LNA-mediated silencing of microrna-122 in African green monkeys

Transcription

1 LNA-mediated silencing of microrna-122 in African green monkeys Supplementary information for Elmen and Lindow et al. February 25, 2008 This document contains details about the clinical parameters measured in African green monkeys treated with an LNA-antimiR against mir-122. Contents 1 Methodology Animal experiments Plasma lipid and lipoprotein analysis Hematology and blood chemistry Liver biopsies In situ hybridizations Statistical analysis Results - biopsied animals Total plasma cholesterol Cholesterol distribution Apolipoprotein profiles Glucose and triglycerides Coagulation profile Electrolytes Liver parameters Tissue damage parameters Kidney parameters Pancreas parameters Histopathology In situ hybridization Results - no biopsies taken Liver parameters Tissue damage parameters Kidney parameters Pancreas parameters

2 1 Methodology 1.1 Animal experiments All primate studies were performed at the St. Kitts Biomedical Research Foundation, St. Kitts, West Indies. Thirty female drug-naive young adult African green monkeys, ranging in weight from 1.8 to 2.8 kg, were employed in the study. Baseline clinical exams including clinical chemistries were collected to confirm good health and suitability for study enrollment. African green monkeys were employed for size and homology considerations. Young females of this species are small (1.8-3 kg), requiring smaller amounts of test compound. The animals were assigned to six treatment groups of 5 females each and were dosed once daily on days 1, 3, and 5 by intravenous infusion over 10 min at a rate of 24 ml/kg/h. Treatment groups consisted of a vehicle control (phosphate buffered saline, PBS) and low, mid and high dose LNA-antimiR groups, all of which received liver biopsies, and a vehicle control and high dose LNA-antimiR group that did not receive liver biopsies to control for perturbations associated with this procedure. Allocation was determined by means of a stratified randomization procedure based on bodyweight and serum cholesterol levels. The animals were sedated with ketamine (7.5 mg/kg) and xylazine (1.5 mg/kg) prior to and during the dosing procedure and at all phlebotomy time points. For dosing a venous catheter was inserted into the saphenous vein. Group assignments, the dose for each group and the concentration of the formulations are indicated in Table 1. Dosing occurred in each monkey between 7:00 and 10:00 a.m. All visible signs of reaction to treatment were recorded on each day of dosing. In addition, the animals were examined at each blood sampling point. Bodyweights were recorded prior to randomization and initial dosing, and at weekly intervals. The humidity and temperature of the housing enclosure was monitored for the duration of the study. Blood samples were obtained for the biopsied animals prior to treatment and 24h post treatment via superficial venipuncture (i.e. study days 1 through 7). Additional blood samples for the four biopsied groups were collected on days 9, 11, 13, 15, 17, 19, 21, 23, 27, 32, 36, 42, 49, 56, 63, 70 and 96. Blood samples for the non-biopsied high dose and vehicle control groups were collected on day 1, 3, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 26, 29, 33, 37, 42, 52, 59, 66, 73 and 80. All samplings were performed prior to feeding after a period of 12 hours without access to food to minimize dietary effects on cholesterol measurements. 1.2 Plasma lipid and lipoprotein analysis Total plasma cholesterol was determined enzymatically in microtiter plates. Lipoprotein cholesterol distribution was determined by fast protein liquid chromatography (FPLC). Apo A-I, Apo A-II, Apo B, Apo E, and Apo Lp(a) apolipoproteins were determined by ELISA. All assays were performed by Dr. Martha Wilson at Wake Forest University. 1.3 Hematology and blood chemistry Prothrombin time, partial thromboplastin time (PTT), fibrinogen and platelet count were measured by Antech Diagnostics by optical and mechanical methodologies and automated cell counter. Glucose, blood urea nitrogen, creatinine, total protein, albumin, total 2 2

