current generation therapeutic antisense oligonucleotides ( aons ) are single - stranded oligomers of synthetic nucleotides designed to hybridize to nucleic acids according to watson - crick base pairing . to improve drug - like properties , including protein binding
part of the nucleosides can also bear modifications such as locked nucleic acids ( lna ) , 2-o - methoxyethyl ( moe ) , and 2-o - ethyl ( cet ) , that further increase biostability and target affinity .
one of the most affinity - enhancing sugar modifications is the locked nucleic acid ( lna ) .
aons with two chemically modified wings and a dna gap in - between are known as gapmers .
these are able to hybridize to a transcript and recruit ribonuclease h ( rnaseh ) , resulting in degradation of the target .
accumulation in renal proximal tubules is a well - known component of the safety profile of aons across chemical modifications.3 , 4 cell morphology changes observed include basophilic cytoplasmic granules or vacuoles in the proximal tubular epithelial cells shown to reflect accumulation of oligonucleotide in phagolysosomes . above a concentration threshold variable from molecule to molecule , degenerative / regenerative changes may be observed.5 , 6 these findings typically consist of tubular degeneration with sloughing of individual cells into the lumen , tubular cell atrophy , and tubular regeneration / diffuse basophilia .
consistent with the long half - life of these molecules , toxicity generally takes weeks to develop .
kidney injury biomarkers detectable in urine , such as kidney injury molecule 1 ( kim-1 ) , could potentially detect tubulotoxicity during pre - clinical development although their value remains to be validated for aons . as of today ,
the mechanisms of aon - related tubulotoxicity are poorly understood and may differ across species .
however , there are high accumulation compounds that are well tolerated and conversely , low accumulation compounds in the kidney cortex that are toxic
. cell uptake , intracellular trafficking , and localization may differ across aon molecules and explain some of the observed diversity .
similar to putative mechanisms in the liver , aons may interact with critical intracellular proteins to disrupt their function or may hybridize to unintended transcripts with a detrimental impact on pathways involved with cell maintenance.9 , 10 the lack of in  vitro models that mimic the in  vivo toxicity of aons has considerably limited the mechanistic understanding of the effect of these compounds at a cellular level .
some lna - aons exhibited acceptable safety profiles , but others were limited by their toxicity , including renal toxicity .
a well - known case of the latter was spc5001 , a pcsk9-targeting drug candidate for hypercholesterolemia.12 , 13 in this example , a dose - related renal tubular toxicity in a clinical phase 1 study led to the termination of this program while the pre - clinical toxicity package , conducted in mice and non - human primates , had not detected a specific renal cause for concern .
five days after receiving the third weekly dose , one high - dose subject incurred clinical acute tubular necrosis in this trial and underwent kidney biopsy .
histopathology showed normal glomeruli , no interstitial involvement , but multifocal tubular necrosis.12 , 13 follow - up non - clinical investigations on this molecule revealed that the rat , at higher doses , would have detected this signal .
while short - term studies in rodents still bring value in late discovery on a limited number of leads , there is a high demand for in  vitro assays of human relevance that could be used upstream to de - select aons within short testing cycles and rapidly inform the design of new molecules with improved therapeutic window . to this end , we established an in  vitro assay using human primary proximal tubule epithelial cells ( ptec ) that fully recapitulates , for  the first time , the in  vivo nephrotoxicity of a panel of aons , including the clinically - annotated spc5001 .
we identified extracellular epidermal growth factor ( egf ) as a robust in  vitro predictor of lna - aon - mediated tubulotoxicity and linked its elevation to a time - dependent reduced uptake by the egf receptor ( egfr ) .
we further explored the regulation of egfr signaling by lna - aons and uncovered the down - modulation of egfr activity and downstream transcriptional signature by tubulotoxic aons .
we first focused on establishing a human cell model capable of capturing the tubulotoxicity of a pcsk9-targeting lna - aon drug ( spc5001 , herein aon - b ) terminated in clinical phase i after the occurrence of overt tubulotoxicity in a healthy volunteer.12 , 13 because aon - b triggered subtle toxicity signals in the remainder of the clinical cohort and is considered moderately toxic in short - term rat studies , we selected for comparison a second pcsk9-targeting lna - aon ( aon - c ) classified as severely nephrotoxic in rats .
a scramble lna - aon ( herein aon - a ) was shown to have an innocuous profile in rodent studies and was thus selected as a negative control ( figure  1a ) .
rat kidney histopathology findings for these three test aons are summarized in table 1 , and examples of renal histological alterations induced by aon - b and aon - c are shown in figures 1b and s1 . because aon accumulation in the kidney occurs mainly in the proximal tubule , we tested the responsiveness of human primary and telomerase reverse transcriptase 1 ( tert1)-immortalized ptec to selected aons . to mimic the physiological exposure of renal tubules to circulating naked aons ( i.e. , without assistance of delivery technology ) , non - dividing confluent monolayers of ptec and ptec - tert1
were exposed to an aqueous solution of aon ( herein referred to as gymnotic delivery ) .
aon were administered at concentrations of 1 to 100  m , which approximate the renal concentrations of aon detected in cynomolgus monkeys after repeated dosing ( see the materials and methods ) .
medium with aons was changed every 3  days and intracellular atp was measured as an indication of cell metabolism and viability ( figure  1c ) . in accordance with the relatively late onset renal failure in human and histopathological manifestations in rodents , our preliminary analyses showed that aon b - mediated effects on intracellular atp in ptec - tert1 became evident after several days of treatment ( figure  s2 ) .
the atp readout was therefore performed at day 9 throughout this study ( figure  1c ) .
using this cell model and experimental design , the toxicity grade of aons a , b , and c could be accurately recapitulated , i.e. , aon - c was more cytotoxic than aon - b , and aon - a appeared innocuous ( figure  1d ) .
measurement of the levels of targeted and untargeted mrnas , namely pcsk9 and malat1 , respectively , confirmed efficient and specific target knockdown by both pcsk9-targeting aons after 3  days of exposure ( figure  1e ) .
these findings ( 1 ) rule out that lower cytotoxicity of aon - b compared to aon - c is a consequence of inferior pharmacology , ( 2 ) demonstrate that gymnotically delivered aons enter the  productive compartment  of ptec ( i.e. ,  reach their rna target ) , and ( 3 ) reveal that efficacy largely precedes cytotoxicity as measured by intracellular atp .
thus , the mode of toxicity described here seemingly requires time - dependent intracellular accumulation of aon or accumulation of aon - induced cellular and molecular alterations .
cell morphological changes induced by aon - b appeared different than those by aon - c under bright field microscopy ( figure  1f , upper panels ) .
aon b - treated ptec - tert1 formed irregular domes and vacuoles whereas aon - c - treated ptec - tert1 adopted a flattened and stable appearance .
