Table 1.

Summary of the Main Recommended Predictive Assays and the Associated References for Detailed Protocols

OND toxicity	Predictive assays	In vitro/in vivo	References for protocols
Off-target effects	In silico evaluation/in vitro validation	In silico and in vitro	Guidelines published by the OSWG in 2012 (Lindow et al. [16]; Michel et al. [146]; Yoshida et al. [147])
Immunostimulatory effects	• Quantification of cytokines/chemokines release in human PBMC or WBA by ELISA • Quantification of cytokines as well as CCL22 mRNA levels in BJAB cells by qRT-PCR	In vitro	Coch et al. [81]; Lankveld et al. [82]; Sewing et al. [104]; Anderson et al. [83]
Toxicities in high exposure organs	• Cytotoxicity by caspase assay Predictive assays for hepatotoxicity: • Quantification of LDH and ATP in primary hepatocytes • Caspase assay in transfected mouse 3T3 fibroblasts or human HepG2 cells • Evaluation of liver enzymes in mice/NHP Predictive assays for nephrotoxicity: • Quantification of EGF in human kidney tubule epithelial cells • Quantification of kidney injury biomarkers using chip-cultured HRPTEC • Quantification of urinary biomarkers (eg, β2-microglobulin and KIM-1) by ELISA	In vitro In vitro In vitro In vivo In vitro In vitro In vivo	Anderson et al. [83] Sewing et al. [104,105] Dieckmann et al. [94] Burel et al. [95] Moisan et al. [114] Nieskens et al. [115] Echevarría and Goyenvalle [117]
Thrombocytopenia	• Evaluation of platelet activation in human or NHP platelet-rich plasma or whole blood by flow cytometry (activation of CD62P and PAC-1)	In vitro and in vivo	Narayanan et al. [90]; Sewing et al. [71]; Slingsby et al. [85]
Inhibition of coagulation	• Quantification of PT and aPTT in vitro in human/mouse/NHP citrated plasma	In vitro	Echevarría et al. [122]; Relizani et al. [125]
Complement activation	• Quantification of split products of the APC (C3a, Bb, and C5a) in vitro in human/NHP/mouse serum	In vitro	Aupy et al. [123]; Henry et al. [129]; Sewing et al. [71]
CNS-specific toxicities	• Prediction of neurotoxicity from sequence features • Quantification of spontaneous calcium oscillations in primary cortical neuronal cultures	In silico In vitro	Hagedorn et al. [27] Hagedorn et al. [27]

APC, alternative pathway of the complement; aPTT, activated partial thromboplastin time; ATP, adenosine triphosphate; BJAB, EBV-negative Burkitt-like lymphoma cell line; CCL22, C-C Motif Chemokine Ligand 22; CNS, central nervous system; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; HRPTEC, human renal proximal tubule epithelial cells; KIM-1, kidney injury molecule-1; LDH, lactate dehydrogenase; mRNA, messenger RNA; NHP, non human primate; OND, oligonucleotide drug; OSWG, Oligonucleotide Safety Working Group; PBMC, peripheral blood mononuclear cell; PT, prothrombin time; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; WBA, whole blood assay.