In vitro toxicity screening assay
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. culturing the cells in vitro in the cell culture media for a period of time; and
c. 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 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 , 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.
4. The method according to claim 1 , wherein step c) further comprises the measurement of the level of microRNA-122 released into the culture media.
5. The method according to claim 1 , 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 hepatotoxic in vivo in the mammal.
6. The method according to claim 1 , 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 claim 1 , wherein the level of cellular ATP is at least 20% lower that the reference value.
8. The method according to claim 1 , wherein the primary mammalian hepatocyte cells are selected from the group consisting of rodent primary hepatocyte cells; primate primary hepatocyte cells pig primary hepatocyte cells and dog primary hepatocyte cells.
9. The method according to claim 1 , wherein the cells are cultured in step b) for a period of between 1 to 7 days.
10. The method according to claim 1 , wherein the oligonucleotide comprises LNA or 2′ modified nucleosides.
11. The method according to claim 1 , wherein the oligonucleotide is a gapmer oligonucleotide.
12. The method according to claim 1 , wherein the toxicity in vivo is hepatotoxicity.
13. 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. administering at least one member of the library of oligonucleotides to a population of primary mammalian hepatocyte cells in vitro via gymnosis;
c. culturing the cells in vitro for a period of time;
d. measuring the amount of at least one biomarker of toxicity for each oligonucleotide
e. selecting one or more oligonucleotides which is or is predicted to be not toxic and optionally administering the selected oligonucleotides in vivo to the mammal.
14. The method according to claim 13 , wherein the library of oligonucleotides is a library of oligonucleotide variants (child oligonucleotides) of a parent oligonucleotide, wherein the parent oligonucleotide is toxic, 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.
15. The method according to claim 14 , wherein the library of oligonucleotide variants comprises a population of child oligonucleotides which differ by virtue of the design of nucleoside modifications.
16. The method according to claim 15 , 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.