cytoskeleton and nuclei staining indicated that the decrease in cellular atp induced by aons b and c is not associated with a significant decrease in cell numbers ( figure  1f , lower panels ) .
quantification of cellular protease activity in cell supernatant suggested that disruption of cell membrane integrity occurred after treatment with aon - b but not aon - a nor c ( figure  1 g ) .
finally , no sign of aon - induced caspase activation was detected in contrast to significant activation by the control tubulotoxicant staurosporine , suggesting that none of the three test aons triggered apoptosis in the assay conditions ( figure  1h ) .
intriguingly , except for reducing intracellular atp , the severely toxic aon - c appeared inert in the cellular assays described in figure  1 .
altogether , these data illustrate the diverse nature of aon - induced toxicities and the challenge of identifying a universal , predictive molecular marker . because intracellular atp as a readout for aon safety screening requires a long ( 9  days ) exposure period and
can not be translated into a practical molecular biomarker in the clinic , we searched for soluble factors present in the cell supernatant that are elevated after aon treatment in accordance with the severity of in  vivo liability and prior to atp decline .
we used a multiplex elisa technology to quantify the concentrations of 42 cytokines , chemokines , growth factors , and kidney injury biomarkers including kim-1 ( see the materials and  methods for a complete list ) in the supernatant of primary and immortalized ptec treated with aons a , b or c for 3 and 6  days ( figure  2a ) .
of the 42 tested proteins , only egf was elevated in correlation with the degree of aon toxicity in a concentration - dependent manner in both primary and immortalized ptec at days 3 and 6 ( figure  2b , upper panel ) .
fluctuations of cytokine and chemokine levels that were not correlated with the degree of aon toxicity were exemplified by interleukin 6 ( il-6 ) , interleukin 8 ( il-8 ) , and monocyte chemotactic protein-1 ( mcp-1 ) ( figure  s3 ) .
supernatant concentrations of the ptec - specific kidney injury biomarker kim-1 were reduced after 6  days of treatment with high concentrations of both toxic aons and slightly but reproducibly elevated with innocuous aon - a ( figure  2b , lower panel ) .
it is worth noting that kim-1 levels in supernatants of untreated human primary and tert1-immortalized ptecs are readily elevated in conventional culture conditions in comparison to the barely detectable kim-1 expression of healthy renal cortex.16 , 17 to verify that elevated egf is a general marker of aon - induced cytotoxicity and does not depend on pcsk9 knockdown , a validation test was performed with two additional tool aons previously profiled in comparable rat studies : aon - d , a mildly toxic aon and the best tolerated of a pcsk9 series , and myd88-e , a myd88-targeting aon reported to cause kidney pathologies ( figure  2c ; table 1 ) .
a gene expression analysis was performed to confirm that pcsk9 mrna is reduced in cells exposed to aon - d but not to the myd88-targeting aon - e ( figure  s4 ) . immortalized ptec ( herein ptec - tert1 ) were preferred to primary ptec to pursue validation and characterization of egf because of their replicative potential and stable phenotype , providing a more robust cellular model for safety screens .
similar to aon - b and aon - c , intracellular atp was reduced and extracellular egf was elevated after exposure to aon - e in a concentration - dependent manner ( figures 2c and 2d ) .
accumulation of egf after exposure to toxic aons was observed as early as day 3 and was more pronounced at days 6 and 9 despite medium change every 3  days , suggesting that the underlying cellular mechanism is exacerbated over time ( figure  2d ) .
the slight egf elevation observed at day 9 in aon - d - treated ptec - tert1 is consistent with the variable degree of histopathological findings reported in a subset of aon - d - treated rats ( figure  s5 ) confirming the sensitivity of the in  vitro assay .
as observed above ( figure  2b ) , kim-1 profile was variable and not consistently associated with cytotoxicity : subtle elevations were noticeable for aon - a and aon - d at days 6 and 9 and for low concentration of aon - e at day 9 whereas a marked decrease was measured for the severely toxic aon - c ( figure  2d , lower panel ) .
hence , kim-1 upregulation appeared to reflect an early stress response to aons that is no longer perceptible upon aggravated cellular or molecular dysfunction .
altogether the data showed that measurement of extracellular egf offers a promising assay to predict aon liabilities and prompted us to investigate the nature and significance of egf accumulation in response to aon treatment .
mechanistic investigations of egf as a biomarker of aon - induced toxicity should take into account that ( 1 ) the medium of ptec is supplemented with human egf as a growth factor for the cells , and ( 2 ) proximal tubule cells are known to express the egf receptor ( egfr ) and respond to egf .
more importantly , in  vivo , egf is produced by distal but not proximal tubules .
therefore , the elevation of egf in the supernatant of ptec - tert1 is most likely a result of dysfunctional uptake by egfr rather than a stimulated production and secretion of egf .
we verified this hypothesis by confirming that ptec - tert1 efficiently deplete egf from the medium within 72  hr ( figure  3a ) , express egfr , and do not express detectable level of egf even after exposure to aons ( figure  3b ) .
in addition , we compared the egf profile of aon - treated ptec - tert1 in the presence and absence of extracellular egf in the medium and observed that accumulation of soluble egf occurred only when the medium was supplemented with egf ( figure  3c ) . to visualize the capacity of aon - treated cells to internalize extracellular egf , ptec - tert1 were exposed to aons for 6  days , incubated overnight without egf , and incubated 10  min at 37c with a fluorescently labeled egf ( egf-488 ) in aon - free medium ( scheme figure  3d ) .
egf uptake and intracellular accumulation was visible in the form of green foci in vehicle - treated cells and was fully blocked by simultaneous incubation with cetuximab , a monoclonal antibody that blocks egf binding to egfr ( figure  3d , upper panels ) .
aon - a - treated cells normally accumulated egf-488 , whereas pre - exposure to toxic aon - d , aon - b , aon - c , and aon - e significantly reduced the number of egf-488 foci ( figure  3d , lower panels ; see figure  s6 for quantification ) .
the decreased number of intracellular egf foci together with the accumulation of egf in the medium suggest that egf uptake is dysfunctional in toxic aon - treated ptec - tert1 cells . to determine whether aon - mediated alterations in egf transport are restricted to a renal epithelial cell model , we replaced ptec - tert1 with human primary hepatocytes , a cell model that is competent for gymnotic uptake of aons , expresses egfr but is not dependent on egf for in  vitro proliferation and survival ( figure  s7 ) .
human hepatocytes cultivated and seeded in a conventional egf - free medium were exposed to safe and tubulotoxic aons along with 10  ng / ml of egf .
intracellular atp and extracellular egf were measured concomitantly after 3  days of aon exposure .
