In vitro nephrotoxicity screening assay

Abstract

The invention relates to methods for predicting the in vivo nephrotoxicity of a nucleic acid molecule, in particular a nucleic acid molecule such as a siRNA or an antisense oligonucleotide using an in vitro cell based assay measuring the levels of EGFR as toxicity biomarker, potentially in combination with other biomarkers like ATP and KIM-1.

1 . An in vitro method for predicting in vivo nephrotoxicity of a nucleic acid molecule in a mammal, said method comprising the steps of:
 a. culturing cells expressing epidermal growth factor receptor (EGFR) in a suitable cell culture media;   b. administering the nucleic acid molecule to said cell culture;   c. incubating the cells for a period of time; and   d. subsequently measuring the EGFR mRNA level in the cells;   
       wherein a decrease in EGFR mRNA levels is indicative of a nucleic acid molecule which is, or is predicted to be, associated with nephrotoxicity. 
     
     
         2 . The method according to  claim 1 , wherein EGFR mRNA level is compared to a reference value obtained from cells treated with vehicle control or a non-toxic reference nucleic acid molecule, where the non-toxic reference nucleic acid molecule has been validated as non-toxic in vivo. 
     
     
         3 . The method according to  claim 2 , wherein the non-toxic reference nucleic acid molecule is an antisense oligonucleotide compound consisting of CGTcagtatgcgAATc (SEQ ID NO: 1), wherein lower case letters represent DNA units, bold upper case letters represent beta-D-oxy-LNA units, all LNA C are 5′methyl C and all internucleoside linkages are phosphorothioate linkage. 
     
     
         4 . The method according to  claim 3 , wherein a level of EGFR mRNA below 80% relative to the vehicle control or non-toxic reference value is predicative of nephrotoxicity of the nucleic acid molecule. 
     
     
         5 . The method according to  claim 2  wherein EGFR mRNA level is further compared to a second reference value obtained from cells treated with a nephrotoxic reference nucleic acid molecule, where the nephrotoxic reference nucleic acid molecule has been validated to cause nephrotoxicity in vivo. 
     
     
         6 . The method according to  claim 5 , wherein the toxic reference nucleic acid molecule is an antisense oligonucleotide compound consisting of GCtgtgtgagcttGG (SEQ ID NO: 4), wherein lower case letters represent DNA units, bold upper case letters represent beta-D-oxy-LNA units, all LNA C are 5′methyl C and all internucleoside linkages are phosphorothioate linkage. 
     
     
         7 . The method according to  claim 1 , wherein step d) further comprises the measurement of extracellular kidney injury molecule-1 (KIM-1) protein or intracellular mRNA levels, wherein an increase in KIM-1 levels are indicative of a nucleic acid molecule which is, or is predicted to be, associated with nephrotoxicity. 
     
     
         8 . The method according to  claim 7 , wherein a level of KIM-1 protein above 200% relative to the saline or non-toxic reference value is predicative of nephrotoxicity of the nucleic acid molecule. 
     
     
         9 . The method according to  claim 7 , wherein a level of KIM-1 mRNA above 1000% relative to the saline or non-toxic reference value is predicative of nephrotoxicity of the nucleic acid molecule 
     
     
         10 . The method according to  claim 7 , wherein the increase in KIM-1 is predicative of nephrotoxicity for a nucleic acid molecule even if the EGFR mRNA level is not decreased. 
     
     
         11 . The method according to  claim 1  wherein the culture media in step a) comprises at least 4 ng/ml of epidermal growth factor (EGF) and step d) further comprises the measurement of intracellular adenosine triphosphate (ATP) levels; wherein a decrease in intracellular ATP levels is indicative of a drug substance which is, or is predicted to be, associated with nephrotoxicity. 
     
     
         12 . The method according to  claim 11 , wherein a level of intracellular ATP below 80% relative to the saline or non-toxic reference value is predicative of nephrotoxicity of the drug substance. 
     
     
         13 . The method according to  claim 1 , wherein the cells expressing EGFR is selected from the group consisting of epithelial cell, endothelial cell and mesenchymal cells, and neuroectodermal cells. 
     
     
         14 . The method according to  claim 13 , wherein the cell culture is a primary kidney epithelial cell culture selected from the group consisting of proximal tubule epithelial cells, distal tubule epithelial cells and collecting duct epithelial cells, in particular primary human PTEC or rat PTEC cells. 
     
     
         15 . The method according to  claim 13 , wherein the cells expressing EGFR are cultured from an immortalized cell line, such as of human PTEC-TERT-1, ciPTEC, CACO2, HK-2, NKi-2 or human A549 cell lines. 
     
     
         16 . The method according to  claim 1 , wherein the period of incubation with the nucleic acid molecule is between 2 and 6 days, such as around 3 days. 
     
     
         17 . The method according to  claim 1 , wherein the nucleic acid molecule is selected from a RNAi agent, an antisense oligonucleotide or an aptamer. 
     
     
         18 . The method according to  claim 1 , wherein the nucleic acid molecule comprises one or more 2′ sugar modified nucleosides, independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides. 
     
     
         19 . (canceled) 
     
     
         20 . The method according to  claim 1 , wherein the nucleic acid molecule is an antisense oligonucleotide capable of recruiting RNase H. 
     
     
         21 . A method for selecting one or more nucleic acid molecules for in vivo administration to a mammal, from a library of nucleic acid molecules, said method comprising the steps of
 a. obtaining a library of nucleic acid molecules;   b. administering each member of the library of nucleic acid molecules to a cell culture expressing epidermal growth factor receptor (EGFR);   c. culturing the cells in vitro for a period of time;   d. measuring the amount of intracellular EGFR mRNA, and optionally additional biomarkers, for each nucleic acid molecule, and   e. selecting one or more nucleic acid molecules wherein the % reduction in EGFR relative to a reference value is above 80%.   
     
     
         22 .- 27 . (canceled)