Preclinical Evaluation of the Renal Toxicity of Oligonucleotide Therapeutics in Mice.

Antisense oligonucleotides (ASO) therapeutics hold great promise for the treatment of numerous diseases, and several ASO drugs have now reached market approval, confirming the potential of this approach. However, some candidates have also failed, due to limited biodistribution/uptake and poor safety profile. In pursuit of better delivery and higher cellular uptake, ASO are being optimized, and new chemistries are developed or conjugated with various ligands. While these developments may lead to candidates with higher potency, it is important to keep the safety aspects in sight and screen for potential toxicity in early phases of preclinical development to avoid subsequent failure in clinical development. Our understanding of ASO-mediated toxicity keeps improving with increased preclinical and clinical data available. In this chapter, we will focus on the assessment of renal toxicity in mice and describe methods to measure the levels of general urinary biomarkers as well as acute kidney injury biomarkers following ASO treatment.


Introduction
The field of synthetic antisense oligonucleotide (ASO) has advanced remarkably in the last decade, and ASOs represent a very promising therapeutic platform that keeps evolving rapidly, in particular in pursuit of delivery improvement. Many preclinical studies in the antisense area focus on improving ASO delivery and assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. However, safety assessment is particularly important when developing new generations of ASOs or novel delivery systems to avoid the potential failure of a new drug in further toxicological studies, as it happened with a peptide conjugated PMO (PPMO) targeting the human dystrophin exon 50 which was found to cause mild tubular degeneration in the kidneys of cynomolgus monkeys [1]. Toxicological properties of ASO have been comprehensively and extensively summarized previously [2,3], and our understanding of them has allowed the development of predictive tests to select the best preclinical candidates.
Following systemic administration, the highest concentrations of ASO, independently of their chemistry, are found in liver and kidney, which are therefore considered as high-exposure organs. Importantly, tissue concentration does not further increase upon re-administration once steady-state is reached [2]. Accumulated ASOs can often be visualized at the histopathology level as basophilic granules on tissue sections stained with hematoxylin and eosin, but these effects are regarded as nonadverse because of their reversible nature upon treatment cessation. In rodents treated with high dose of PS-ASO, it is also frequent to observe tissue macrophages with a foamy appearance, referred to as histiocytes, which store cytokines in response to an activated state [3].
Considering the high concentrations of ASO accumulating in the kidneys, including the charge-neutral backbone such as PMO [4,5], they are regarded as a common organ for toxicity. The highest uptake is generally observed in the proximal tubular epithelial cells of the convoluted tubule, whereas uptake in tubular cells in kidney medulla is much lower [6,7]. Renal effects therefore tend to be more tubular than glomerular, apart from the reported glomerulopathies in mouse and monkey studies with the 2 0 OMe PS Drisapersen developed for the treatment of DMD [8]. However, it appears that this toxicity was linked to the chronic complement activation and inflammatory effects of the ASO and therefore overpredicted in animal studies since humans are less susceptible to these effects. Much more common are the lesions observed in the proximal tubules, which typically appear in animals treated with much higher doses of ASOs than the clinically relevant doses. Renal toxicity is mostly regarded as an accumulation-related toxicity and mostly sequence unspecific, except for more acute tubular lesions reported with high-affinity ASO such as locked nucleic acid (LNA) [9]. These effects might be related to excessive accumulation of RNase H-dependent off-target transcripts and/or specific protein binding [2] and a predictive EGF-based assay has recently been developed to exclude this type of kidney-toxic candidates [10].
Besides this EGF-based assay, several specific and early biomarkers of toxicity can be evaluated in mice (treated with high doses of ASO) to predict toxicity in preclinical development and exclude nephrotoxic candidates [8]. Evaluation of renal toxicology typically includes macroscopic examination of the kidneys upon necropsy of the animals followed by microscopic examination and careful histopathology analysis. General biomarkers of renal toxicity can be measured in the serum or plasma of treated mice such as urea, albumin, creatinine, and total protein. In this chapter, we focus on urinary biomarkers of kidney toxicity and describe the methods to measure the levels of total protein, albumin, creatinine as well as specific kidney injury biomarkers as a way of evaluating the potential renal toxicity of antisense oligonucleotides in mice. For this assessment, urines are collected from ASO-treated mice either shortly after ASO injection to evaluate the potential acute kidney toxicity or after several weeks of repeated treatment to evaluate the potential long term renal toxicity induced by the accumulation of ASO in kidneys.