aon - b and aon - c showed significantly elevated egf levels whereas aons d and e had minimal changes in egf ( figure  3e ) .
aon - c did not lead to a substantial reduction in intracellular atp in human hepatocytes despite strong egf elevation , thus uncoupling the two readouts and implying that reduced egf uptake is not solely caused  by a general cellular deterioration .
in addition , we exposed the arpe-19 retinal pigment epithelial cell line to aons in cell medium supplemented with egf .
we observed an accumulation of egf in the supernatant of aon - c - treated arpe-19 associated with a decrease in intracellular atp ( figure  3f ) .
interestingly , arpe-19 did not show signs of cytotoxicity in response to aon - b and aon - e , thus highlighting the importance of choosing the most appropriate and responsive cell model to fully capture the toxicity potential of aons with correlation to histopathology findings . following the finding that tubulotoxic aons impair egf uptake , we assessed whether tubulotoxicity is correlated with the amount of extracellular egf in the medium .
ptec - tert1 were grown until confluence in regular egf - containing medium ( 10  ng / ml ) and measurements of intracellular atp and extracellular egf accumulation were performed after simultaneous incubation with aon along with 0 , 1 , 3 , 10 , 30 , or 100  ng / ml of egf .
this experiment showed that regulation of intracellular atp levels in confluent , non - dividing ptec - tert1 is dependent on egf concentration ( figure  4a ) .
remarkably , the toxicity profile of aons d , b , and c was no longer observed at 0 and 1  ng / ml of egf , and noticeable differences between innocuous and toxic aons required 10  ng / ml or higher concentrations of egf ( figure  4a ) .
importantly , aon target reduction was not affected by the presence or absence of egf ( figure  4b ) .
these data suggest that toxic aons may block egf - dependent cellular energy production . to further investigate this hypothesis , we monitored the effect of aons after inhibition of egfr signaling with cetuximab .
similar to toxic aons , cetuximab alone reduced egf - induced intracellular atp ( figure  4c ) .
the combination of aons and cetuximab had little synergistic or additional effects on atp levels , suggestive of a common mode of action via inhibition of egfr signaling . to corroborate these observations , the toxicity profile of aons
was assessed in the presence of erlotinib , a small molecule inhibitor of egfr kinase activity . in agreement with our hypothesis , the atp profile of innocuous and toxic aons was undistinguishable in egfr - inactivated cells via erlotinib ( figure  4d ) .
altogether , these data provide strong evidence that inhibition of egfr signaling may be a central mechanism of aon - induced cytotoxicity in ptec models .
aons are poly - anionic molecules capable of binding to cell surface proteins and growth factors,21 , 22 and it is conceivable that direct aon / egf or aon / egfr interactions initiate a negative regulation of egfr that progressively leads to weakening of egfr activity and decreased cellular activity .
we assessed the direct interactions of five selected aons with egf and the extracellular domain of egfr , separately and in combination , using label - free surface plasmon resonance ( spr ) . despite robust interaction between egf and egfr
, no direct aon / protein interaction could be detected between any of the five tested aons and egf or egfr ( figure  s8 ) .
moreover , the data suggest that aons did not interfere with the binding of egf to egfr in a cell - free system .
we therefore used a fluorescence - based cellular assay that preserves the native conformation of membrane - bound receptors to visualize the binding of egf to egfr and the internalization of the egf / egfr complex .
briefly , egf - starved ptec - tert1 were exposed to innocuous and toxic aons in the presence of a fluorescently labeled egf ( egf-488 ) for 10  min on ice to prevent internalization from the plasma membrane .
binding of egf-488 to the cellular membrane was evident for all conditions including ptec - tert1 exposed to toxic aons ( figure  5a , upper panels ) .
addition of cetuximab completely abolished the binding of egf-488 to the cell membrane and thereby confirmed the specific and effective binding of egf-488 to egfr for all treatments ( figure  5a , lower panels ) .
next , to visualize the internalization of egf / egfr , ptec - tert1 were incubated with egf-488 at 37c
. cellular uptake of egf-488 was evident in both the vehicle and aon - exposed ptec - tert1 ( figure  5b , upper panels ) and exclusively mediated by egfr because the addition of cetuximab completely abolished the internalization of egf-488 in all conditions ( figure  5b , lower panels ) .
in agreement with this image - based assay , quantification by flow cytometry showed similar egf-488 uptake by ptec - tert1 in the presence of innocuous and toxic aons ( figure  s9 ) .
we conclude that these aons do not significantly interfere with egf binding to egfr and that simultaneous acute incubation with egf and aons does not directly block egf uptake .
intriguingly , in cells exposed to aons d , b , c , and e , egfr was internalized in the presence of cetuximab and was not associated with any detectable egf-488 signal ( figures 5b and 5c , arrows ) . in all conditions except vehicle and aon - a ,
a subpopulation of cells showed a perinuclear distribution of egfr ( figure  5c , arrows ) .
we developed a method to quantify the presence of egfr foci located at a fixed distance from the nucleus ( perinuclear egfr ) and determined the mean intensity of perinuclear egfr per cell . in vehicle - treated cells ,
co - incubation with egf and cetuximab maintained egfr at the cell membrane ( figure  5c , arrow ) , which is translated as a left shift on the quantification graph of perinuclear egfr ( figure  5d ) .
incubation with toxic aons significantly disrupted the cetuximab - dependent distribution of egfr between the plasma membrane and the perinuclear compartment : the amount of egfr at the cell membrane was diminished and accumulation of perinuclear egfr was increased ( figures 5d and s10 ) .
the molecular nature of perinuclear egfr foci caused by toxic aons , unveiled here by the use of cetuximab , remains to be elucidated in future studies .
nevertheless , these observations suggest that toxic aons induce ligand - independent internalization of egfr .
next , the acute effect of aons on egfr kinase activity was determined by immunoblot analysis of akt and erk1/2 phosphorylation .
ptec - tert1 were exposed to egf and aons and harvested after 10  min of incubation .
downregulation of phosphorylation of akt by toxic aon - c and e was readily apparent after 10  min of exposure ( figures 5e , left panel , and s11 ) .
the effects of toxic aon - b , aon - c , and aon - e on phosphorylation of akt and erk1/2 were more pronounced after 24 and 72  hr of aon exposure ( figures 5e , middle and right panels , and s11 ) , likely reflecting a reduced steady - state level of egfr signaling in toxic aon - exposed cells .
such reduction is not caused by a general toxicity effect , since the nephrotoxicant cyclosporine a ( cysa ) did not reduce the phosphorylation level of akt and erk1/2 relative to the gapdh protein loading control ( figure  s12 ) .
the effect of aon - d on egfr activity could not be detected by measurement of akt and erk1/2 phosphorylation level , hypothetically due to compensatory signals or feedback mechanisms , which would also explain the variable profile of aon - d across in  vivo studies in rodents ( table 1 ; figure  s5 ) .
