The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides

Abstract

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

The Neonatal and Juvenile Pig in Pediatric Drug Discovery and Development

“…Aside from accumulation-related toxicities, nonspecific binding of ASOs to platelet-related proteins (i.e., platelet factor 4 and platelet collagen receptor VI) with corresponding platelet activation induces thrombocytopenia [ 149 ]. Similar effects have been observed in the adult minipig [ 143 ]. As fluctuations in platelet counts and coagulation parameters (prothrombin time, activated partial thromboplastin time) occur in the juvenile conventional pig and Göttingen Minipig [ 150 , 151 ], these factors may be of practical importance during pediatric safety testing of ASO drug candidates.…”

“…However, with the sequencing of the minipig genome [ 83 , 142 ], designing cross-reactive ASOs and evaluating pharmacology-related adverse effects became feasible in this porcine strain. The adult Göttingen Minipig has already been characterized to be a suitable alternative model for NHPs in the adult safety assessment of ASO drugs [ 143 ]. Target binding by ASOs was demonstrated and the toxicokinetic behavior in plasma, kidney and liver was similar compared to NHPs [ 143 ].…”

“…The adult Göttingen Minipig has already been characterized to be a suitable alternative model for NHPs in the adult safety assessment of ASO drugs [ 143 ]. Target binding by ASOs was demonstrated and the toxicokinetic behavior in plasma, kidney and liver was similar compared to NHPs [ 143 ]. ASOs are well-absorbed in the circulation following subcutaneous administration.…”

Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice

“…One pair, P1 and P2, targeted mouse Pcsk9 in non-overlapping but fairly close target regions, while the other pair, A1 and A2, targeted mouse Apob in widely distant target regions (Figure 1 and Table 1 ). Gapmers P2 ( 30 ), A1 ( 31 ) and A2 ( 32 ) have previously been investigated. To reduce the potential for mismatched binding of either P1 or P2 to Apob or for A1 or A2 to Pcsk9 , the target regions of P1 and P2 in Pcsk9 were chosen so that there were at least three mismatches or bulges to any target region in Apob .…”

Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop

“…Macrophages that have engulfed ASOs generally are enlarged and have abundant granular or finely vacuolated cytoplasm that is considered to primarily reflect the lysosomal accumulation of ASO material (Henry et al 2008;Braendli-Baiocco et al 2017). However, accumulation of cytokines and/ or other secretory substances must also be considered due to tinctorial variations in cytoplasmic staining from pale blue to clear (Engelhardt et al 2015).…”