3 bilirubin, alkaline phosphatase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, calcium, phosphorus, sodium, potassium, chloride, a/g ratio, bun/creatine (calculated), globulins (calculated), lipase, amylase, triglycerides, creatine phosphokinase (CPK), lactate dehydrogenase, gamma glutamyl transferase (GGT), and magnesium were measured by Antech Diagnostics by an Hitachi 747 analysis system. 1.4 Liver biopsies A percutaneous liver biopsy was performed on the study monkeys one day and 90 days post LNA-antimiR treatment, respectively, employing an INRAD 14 gauge biopsy needle to obtain 2 core biopsies ( 1.5 cm in length) from both the right and left lobe of the liver (4 total biopsies). Half of each biopsy was immediately immersed in a labelled cryotube containing 2 mls of RNAlater (Qiagen) and incubated at 4 overnight, following which the RNAlater was aspirated and the sample tube flash frozen in liquid nitrogen. Following transportation in liquid nitrogen total RNA was isolated using the Trizol method. The remaining biopsy tissue was divided, one section fixed in paraformaldehyde for hematoxylin and eosin (HE) staining and one section cryopreserved for frozen section preparation for LNA-antimiR in situ analysis. Sectioning and mounting was performed by James Staruk at Mass Histology. 1.5 In situ hybridizations In situ detection of LNA-antimiR compound was performed on 10 µm frozen liver sections of LNA-antimiR treated and control monkeys. Slides were thawed, fixed in 4% paraformaldehyde for 10 min at room temperature and treated in acetic anhydride / triethanolamine followed by rinsing in PBS. Slides were pre-hybridized at 48 for 30 min in 50 % formamide, 5x SSC, 500 µg/ml yeast trna, 1x Denhardt s solution. LNA-antimiR was detected with a complementary FAM-labelled LNA probe hybridized to the liver sections for 30 minutes at 48 followed by three 10 min post-hybridization washes in 0.1x SSC at 52. After exposure for 10 min to 3% H 2 O 2, slides were preincubated for 15 min TN buffer (0.1 M Tris-HCl, ph 7.5, 0.15 M NaCl) containing 1% blocking reagent (TNB buffer, TSA kit; Perkin-Elmer) and subsequently with polyclonal rabbit anti-fluorescein isothiocyanate antibodies conjugated to HRP (diluted 1:500 in TNB; Dako) for 30 min. Slides were rinsed with TN buffer containing 0.3% Triton X-100, and incubated with Cyanine 3-Plus tyramide (diluted 1:100 in amplification buffer, Perkin-Elmer). The slides were rinsed and mounted in Vectashield containing DAPI (Vector Laboratories) and analysed on a Leica epifluorescence microscope equipped with a charge-coupled device camera (Leica Microsystems) and NIS-Elements software. 1.6 Statistical analysis For clinical chemistry, coagulation and apolipoproteins A-I and B the data are visualized by a set of three diagrams, showing different values as a function of time. ˆ Absolute value of the measurement in the relevant physical unit. The mean value from all animals in the group is plotted (n=5 with an average of 2.7% missing data 3 3

4 over all animals, time points and parameters). Both the PBS (black dashed) and high LNA-antimiR dosed (10 mg/kg, red) groups are shown. ˆ. At each point the log 2 -ratio between the untreated (PBS) animals and the 10 mg/kg LNA-antimiR group is calculated and the data are lowess smoothed. This display attempts to capture qualitative trends in the data, i.e. how does the treatment affect the parameter in question. The horizontal dotted line indicates no change between treated and untreated. ˆ. At each time point a two-tailed t-test was carried out testing the null hypothesis that the mean values of the PBS and 10 mg/kg group are equal. A horizontal dotted line indicates

5 Group Monkey Test Substance Concentration Dose Volume Dose Biopsy 1 R006 vehicle - 4 ml/kg - yes 1 R051 vehicle - 4 ml/kg - yes 1 R108 vehicle - 4 ml/kg - yes 1 R143 vehicle - 4 ml/kg - yes 1 X493 vehicle - 4 ml/kg - yes 2 R023 LNA-antimiR 0.25mg/ml 4 ml/kg 1 mg/kg yes 2 R171 LNA-antimiR 0.25mg/ml 4 ml/kg 1 mg/kg yes 2 X611 LNA-antimiR 0.25mg/ml 4 ml/kg 1 mg/kg yes 2 X783 LNA-antimiR 0.25mg/ml 4 ml/kg 1 mg/kg yes 2 X809 LNA-antimiR 0.25mg/ml 4 ml/kg 1 mg/kg yes 3 R010 LNA-antimiR 0.75mg/ml 4 ml/kg 3 mg/kg yes 3 R160 LNA-antimiR 0.75mg/ml 4 ml/kg 3 mg/kg yes 3 X398 LNA-antimiR 0.75mg/ml 4 ml/kg 3 mg/kg yes 3 X400 LNA-antimiR 0.75mg/ml 4 ml/kg 3 mg/kg yes 3 X464 LNA-antimiR 0.75mg/ml 4 ml/kg 3 mg/kg yes 4 R070 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg yes 4 R079 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg yes 4 X293 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg yes 4 X781 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg yes 4 X812 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg yes 5 X100 vehicle - 4 ml/kg - no 5 X838 vehicle - 4 ml/kg - no 5 X881 vehicle - 4 ml/kg - no 5 X915 vehicle - 4 ml/kg - no 5 X930 vehicle - 4 ml/kg - no 6 X450 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg no 6 X764 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg no 6 X791 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg no 6 X848 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg no 6 X922 LNA-antimiR 2.5mg/ml 4 ml/kg 10 mg/kg no Table 1: African green monkey group assignments and treatments