together , these results reveal acute effects of toxic aons on egfr cellular distribution and kinase activity and suggest that reduced egf uptake in toxic aon - exposed ptec - tert1 occurs progressively as a consequence of dysfunctional egfr trafficking and signaling . in order to further investigate the effects of aons on egf - dependent responses at early time points , we used molecular phenotyping , a recently introduced technology to infer human pathway activities by quantifying expression of pathway - reporter genes with next - generation sequencing .
ptec - tert1 were treated with innocuous and toxic aons in the presence or absence of egf for 6  hr , total rna was isolated and pathway reporter genes were quantified based on sequencing of predefined amplicons . to derive a list of genes that are modulated by extracellular egf in ptec - tert1 ( egf signature herein )
, we compared expression profiles of vehicle - treated cells with and without egf and identified 66 genes that are consistently positively or negatively modulated by egf .
we performed functional enrichment analysis of egf signatures and observed that genes induced by egf have versatile biological functions such as signal transduction , cytokine production , response to oxidative stress and hypoxia , defense response , and fatty acid metabolism ( figure  6a ) .
it is worth mentioning that no gene set directly associated with cell cycle or apoptosis passed the significance threshold .
cluster analysis of egf - induced changes of the signature revealed that treatment with toxic aons altered the downstream expression output of egf signaling as early as 6  hr compared with vehicle control . at 30 and 100 
m , the innocuous aon - a and vehicle clustered together while aon - b , aon - c , aon - d , and aon - e displayed substantially different profiles ( figure  6b ) .
next , we used heatmap visualization of expression changes to compare the effects of aons on the egf signature after 6  hr of exposure at 30  m .
the vehicle control highlights positively and negatively regulated genes in the egf versus no - egf condition ( figure  6c ) . the egf - induced signature ( red signals ) is preserved in aon a - treated cells consistent with the innocuous profile observed in  vitro and in  vivo under our experimental conditions .
interestingly , several genes strongly downregulated by the presence of egf ( blue signals ) were less modulated in the presence of aon - a , suggesting a general effect of aons that is not deleterious for normal cellular function .
remarkably , aon - b , aon - c , aon - d , and aon - e almost abolished the egf signature of both positive and negative egf - dependent modulation ( figure  6c ) .
interestingly , all four potentially toxic aons showed a similar profile in this analysis , including aon - d .
therefore , molecular phenotyping analysis of egf - induced signature is a sensitive and powerful approach to detect the tubulotoxicity potential of aons .
overall , molecular phenotyping data revealed that only known toxic aons inhibit egf signaling as early as 6  hr after treatment , consistent with molecular and cellular analyses of egfr signaling . to validate the use of egf as a biomarker in discovery and pre - clinical kidney safety screens , we reviewed pre - clinical studies available to our group and selected an additional 12 lna - aon compounds targeting myd88 or bcl11a with reported gross anatomical changes in mouse kidneys or histopathological findings in rat kidneys ( table s1 ) .
for all 14 aons , supernatant concentrations of egf and kim-1 at day 6 were measured and reported in percentage of vehicle control .
elevation of egf in the supernatant of ptec - tert1 accurately matched the in  vivo liabilities of 12 of the 14 aons , a score superior to elevation of kim-1 ( only 3/14 at 100  m ) ( figure  7a ) .
as observed previously for aon - b , aon - c , and aon - e ( figure  2d ) , increasing doses of toxic aons j , k , l , o , p , and s lead to a decrease in kim-1 level .
interestingly , elevated kim-1 predicted the toxicity of myd88-i and myd88-m , the only two aons that the egf readout had failed to identify as toxic . in a recent clinical study , administration of a 2-o - methoxyethyl - modified aon targeting the sodium - glucose cotransporter 2 ( herein sglt2-moe ) resulted in increases in serum creatinine levels at the highest dose level with concomitant induction of other markers indicative of renal damage or dysfunction .
this sglt2-moe was demonstrated to be safe and effective in preclinical studies in several species .
these findings gave us the opportunity to challenge our in  vitro assays with potentially toxic aon therapeutics of chemistries other than lna .
ptec cells were exposed to sglt2-moe following the experimental scheme established for lna - aons ( figure  2a ) with doses up to 500  m . in accordance with the reported kidney liabilities in human , significant accumulation of egf
was detected in the supernatant of sglt2-moe - treated primary ptec and ptec - tert1 ( figure  7b ) .
kim1 was elevated in ptec - tert1 but not in primary ptec ( figure  s14 ) , consistent with the inferior predictability of kim1 compared to egf documented with lna - aons .
these results suggest that the proposed in  vitro screen is a promising strategy to advance aon therapeutics of various chemistries . in light of the data presented here
, we propose an egf - based assay that can readily be implemented as an early safety screen in replacement of large - scale rat studies dedicated to identify nephrotoxicity prior to clinical lead selection ( figure  7c ) . in summary ,
a library of aons derived from in  silico efficacy and safety prediction tools is applied to a confluent monolayer of ptec - tert1 without delivery assistance to mimic physiological conditions .
measurement of egf in the cell supernatant serves as the principal toxicity biomarker and kim-1 can be monitored as a complementary readout .
aons for which extracellular egf and kim-1 remain unchanged are prioritized for in  vivo safety studies in rodents and non - human primates .
finally , further investigations of the egf transcriptional signature in those cells and later on in aon - treated animals may unveil molecular biomarkers that could be translational .
antisense oligonucleotide ( aon ) drugs have already demonstrated therapeutic value for patients with cancer and metabolic diseases undergoing clinical trials .
however , the occurrence of renal failure in a healthy volunteer after the third dose of spc5001 , a locked nucleic acid ( lna)-modified aon , pointed out a crucial need to improve safety assessment of aon drug candidates prior to clinical trials . to date , safety profiling of aons has typically relied on rodent studies with resource- , ethical- , and human - relevance matters . here , we established a human cell model capable of recapitulating the renal toxicity caused by spc5001 and a series of aons poorly tolerated in  vivo .
we identified elevation of extracellular egf as a robust in  vitro biomarker of aon tubulotoxicity and uncovered the downregulation of egfr signaling as central mode of toxicity of aons in renal tubular cells .
the in  vitro assay described here predicts for the first time the kidney safety risk of aons , and we propose its implementation as an early safety screen before in  vivo studies are initiated .
importantly , our mechanistic investigations revealed that the elevation of egf levels in the supernatant of toxic aon - treated ptec results from a progressive reduction of egf uptake .
thus , the nature of egf as an in  vitro biomarker described here contrasts with the elevated expression of egf observed upon kidney injury of distal tubule origin and may complicate its direct translation in the clinic given the abundance of circulating egf in plasma and urine .
