In vitro toxicity screening assay

Abstract

The invention relates to methods for predicting the in vivo toxicity of oligonucleotides, such as antisense oligonucleotides using in vitro cell based assays based on gymnotically administering oligonucleotides to primary mammalian hepatocytes and subsequently measuring the levels of toxicity biomarkers such as the release of LDH into the cell culture media and/or intracellular ATP.

1 . A method for predicting the in vivo toxicity of an oligonucleotide in a mammal, said method comprising the steps of:
 a. administering the oligonucleotide to a population of primary mammalian hepatocyte cells (or population of hepatocytes derived from induced pluripotent stem cells) in vitro in a cell culture media;   b. and culturing the cells in vitro in the cell culture media for a period of time;   c. and subsequently measuring the level of lactate dehydrogenase (LDH) released into the culture media, or measuring the level of cellular ATP levels;   
       wherein an increase in lactate dehydrogenase in the cell culture media, or a decrease in cellular ATP levels is indicative of an oligonucleotide which is, or is predicted to be, toxic e.g. hepatotoxic in vivo in the mammal. 
     
     
         2 . The method according to  claim 1 , wherein the oligonucleotide is administered to the population of primary mammalian hepatocyte cells via gymnosis. 
     
     
         3 . The method according to  claim 1  or  2 , wherein level of lactate dehydrogenase in the cell culture media, or the level of intracellular ATP levels, is compared to a reference value obtained from negative control sample, e.g. a non-toxic oligonucleotide or a no-oligonucleotide control. 
     
     
         4 . The method according to any one of  claims 1 - 3 , wherein step c) further comprises the measurement of the level of microRNA-122 released into the culture media. 
     
     
         5 . The method according to any one of  claims 1 - 4 , wherein step c) further comprises the measurement of intracellular glutathione (GSH) levels, wherein a reduction in GHS levels are indicative of an oligonucleotide which is, or is predicted to be, hepatotoxic in vivo in the mammal. 
     
     
         6 . The method according to any one of  claims 2 - 5 , wherein the level of LDH present in the culture media is at least 20% higher as compared to the reference value. 
     
     
         7 . The method according to any one of  claims 2 - 6 , wherein the level of cellular ATP is at least 20% lower that the reference value. 
     
     
         8 . The method according to any one of  claims 1 - 6 , wherein the primary mammalian hepatocyte cells are selected from the group are selected from the group consisting of rodent primary hepatocyte cells, such as mouse, rabbit or rat primary hepatocyte cells; primate primary hepatocyte cells, such as monkey or human primary hepatocyte cells; or pig (e.g. minipig), dog, or monkey (e.g. cynomolgus monkey) primary hepatocytes cells. 
     
     
         9 . The method according to any one of  claims 1 - 8 , wherein the mammal is a rodent, such as a mouse, rabbit or rat, or is a human. 
     
     
         10 . The method according to  claim 9  wherein the mammal is a mouse or a human. 
     
     
         11 . The method according to any one of  claims 1 - 10 , wherein the mammalian primary hepatocyte cells are mouse primary hepatocyte cells, and the mammal is either a mouse or a human. 
     
     
         12 . The method according to any one of  claims 1 - 10 , wherein the cells are cultured in step b) for a period of between about 1-about 7 days, such as between about 2-about 4 days, such as about 3 days,. 
     
     
         13 . The method according to any one of  claims 1 - 11 , wherein the oligonucleotide comprises LNA or 2′ modified nucleosides. 
     
     
         14 . The method according to any one of  claims 1 - 12 , wherein the oligonucleotide is a gapmer oligonucleotide, such as a gapmer oligonucleotide which comprises LNA nucleosides or 2′ modified nucleosides. 
     
     
         15 . The method according to any one of  claims 1 - 14 , wherein the toxicity in vivo is hepatotoxicity, such as acute hepatotoxicity. 
     
     
         16 . A method for selecting one or more oligonucleotides suitable for in vivo administration to a mammal, from a library of oligonucleotides, said method comprising the steps of
 a. Obtaining a library of oligonucleotides   b. Administer each member of the library of oligonucleotides to a population of primary mammalian hepatocyte cells in vitro via gymnosis (e.g. as per any one of the  claims 1 - 15 );   c. culturing the cells in vitro for a period of time (e.g. as per any one of the  claims 1 - 15 );   d. measuring the amount of at least one biomarker of toxicity, such as hepatotoxicity for each oligonucleotide (e.g. as per any one of the  claims 1 - 15 )   e. selecting one or more oligonucleotides which is or is predicted to be not toxic e.g. hepatotoxic in vivo in the mammal (e.g. as per any one of the  claims 1 - 15 ).   and optionally administering the selected oligonucleotides in vivo to the mammal.   
     
     
         17 . The method according to  claim 16 , wherein the library of oligonucleotides is a library of oligonucleotide variants (child oligonucleotides) of a parent oligonucleotide, wherein the parent oligonucleotide is toxic, such as hepatotoxic, and wherein step c. identifies one or oligonucleotide variants which are less toxic than the parent oligonucleotide; wherein the oligonucleotide variants retaining the core nucleobase sequence of the parent oligonucleotide. 
     
     
         18 . The method according to  claim 17 , wherein the oligonucleotide variants differ from the parent oligonucleotide by the presence of one or more stereodefined phosphorothioate internucleoside linkages. 
     
     
         19 . The method according to anyone of  claims 1 - 18 , wherein the oligonucleotide is an antisense oligonucleotide, such as a gapmer oligonucleotide. 
     
     
         20 . The method according to  claim 19 , wherein the oligonucleotide is an LNA oligonucleotide. 
     
     
         21 . The method according to any one of  claims 17 - 20 , wherein the library of oligonucleotide variants comprises a population of child oligonucleotides which differ by virtue of the design of nucleoside modifications. 
     
     
         22 . The method according to  claim 21 , wherein the library of child oligonucleotides are or comprise a population of child oligonucleotides with different gapmer designs, optionally including different mixed wing gapmer designs.