Materials
For all analysis described in this chapter, urine is collected using metabolic cages for rodents (e.g., metabolic cages for mice ref.
2. Record the total volume of collected urine.

Evaluation of Creatinine Levels in Urine
Urine creatinine is measured using a creatinine assay kit (in our case R&D Systems), based on the Jaffe reaction where creatinine is treated with an alkaline solution to yield a bright orange-red complex. Intensity of the color at 490 nm corresponds to the concentration of creatinine in samples.
2. Prepare the alkaline picrate solution: for one plate, add 2.5 mL of NaOH to 12.5 mL of picric acid reagent (provided in the kit). Mix well. 100 μL of alkaline picrate solution is required per well.
3. Prepare the standard curve from the stock solution provided at 100 mg/dL. Label 7 microcentrifuge tubes from 20 mg/dL to 0.31 mg/dL. For the first point at 20 mg/dL, add 200 μL of stock into 800 μL of deionized or distilled water. The 20 mg/ dL standard serves as the high standard. For the six subsequent tubes, serial dilute (1:2) by pipetting 500 μL of standard into 500 μL of deionized or distilled water made of 7 points from 20 mg/dL to 0.31 mg/dL. Mix each tube thoroughly before the next transfer. Use deionized or distilled water as the zero standard (0 mg/dL).
4. Once all reagents, samples and creatinine standards are ready, remove excess microplate strips from the plate frame, return them to the foil pouch, and reseal.
5. Add 50 μL of standard, control, or sample to each well.
7. Incubate for 30 AE 5 min at room temperature.
8. Determine the optical density of each well using a microplate reader set to 490 nm. 9. Calculate the creatinine concentration in your samples using the optical density measurements and the standard curve.
10. Since samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor (i.e., 20) to obtain the sample concentration. (f) Calculate the total protein concentration in your samples using the optical density measurements and the standard curve.