notwithstanding , understanding the underlying mechanisms of inefficient egf uptake may provide valuable insight toward identification of clinically translatable molecular biomarkers . to this end , we studied physical and functional interactions between aon , egf , and egfr .
we found that the negative regulation of egfr signaling is a central mode of toxicity of aons in ptec - tert1 .
we further uncovered mechanisms of egf / egfr regulation that can contribute to the progressive accumulation of egf in the supernatant of toxic  aons - treated cells .
acutely , toxic aons promoted egf - independent perinuclear accumulation of egfr and transient downregulation of egfr signaling , followed by blockage of an egf transcriptional signature at 6  hr . at steady state , egfr signaling and egf uptake were diminished in toxic aons - treated cells .
it has been shown that gymnotically delivered aons can largely localize in the perinuclear region of the cytoplasm , in distinct bodies that stain positive for markers of the late endosome.27 , 28 hypothetically , toxic aons may distribute to the late endosome more prominently than innocuous aons and interfere with proper trafficking and processing of endocytosed egfr .
beyond reporting a novel detection workflow to predict aon liabilities , our findings shed light on a yet unappreciated contribution of the egfr signaling pathway to the cellular mechanisms of aon - induced cytotoxicity .
egfr is a central player in renal biology from development to homeostasis and injury repair .
genetic and pharmacologic evidence have shown that egfr activation in proximal tubular cells plays an important role in the recovery phase after acute kidney injury and that phospho - egfr is the most markedly upregulated receptor tyrosine kinase in response to renal ischemia / reperfusion injury .
downregulation of egfr expression and activity after repeated injection of an unfavorable aon may cause tubular dysfunction and contribute to a negative outcome as other aon - induced cellular alterations come into play , such as lysosomal accumulation , off - target hybridization , and protein binding .
ultimately , organ function will depend on the cumulative tissue damage and the regenerative capacity of proximal tubular cells , and one can speculate that blockage of egfr signaling by toxic aons will pose a considerable hurdle to tissue repair . the physiological relevance of this mechanism , though , remains to be elucidated in rats and humans .
kim-1 was identified as the most highly upregulated protein in the renal proximal tubule 24 to 48  hr after ischemia in the rat and has since been associated with renal fibrosis , inflammation , and proximal tubular cell dedifferentiation .
the kim-1 profile observed in our in  vitro assays is intriguing in that aon - mediated cytotoxicity in human ptecs correlated mainly with decreases in kim-1 levels except for the rare instances where extracellular egf remained low .
multiple technical and biological reasons may account for the difficulty to reproduce the response of kim-1 to kidney injuries in rat using cell models .
conventional cell culture conditions may not maintain a cellular state where the kim-1 gene is adequately shut down and poised to positively respond to cellular stress and injuries .
it will be relevant to address kim-1 modulation in advanced human tissue - like models to re - evaluate its use as an in  vitro biomarker and investigate a putative functional relationship between egfr and kim-1 and the translatability of this biomarker profile to the in  vivo situation .
overall , implementation of an egf - based in  vitro safety assessment of aons prior to animal testing will have an immediate impact on the development of safer aon drugs .
future studies in rodents and higher species will indicate the usefulness of studying egfr regulation as a predictor of aon - induced kidney damage and hopefully provide insights into the development of translational biomarkers .
all tested antisense oligonucleotides ( aons ) were 1416 nucleotide - long gapmers with gap segments comprised of 2-deoxynucleotides flanked by wing segments comprised of nucleotides with lna sugar modifications .
purpose - bred wistar han crl : wi(han ) male rats obtained from charles river laboratories at 78  weeks of age were divided into groups of four ( table 1 , value a ) or eight ( table 1 , value b ) based on body weight and acclimatized for at least 5  days before dosing . the animals were group - housed under standard environmental conditions ( 22c   2c , relative humidity 50%   20% , a light / dark cycle 12  hr/12  hr , pelleted food and water ad libitum ) , were offered enriched environment in an association for assessment and accreditation of laboratory animal care international ( aaalac)-accredited facility and were regularly and carefully monitored .
all procedures were conducted in strict adherence to the swiss federal ordinance on animal protection and welfare , according to the rules of ( aaalac ) and with the explicit approval of the local veterinary authority ( kantonales veterinramt basel - stadt , switzerland ) .
test compounds were dosed subcutaneously at 40  mg / kg on days 1 and 8 at 2.5  ml / kg in the interscapular region .
control group animals received saline as vehicle control . on day 15 , animals were orally administered tap water ( 10  ml / kg ) , and urine was collected on ice for 6  hr in metabolic cages .
urine protein levels ( aution max ax-4280 , arkray ) and urinary renal injury biomarkers were measured ( rat kidney toxicity magnetic bead panel 2 ; milliplex rktx2mag-37k , millipore ) .
subsequently , on day 15 , the rats were euthanized by an intraperitoneal injection of pentobarbital and exsanguinated .
kidney cortex samples were collected and fixed by immersion in 10% neutral buffered formalin , embedded in paraffin , sectioned to 5  m and stained with h&e .
the h&e sections were scanned at 20 magnification using an aperio scanscope at ( leica biosystems ) scanner and pictures were captured via imagescope software .
c57bl/6jbom female mice ( 20  g body weight ) , were obtained from taconic biosciences , divided into groups of five and acclimatized for at least 1  week before dosing .
test compounds were dosed intravenously at 15  mg / kg on days 1 , 4 , 8 , 11 , and 15 at 10  ml / kg .
groups were injected with saline as vehicle control . on day 17 , animals were anaesthetized with 70% co2 - 30% o2 before collection of blood from the retroorbital sinus .
wistar hannover male rats ( 180  g body weight ) , were obtained from taconic , divided into groups of five and acclimatized for at least 1  week before dosing .
test compounds were dosed subcutaneously at 25  mg / kg on days 1 , 8 , 15 , 22 , and 29 at 10  ml / kg in the flank .