6 2 Results - biopsied animals 2.1 Total plasma cholesterol TPC (mg/dl) To visualize that the effect of the LNA treatment is dose dependent the next figure shows the trend plot for all dose levels

7 Total Plasma Cholesterol PBS baseline 1 mg/kg 3 mg/kg 10 mg/kg 2.2 Cholesterol distribution Total plasma cholesterol can be separated into different fractions: ˆ HDL - High density lipoproteins ˆ LDL - Low density lipoproteins ˆ VLDL - Very low density lipoproteins These fractions were separated using FPLC. The plot below shows amount of each fraction in the different groups

8 HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl) PBS 3 mg/kg 10 mg/kg It is evident that both LDL and HDL are reduced as a consequence of the treatment, however HDL is depressed slightly more than LDL. 2.3 Apolipoprotein profiles Apolipoproteins A-I and B are surrogate, but reliable markers for HDL and LDL and were measured by ELISA. ˆ Apo A-I is a constituent of HDL particles. ˆ Apo B is a constituent of LDL particles. ˆ The ratio between the two in each individual animal is used as marker to assess the balance of lipoproteins

9 Apo B Apo A I Apo A I / Apo B

10 Additional apolipoproteins were assayed at four time points and are shown in barplots (mean +/- sem) below. Shown are PBS (black), 1 mg/kg (green), 3 mg/kg (orange) and 10 mg/kg LNA-antimiR (red). Apo A II (AU) Apo E (AU) Apo Lp(a) (AU) Day Day Day 2.4 Glucose and triglycerides Triglycerides (mg/dl) Glucose (mg/dl)

11 2.5 Coagulation profile PTT (s) Prothrombin Time (s) Platelet Count (1E9) Fibrinogen (mg/dl)

12 2.6 Electrolytes Sodium (mmol/l) Potassium (mmol/l) Phosphorus (mg/dl)

13 Chloride (mmol/l) Calcium (mg/dl)

14 2.7 Liver parameters Albumin (g/dl) ALT (IU/L) AST (IU/L)

15 Alkaline Phosphatase (IU/L) Total Bilirubin (mg/dl) GGTP (IU/L)

16 2.8 Tissue damage parameters LDH (IU/L) CPK (IU/L)

17 2.9 Kidney parameters Creatinine (mg/dl) Urea Nitrogen (mg/dl) Total Protein (mg/dl)

18 BUN/Creatinine Ratio Albumin/Globulin Ratio Globulin

19 2.10 Pancreas parameters Amylase (IU/L) Lipase (IU/L)

20 2.11 Histopathology Photomicrographs (100x) of hematoxylin and eosin stained sections from liver biopsies. Biopsies were obtained 24 hours after the last administration of LNA-antimiR. Images from control monkeys are depicted in the left column. Images from 1 mg/kg LNA-antimiR treated animals are depicted in the 2nd column, images from the 3 mg/kg LNA-antimiR treated animals are depicted in the 3rd column, and images from the 10 mg/kg LNAantimiR treated animals are depicted in the 4th column. Images were framed to include hepatic portal triad architecture. No histopathological abnormalities beyond the limits of normal variation were appreciated in any animals by microscopy

21 2.12 In situ hybridization Sections were probed with a FAM-labelled LNA probe complementary to the LNAantimiR as decribed in the methods section. Images from control monkeys are depicted in the left column. Images from 1 mg/kg LNA-antimiR treated animals are depicted in the 2nd column, images from the 3 mg/kg LNA-antimiR treated animals are depicted in the 3rd column, and images from the 10 mg/kg LNA-antimiR treated animals are depicted in the 4th column. Although in situ hybridization is not a fully quantitative technique, it can be appreciated that higher dose level of LNA-antimiR correlates with stronger in situ signals

22 3 Results - no biopsies taken Some of the clinical biochemistry data presented above have transiently increased levels coinciding with the biopsy-taking at day 6. To control for this a second study was carried out without taking biopsies, addressing only the toxicological parameters. 3.1 Liver parameters Albumin (g/dl) ALT (IU/L) AST (IU/L)

23 Alkaline Phosphatase (IU/L) Total Bilirubin (mg/dl) GGTP (IU/L) Gamma-glutamyl transpeptidase (GTTP), a sensitive marker for liver stress, is elevated in the treated group, but is still within the normal human reference range of 0-51 IU/L

24 3.2 Tissue damage parameters LDH (IU/L) CPK (IU/L)

25 3.3 Kidney parameters Creatinine (mg/dl) Urea Nitrogen (mg/dl) Total Protein (mg/dl)

26 BUN/Creatinine Ratio Albumin/Globulin Ratio Globulin

27 3.4 Pancreas parameters Amylase (IU/L) Lipase (IU/L)