Evaluation of Albumin Levels in Urine
Albumin levels from urine samples are measured using an albumin ELISA kit. All reagents must be at room temperature before use. We describe the method below using the albumin ELISA kit from Bethy Laboratories: 17. Calculate the albumin concentration in your samples using the optical density measurements and the standard curve.
6. Perform the immunoassay procedure making sure that all reagents are warmed at room temperature (20-25 C).
(a) Add 200 μL of assay buffer into each well of the plate. Seal and mix on a plate shaker for 10 min at room temperature (20-25 C).
(b) Decant assay buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
(c) Add 25 μL of each standard or control into the appropriate wells. Assay buffer should be used for 0 pg/mL standard (background).
(d) Add 25 μL of sample (diluted) into the appropriate wells.
(e) Add 25 μL of assay buffer to all wells.
(f) Add beads: vortex mixing bottle and add 25 μL of the mix to each well (see Note 7).
(g) Seal the plate with a plate sealer.
(h) Wrap the plate with foil and incubate with agitation on a plate shaker (~700 rpm) overnight (16-18 h) at 2-8 C.
(i) Place the plate on magnetic holder (handheld magnet, EMD Millipore Catalog #40-285) and rest plate on magnet for 60 s to allow complete settling of magnetic beads.
(j) Remove well contents by gently decanting the plate in an appropriate waste receptacle and gently tapping on absorbent pads to remove residual liquid.
(k) Wash plate with 200 μL of wash buffer by removing plate from magnet, adding wash buffer, shaking for 30 s, reattaching to magnet, letting beads settle for 60 s, and removing well contents as previously described after each wash. Repeat wash steps three times.
(l) Add 25 μL of detection antibodies into each well. Allow the detection antibodies to warm to room temperature prior to addition.
(m) Seal, cover with foil and incubate with agitation on a plate shaker (~900 rpm) for 1 h at room temperature (20-25 C). Do not aspirate after incubation.
(n) Add 25 μL streptavidin-phycoerythrin to each well containing the 25 μL of detection antibodies.
(o) Seal, cover with foil and incubate with agitation on a plate shaker for 30 min at room temperature (20-25 C).
(p) Gently remove well contents (after placing the plate on magnetic holder) and wash plate three times following instructions listed above (Step k). 1. Allow all reagents to warm to room temperature (20-25 C) before use in the assay (except antibodies and beads). (e) The 0 pg/mL standard (background) will be assay buffer.
6. Perform the immunoassay procedure making sure that all reagents are warmed at room temperature (20-25 C).
(a) Add 200 μL of assay buffer into each well of the plate. Seal and mix on a plate shaker for 10 min at room temperature (20-25 C).
(b) Decant assay buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
(c) Add 25 μL of each standard or control into the appropriate wells. Assay buffer should be used for 0 pg/mL standard (background).
(d) Add 25 μL of sample (diluted) into the appropriate wells.
(e) Add 25 μL of assay buffer to all wells.
(f) Add beads: Vortex mixing bottle and add 25 μL of the mix to each well. During addition of beads, shake bead bottle intermittently to avoid settling.
(g) Seal the plate with a plate sealer.
(h) Wrap the plate with foil and incubate with agitation on a plate shaker (~700 rpm) overnight (16-18 h) at 2-8 C.
(i) Place the plate on magnetic holder (handheld magnet, EMD Millipore Catalog #40-285) and rest the plate on magnet for 60 s to allow complete settling of magnetic beads.
(j) Remove well contents by gently decanting the plate in an appropriate waste receptacle and gently tapping on absorbent pads to remove residual liquid.
(k) Wash plate with 200 μL of wash buffer by removing plate from magnet, adding wash buffer, shaking for 30 s, reattaching to magnet, letting beads settle for 60 s, and removing well contents as previously described after each wash. Repeat wash steps three times.
(l) Allow the detection antibodies to warm to room temperature.
(m) Add 25 μL of detection antibodies into each well.
(n) Seal, cover with foil, and incubate with agitation on a plate shaker (~900 rpm) for 1 h at room temperature (20-25 C). Do not aspirate after incubation.
(o) Add 25 μL streptavidin-phycoerythrin to each well containing the 25 μL of detection antibodies.
(p) Seal, cover with foil and incubate with agitation on a plate shaker for 30 min at room temperature (20-25 C).
(q) Gently remove well contents (after placing the plate on magnetic holder) and wash plate three times following instructions listed above (Step k).
(r) Add 150 μL of sheath fluid (or drive fluid if using MAG-PIX ® ) to all wells. Resuspend the beads on a plate shaker for 5 min.
7. Analysis: Save and analyze the median fluorescent intensity (MFI) data using a five-parameter logistic or spline curvefitting method for calculating analyte concentrations in samples. For diluted samples, final sample concentrations should be multiplied by the dilution factor (1000 as per protocol instructions). If using another dilution factor, multiply by the appropriate dilution factor (see Note 8).

Notes
7. During addition of beads, shake bead bottle intermittently to avoid settling.
8. All the quantification methods described here measure the concentration of analyte (Albumin, KIM-1, etc.) in the collected urines (e.g., as ng/μL). Data can also be normalized to the levels of creatinine. Alternatively, data can be expressed as total quantity/24 h when the volume of urine collected in the metabolic cage (per 24 h) is taken into account.
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