, animals were euthanized by cervical dislocation following anesthesia , blood was collected for serum creatinine , kidneys were weighed , and tissue was collected for histopathology and mrna kidney biomarker analysis ( kidney cortex in ice - cold mrna later , then stored at 4c ) .
all test compounds were formulated in isotonic sterile saline , sterile filtered ( 0.22  m ) .
primary ptec ( hrptepic , # 4100 , sciencell ) and ptec - tert1 ( rptec / tert1 , cht-003 - 0002 , evercyte gmbh ) were cultured according to the manufacturer s instructions in ptec medium ( dmem / f12 without phenol red [ 110390 - 021 , thermofisher scientific ] ) containing 1% penicillin / streptomycin [ gibco , 15140 - 122 ] , 10  mm hepes [ gibco , 15630 - 056 ] , 5.0  g / ml human insulin [ gibco , 41400 - 045 ] , 5.0  g / ml human transferrin [ gibco , 41400 - 045 ] , 8.65  ng / ml sodium selenite [ gibco , 41400 - 045 ] , 0.1  m hydrocortisone [ h6909 , sigma - aldrich ] , 10  ng / ml human recombinant epidermal growth factor [ 236-eg-200 , r&d systems ] , 3.5  g / ml ascorbic acid [ sigma - aldrich , a4544 powder ] , 25  ng / ml prostaglandin e1 [ sigma - aldrich , p5515 ] , 3.2 pg / ml triiodo - l - thyronine [ sigma - aldrich , t-5516 ] , and 100  g / ml geneticin [ gibco , 10131 - 027 ] ) . for aon toxicity assessment , primary ptec and ptec - tert1 were seeded into 96-well plates ( 353219 , corning ) at a density of 40,000 and 20,000 cells / well respectively in ptec medium and grown until confluence prior to treatment with aons .
lna - aons were dissolved in pbs and added to the cell culture at a final concentration of 1 , 3 , 10 , 30 , or 100  m in a final volume of 100  l .
sglt2-moe - aon was dissolved in pbs and added to the cell culture at a final concentration of 30 , 100 , 300 , 400 , or 500  m in a final volume of 100  l .
medium was changed , stored at 20c for kidney injury biomarker and cytokine analysis and refreshed along with aons every 3  days .
importantly , lna - aon concentrations used in  vitro approximate the concentration of lna - aon detected in the renal cortex of cynomolgus monkeys after repeated dosing .
the later study reported renal concentrations of aon exceeding 2,500  mg / kg ( 350  m ) after seven doses of lna - aons , associated with minimal degeneration of proximal tubule epithelial cells .
cell viability was determined by measurement of intracellular atp levels after 9  days of aon treatment using the celltiter - glo luminescent cell viability assay ( g7571 , promega ) according to manufacturer s instructions . for gene expression analysis ,
cells were collected in panomics lysis mixture ( quantigene sample processing kit , qs0101 , affymetrix ebioscience ) at day 6 of aon treatment unless otherwise indicated in the figure legends and stored at  80c until analysis ( described below ) . for cellular integrity , supernatants were collected at day 9 and measured for protease release using the cytotox - glo cytotoxicity assay ( promega , g9290 ) according to manufacturer s instructions . as positive control of drug - induced cell death ,
ptec - tert1 were treated with 30 and 100  m of cyclosporine a ( sigma - aldrich , c3662 ) for 24  hr . for apoptosis assay , cell lysates were prepared and monitored for caspases 3 and 7 activation at day 3 of aon exposure using the caspase - glo 3/7 assay kit ( promega , g8090 ) according to manufacturer s instructions . as positive control of drug - induced apoptosis , ptec - tert1 were treated with 1 , 3 , and 10  m of staurosporine ( sigma - aldrich , s4400 ) for 24  hr . bright field images documenting ptec - tert1 morphological changes were taken at day 7 of aon treatment on living cells at 20 magnification . for cell count assessment at end point , cells exposed to aons for 9  days were washed with pbs ( gibco , 14190 - 094 ) , fixed with 37c - heated 4% paraformaldehyde ( alfa aesar , 43368 ) , permeabilized ( 4% cold - water fish gelatin [ sigma - aldrich , g7041 ] , 0.1% saponin [ sigma - aldrich , 47036 ] in pbs ) and stained with phalloidin - atto 488 ( sigma - aldrich , 49409 ) , 1:200 in staining buffer ( 4% cold - water fish gelatin , 0.01% saponin in pbs ) , and dapi ( sigma - aldrich , d9542 , 1  mg / ml ) . image acquisition was performed on 6 wells per conditions ( 3565 fields per well ) using the operetta high content imager ( perkinelmer ) at 20 magnification and analyzed using harmony software ( perkinelmer ) .
cryopreserved human hepatocytes were suspended in william s medium e ( wme ) without phenol red ( sigma - aldrich , w-1878 ) supplemented with 10% fetal calf serum , penicillin ( 100  u / ml ) , and streptomycin ( 0.1  mg / ml ) at a density of approximately 5    10 cells / ml and seeded into collagen - coated 96-well plates ( becton dickinson ag ) at a density of 40,000 cells / well .
cells were pre - cultured for 34  hr allowing for attachment to cell culture plates before start of treatment with aons .
seeding medium was replaced by 90  l of serum - free wme , and 10  l of aon stock solutions in pbs were added to the cell culture and left on the cells for 3  days , before egf levels in the supernatant and cellular atp levels were determined as described for ptec - tert1 .
the retinal pigment epithelial cell line arpe-19 ( crl-2302 , atcc ) was cultivated according to manufacturer s instructions in arpe-19 medium ( atcc 30 - 2006 ) .
aon treatment was performed at confluency in arpe-19 medium supplemented with 5  ng / ml of egf and intracellular atp was measured at day 7 as described for ptec - tert1 . for kidney injury biomarker and cytokine profiling shown in figures 2b and s2 , supernatants were thawed on ice , diluted 1:2 and 1:10 in sample dilution buffer ( m60 - 009rdpd , biorad ) , and analyzed by multiplex elisa using human kidney tox panel i ( bio - plex,171-atr1ck , analytes : calbindin , clusterin , gst- , il-18 , kim-1 , mcp-1 ) , human kidney tox panel ii ( bio - plex,171-atr2ck , analytes : albumin , beta2 microglobulin , cystatin c , ngal , opn , tff3 ) , and human cytokine and chemokine panel ( milliplex , hcytomag-60k - px30 , analytes : egf ,
g - csf , gm - csf , ifn-2 , ifn- , il-1 , il-1 , il-1ra , il-2 , il-3 , il-4 , il-5 , il-6 , il-7 , il-8 , il-10 , il-12 [ p40 ] , il-12 [ p70 ] , il-13 , il-15 , il-17 , ip-10 , mcp-1 , mip-1 , mip-1 , tnf- , tnf- , vegf , rantes , and eotaxin / ccl11 ) followed by analysis using the bio - plex 200 systems ( biorad ) according to manufacturer s instructions . for analysis of human egf and kim-1 from figures 2d , 3 , 4 , 5 , 6 , and 7 ,
supernatants were thawed on ice , diluted 1:2 and 1:10 in sample dilution buffer ( biorad , m60 - 009rdpd ) , and analyzed by multiplex elisa using human kidney injury panel 4 ( millipore , hki4mag-99k ) . mean concentrations and sds of triplicate wells were calculated and normalized on vehicle control .
cells grown in 96-well plates format were collected by replacing culture medium with 100  l / well of 1 rna lysis mixture ( quantigene sample processing kit , qs0101 ) .
