nephrotoxicity in rodents or monkeys related to aptameric binding to renal epithelial cytosolic proteins. 5,54 These include renal tubule toxicity associated with some locked nucleic acid (LNA) ASOs, which appear to lack a clinical correlate. 54 Fortunately, drug candidates of either class that are inherently nephrotoxic or hepatotoxic are identified in nonclinical toxicity studies and terminated early in development., 53,55 Other instances of renal tubular and glomerular toxicities with some 2-oMe and 2-MOE PS ASOs have been reported in rodents but similarly do not appear to translate clinically to patients.…”

“…5,54 These include renal tubule toxicity associated with some locked nucleic acid (LNA) ASOs, which appear to lack a clinical correlate. 54 Fortunately, drug candidates of either class that are inherently nephrotoxic or hepatotoxic are identified in nonclinical toxicity studies and terminated early in development., 53,55 Other instances of renal tubular and glomerular toxicities with some 2-oMe and 2-MOE PS ASOs have been reported in rodents but similarly do not appear to translate clinically to patients. 5,55,56 Glomerulopathy and mesangial hyperplasia have been noted in monkeys with the same class of PS ASOs, but this toxicity is related to monkey sensitivity to complement and enhanced complement responsiveness, thus it also has very little clinical relevance.…”