a total of 20  l of lysates were mixed with mrna - capture magnetic beads sets ( panomics quantigene plex sets , 12697 and 12812 ) , incubated overnight , processed for branched dna amplification , and analyzed according to manufacturer s instructions ( panomics quantigene plex assay kit , qp1015 ) .
average fluorescence intensity ( fi ) and sd of three biological replicates were calculated and normalized on vehicle control .
the medium of confluent ptec - tert1 maintained in 96-well plates as described above was replaced with fresh ptec medium , and 10  l of supernatant was collected after 8 , 24 , 48 , and 72  hr for egf analysis by luminex elisa .
six wells were devoid of cells and served as no - cell control to account for egf stability over time at 37c .
data are reported as egf concentration in the supernatant of ptec - tert1 normalized on the no - cell control at the corresponding time point .
ptec - tert1 cells were plated at 500,000 cells / well in 6-well plates in ptec medium and grown until confluency prior to aon treatment and protein analysis .
for steady - state experiments , cells were exposed  to 100  m of aons as described above for human ptec , incubated for 24 and 72  hr , respectively , washed with cold pbs , and lysed in 100  l of ice - cold ripa buffer ( 60  mm tris - hcl at ph  7.4 , 150  mm nacl , 0.25% sds , 1% np40 , 1 phosstop [ 04906845001 , roche diagnostics ] and 1 complete protease inhibitor [ roche , 04693124001 ] ) . to measure effects of non - aon nephrotoxicant ,
ptec - tert1 were treated with 10  m of cyclosporine a for 24  hr prior to lysis as described above . for acute experiments ( figure  5d , 10  min )
, cells were incubated in ptec medium without egf for an overnight period followed by exposure to 100  m aons or vehicle in ptec medium containing 10  ng / ml of egf .
plates were transferred on ice after 10  min of aon and egf exposure , washed with cold pbs , and lysed in 100  l of ice cold ripa buffer .
protein content in cell lysis was measured by bca kit ( thermofisher , 23225 ) , and 20  g of total proteins were separated by electrophoresis using a 5%20% polyacrylamide gel .
western blot analyses was carried out using the following antibodies : phospho - akt - ser473 ( cell signaling , 4060 , 1:1,000 ) , phospho - erk1/2-thr202/tyr204 ( cell signaling , 4370 , 1:2,000 ) , and gapdh ( sigma - aldrich , g8795 , 1:2,000 ) . the chemidoc mp imaging system ( biorad ) was used to scan the membranes .
ptec - tert1 were seeded into 96-well plates ( corning , 353219 ) at a density of 20,000 cells / well in ptec medium and grown until confluence prior to treatment .
compounds and vehicle controls were diluted in ptec medium along with aons and added to cells according to the experimental layout in figure  1a .
the following final concentrations were used : 50 , 250 , and 500  g / ml cetuximab ( merk serono , 5  mg / ml in 0.9% nacl ) and 5  m erlotinib ( sc-202154 , santa cruz biotechnology ) .
surface plasmon resonance ( spr ) experiments were performed on the biacore instruments 3000 and 2000 ( ge healthcare , uppsala , sweden ) at 25c .
pbs buffer composed of 10  mm phosphate , 137  mm nacl , and 27  mm kcl with or without 0.05% p20 ( w / v ) , ph 7.4 was used as running buffer to perform protein immobilization and binding assays . prior to spr analysis
, the running buffer was filtered with expressplus steritop filters with 0.22  m cut off ( millipore ) and degassed .
immobilization and binding assays were performed at flow rate of 5 or 50  l / min , respectively .
tris - nta - biotin conjugate ( biotechrabbit gmbh ) was captured on streptavidin pre - coated sa sensors ( ge healthcare , br-1000 - 32 ) .
first , streptavidin sensor was conditioned with three consecutive 1-min injections of high salt solution in sodium hydroxide ( 50  mm naoh , 1  m nacl ) .
next , tris - nta - biotin stock solution ( 1  mg / ml ) was diluted 100 in running buffer and applied over the streptavidin sensor surface to achieve saturated immobilization levels of tris - nta of 400500 resonance units ( rus ) .
finally , free biotin solution ( 10  m in running buffer ) was injected once ( 1  min ) over the sensor surface to block remaining binding sites of streptavidin .
egfr receptor ( ectodomain , residues 1645 , thermofisher , 10001-h08h-250 ) was captured on tris - nta- modified sensor surface as follows : first , tris - nta sensor was conditioned with three consecutive 1  min injections of 350  mm edta in distilled water and contacted with a solution of 500  m nicl2 ( 1  min injection ) ; next , human egfr was diluted in running buffer to 5  g / ml and applied over tris - nta sensor surface to achieve egfr immobilization levels of 400 ru .
egf peptide ( sigma - aldrich , e9644 ) was titrated up to 1  m over egfr surface in triplicates .
affinity of egf to egfr was estimated to be 40  nm ( 3.7  nm ) ( data not shown ) . for competition experiments , egf ( 500  nm ) and aons ( 5  m ) were analyzed separately in solution or in a mixture ( 500  nm egf and 5  m aon ) .
for the egf / egfr binding assay ( figure  5a ) , cells were incubated in ptec medium without egf for an overnight period , washed with room temperature pbs ( gibco , 14190 - 094 ) , incubated for 5  min in pbs , followed by a 10-min incubation on ice with 100  m of aons and 10  ng / ml egf-488 ( thermofisher , e13345 ) in egf - free cold ptec medium .
cells were washed in cold pbs , fixed with cold 4% paraformaldehyde for 15  min ( alfa aesar , 43368 ) , and washed again three times with pbs . for the egf uptake assay shown in figure  5b ,
the same steps were performed except that all reagents were kept at room temperature and incubation with aons and egf-488 was done at 37c . for both assays ,
the control with cetuximab was performed by adding 100  g / ml of cetuximab ( merck serono ) 5  min prior to and during exposition to egf-488 and aons .
fixed cells were permeabilized using 4% cold - water fish gelatin ( sigma - aldrich , g7041 ) and 0.1% saponin ( sigma - aldrich , 47036 ) in pbs for 10  min at room temperature and immuno - labeled with anti - egfr ( 2e9 , abcam , ab8465 , 1:200 in staining buffer [ 4% cold cold - water fish gelatin ( sigma - aldrich , g7041 ) and , 0.01% saponin ( sigma - aldrich , 47036 ) in pbs ] ) followed by anti - mouse igg - alexa fluor 647 antibody ( thermofisher , a31571 in staining buffer ) and stained with dapi ( sigma - aldrich , d9542 , 1  mg / ml , 1:1,000 ) .
five images of randomly selected field of view per biological triplicates were acquired on a laser - scanning confocal microscope ( leica tcs sp8 ) using a 63 objective .
settings were adjusted to maximize dynamic range and reduce pixel saturation ( pixel size : 120.56  nm  120.56  nm ; image size : 1,024   1,024 ; bit depth : 12  bit ; number of z sections : 5 ) . for egf uptake shown in figure  3d ,
the same procedure was applied  except that ptec - tert1 were exposed to 100  m aons for 6  days prior to egf uptake assay and aons were not added during the 10  min incubation with egf-488 .
the number of egf foci and nuclei per field was determined using the imagej image processing program .
quantification of egfr intensity within an arbitrarily defined area around the nucleus was performed based on a particle tracking method introduced by balzarini and koumoutsakos .
first , in order to correct for the uneven background intensity , image restoration was performed by using boxcar average over a square region with a user - defined parameter - radius .
second , point locations were estimated by finding local intensity maxima under the condition that the intensity value should be higher than a user - defined absolute intensity value .
third , the point locations were further refined by calculating the offset from the brightness - weighted centroid .
fourth , non - particle discrimination was used to reject spurious detection such as dust or particle aggregates .
in fact this parameter was set as zero in our detection because particle aggregates were aimed to be kept .
in addition , two modifications from the original algorithm were implemented to better fit this work .
first , the level of radius was adapted to detect both large egfr particles with a 5-pixel radius and small particles with a 2-pixel radius .
second , an adaptive intensity threshold based on the distribution of the control image was used to overcome the inter - plate variability .
the following quantification was then performed per nucleus : ( 1 ) a perinuclear area was defined as 50% larger than the nucleus radius , ( 2 ) the number and size in pixel of egfr foci in the perinuclear area per cell were calculated , and ( 3 ) the summed intensities of large egfr ( greater than five pixels ) in the perinuclear area per cell were calculated and named as i.efgr_50%_big_sum .
the mean fluorescence intensity reflects the total perinuclear egfr intensity level regardless of the number of particles . for statistical analysis , a generalized least - squares model ( a weighted linear regression )
specifically , the readout i.efgr_50%_big_sum was log - transformed and regressed on treatment and interactions of treatment and cetuximab status ( yes / no binary variable ) .
the model was validated by means of residual analysis , checking independence from dependent variable and homogeneity of variances with regards to independent variables , as well as overall normal distribution of residuals .
eighty percent confluent ptec - tert1 were incubated overnight in ptec medium without egf , harvested with 0.25% trypsin ( gibco , 25200 - 056 ) , centrifuged at 220   g for 5  min , and distributed at 150200,000 cells per tube in ptec medium without egf with or without 100 ug / ml of cetuximab for 5  min at 37c .
cells were incubated for 10  min at 37c in egf - free or egf-488-containing ( 10  ng / ml ) ptec medium with or without cetuximab and 100  m aons as indicated in figure legends .
cells were re - suspended in ice - cold running buffer ( macsquant , 130 - 092 - 747 ) and analyzed by flow cytometry on a macsquant vyb ( miltenyi biotec ) .
the data of at least 10,000 cells per condition were analyzed with flowjo v10 software and reported as cell number relative to egf-488 fluorescence intensity .
ptec - tert1 were exposed to 10 , 30 , and 100  m of aons in egf - containing ( 10  ng / ml ) or egf - free ptec medium .
after 6  hr of treatment , cells were lysed , total rna was extracted , and pathway reporter genes were quantified following procedures described before . each condition was tested in three biological replicates . using vehicle controls with and without egf
, we identified 66 egf - signature genes in ptec - tert1 by using the r / bioconductor software package edger and the following threshold : 8 reads per million reads in an average sequencing run , absolute log2 fold change 0.5 , and benjamini - hochberg ( bh ) adjusted p value of differential expression no larger than 0.05 . we performed functional enrichment analysis of the 66 egf - signature genes with biological processes defined in gene ontology and biological pathways encoded in kegg using the david .
we identified 115 significantly enriched gene sets ( bh - adjusted p  < 0.05 ) that can be divided into 22 clusters based on composition similarity .
the  gene sets with the lowest p value of enrichment in each cluster are shown .
egf - dependent regulation of egf - signature genes in different aon treatments were quantified by egf response scores , which are defined as |log10(pvalue)|*sign(log2fc ) , namely absolute log-10-transformed p values reported by edger multiplied by the sign of the log-2-transformed fold change .
a positive ( negative ) score of gene g in aon x treatment indicates that g is higher ( lower ) expressed when aon x is applied to cells in the presence of egf than in its absence .
unless otherwise stated in the materials and methods and figure legends , statistical analysis was carried out by anova using dunnett s multiple comparison test at 95% confidence interval and alpha of 0.05 .
aon treatments were analyzed against the vehicle control of similar conditions unless otherwise stated in the figure legends ; p  values were adjusted to account for multiple comparisons .
n indicates number of biological replicates , i.e. , cells from different well plates and passages .
a. moisan conceived the study , supervised experiments , analyzed the data , and wrote the manuscript ; m.g . supervised experiments in primary ptec ; j.d.z . performed data analysis of molecular phenotyping and participated in manuscript writing ; y.t . and k.d.e .
performed post - hoc analysis of rodent studies and participated in manuscript writing ; s.s . supervised experiments in hepatocytes ; r.g . and b.a .
performed image - based quantification of perinuclear egfr ; a. maunz performed data analysis of perinuclear egfr ; r.v .
; t.s . , f.s . , and a.b.r . revised and approved the manuscript .
the funder provided support in the form of salaries for authors but did not have any additional role in the study design , data collection and analysis , decision to publish , or preparation of the manuscript .
 antisense oligonucleotide ( aon ) therapeutics offer new avenues to pursue clinically relevant targets inaccessible with other technologies . advances in improving aon affinity and stability by incorporation of high affinity nucleotides , such as locked nucleic acids ( lna ) , 
 have sometimes been stifled by safety liabilities related to their accumulation in the kidney tubule . in an attempt to predict and understand the mechanisms of lna - aon - induced renal tubular toxicity , we established human cell models that recapitulate in  vivo behavior of pre - clinically and clinically unfavorable lna - aon drug candidates . 
 we identified elevation of extracellular epidermal growth factor ( egf ) as a robust and sensitive in  vitro biomarker of lna - aon - induced cytotoxicity in human kidney tubule epithelial cells . 
 we report the time - dependent negative regulation of egf uptake and egf receptor ( egfr ) signaling by toxic but not innocuous lna - aons and revealed the importance of egfr signaling in lna - aon - mediated decrease in cellular activity . 
 the robust egf - based in  vitro safety profiling of lna - aon drug candidates presented here , together with a better understanding of the underlying molecular mechanisms , constitutes a significant step toward developing safer antisense therapeutics . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest