Fig 1.
Effects of genetic background on hepatic accumulation of Malat1 ASO in the HMDP.
(A) 6-week-old male mice from 100 HMDP strains (n = 5/strain/treatment) were dosed with a single 2 mg/kg dose of either Malat1 ASO (ION 556089) or control ASO (ION 549144). Livers were harvested after 72 hours and PK analysis performed as described. Results are presented as mean ± S.E.M. (B) Correlation of ASO PK with average body weight (BW) in grams and (C) average liver weight (expressed as % BW).
Fig 2.
GWA results for hepatic Malat1 ASO concentrations in the HMDP.
Manhattan plot showing the–log10 of the association p-values (-logp) for hepatic accumulation of Malat1 ASO in 100 HMDP strains. Each chromosome is plotted on the x-axis in alternating light and dark colors. Genome-wide significance threshold line is shown in blue (-logp = 5.39).
Table 1.
List of Peak SNP results for hepatic Malat1 ASO accumulation in the HMDP.
Table 2.
List of genes within the linkage disequilibrium block for identified SNP rs32062485 in chromosome 4.
Table 3.
List of genes within the linkage disequilibrium block for identified SNP rs29364476 in chromosome 10.
Fig 3.
Systems genetics analysis and validation of Stab2 for influencing hepatic accumulation of the Malat1 ASO.
(A) Chromosome 10 plot for Stab2 displaying all significant SNPs (-log10 of p) for ASO uptake on Chromosome 10. Red arrow indicates position of gene. (B) Distribution of Malat1 ASO accumulation based on genotype associated SNP on chromosome 10 (rs29364476). Box and whisker plot depicting mean and distribution. (C) HMDP strains with lower hepatic Stab2 expression (S2 Fig) demonstrate lower hepatic ASO accumulation than those with higher Stab2 expression. Mean ± SEM, * p ≤ 0.05 unpaired t-test, Welch’s correction. (D) Hepatic accumulation of Malat1 ASO was assessed in Stab2-/- and WT mice 72 hours after single Malat1 ASO dose of 2 mg/kg. Hepatic concentration of ASO is significantly lower in livers of Stab2-/- mice as compared to WT mice. Mean ± SEM, * p ≤ 0.05 unpaired t-test. (E) Splenic accumulation of Malat1 ASO was assessed in Stab2-/- and WT mice 72 hours after single Malat1 ASO dose of 3mg/kg. Data shows mean ± S.E.M, * p≤ 0.05 ** p ≤ 0.01, unpaired t-test. (F) Expression of hepatic Malat1 mRNA by QPCR after treatment with Malat1 ASO compared to a control ASO. There is no significant difference between the Stab2+/+ and Stab-/- mice, unpaired t-test. Data shows mean ± S.E.M.
Fig 4.
Systems genetics analysis and validation of Vamp3 for influencing hepatic accumulation of the Malat1 ASO.
(A) Chromosome 4 plot for Vamp3 displaying all significant SNPs (-log10 of p) on Chromosome 4 for ASO uptake. Red arrow indicates position of gene. (B) Distribution of hepatic Malat1- ASO accumulation based on genotype distribution at peak SNP associated with ASO accumulation on chromosome 4 (rs32062485) data shows mean ± SEM, * p ≤ 0.05 unpaired t-test. (C) BXD strains with lower hepatic Vamp3 expression (S3A Fig) displays significantly lower hepatic accumulation of Malat1 ASO. Data shows mean ± S.E.M, * p ≤ 0.05, unpaired t-test. (D) MHT cells transduced with shRNA targeting either scrambled control (shCtrl) or Vamp3 (sh_Vamp3_1, sh_Vamp3_2) were treated with 250 ug Malat1 ASO for 24 hours, then assessed for ASO uptake via LCMS. Cells with reduced Vamp3 expression had significantly lower ASO uptake. Data shows mean ± S.E.M, *** p< 0.0001 with one way ANOVA, Dunnett’s multiple compairisons test.
Fig 5.
Large genetic variation in hepatic potency of the Malat1 ASO among male mice.
(A) Hepatic Malat1 expression in HMDP strains following a single dose of 2mg/kg of the Malat1 and control ASOs. Results are presented as mean ± S.E.M (% Control ASO). (B) Correlation of hepatic Malat1 expression with average body weight (BW) in gm and (C) Average liver weight (expressed as % BW) (D) Correlation of hepatic Malat1 knockdown and basal level of Malat1 expression in liver of the 100 strains (as compared to C57BL/6J).
Fig 6.
GWA results and validation for hepatic Malat1 ASO activity in the HMDP.
(A) Manhattan plot showing the–log10 of the association p-values (-logp) for hepatic expression of Malat1 mRNA in 100 HMDP strains. Each chromosome is plotted on the x-axis in alternating light and dark colors. Genome-wide significance threshold line is shown in blue (-logp = 5.39). (B) Distribution of hepatic Malat1 expression based on genotype distribution at peak SNP rs27459337. Data shows box-and-whisker plot showing mean and distribution, unpaired t-test, ** p≤0.01. (C) Distribution of basal hepatic Malat1 expression based on genotype distribution at rs27459337. Box-whisker-plot showing mean and distribution, unpaired t-test, n.s.
Table 4.
List of peak SNP results for hepatic Malat1 ASO activity in the HMDP.
Table 5.
List of genes within the linkage disequilibrium block for identified SNP rs29210579 in chromosome 12.
Table 6.
List of genes within the linkage disequilibrium block for identified SNP rs2912236 in chromosome 12.
Table 7.
List of genes within the linkage disequilibrium block for identified SNP rs27549337 in chromosome 4.
Fig 7.
Identification and validation of Rock2 in ASO activity.
(A) Distribution of hepatic Malat1 expression based on genotype distribution at rs29210579. Box and whisker plot depicting mean and distribution, unpaired t-test, * p≤0.05 (B) Distribution of basal hepatic Malat1 expression based on genotype distribution at rs29210579. Box and whisker plot depicting mean and distribution. (C) Chromosome 12 plot for Rock2 displaying all significant SNPs (-log10 of p) for ASO efficacy. Red arrow indicates position of gene. (D) MHT cells were incubated with Malat1 ASO and control ASO in presence and absence of 20 μM Y27632. After 24 hrs, Malat1 ASO potency was assessed in all samples. MHT cells exhibit lower reduction in Malat1 expression with Y27632 as compared to PBS. Data shows mean ± S.E.M (% control ASO) with two-way ANOVA and Bonferroni’s multiple comparison test. * p≤ 0.05.
Fig 8.
Identification and validation of Adi1 in ASO activity.
(A) Chromosome 8 plot for Adi1 displaying all significant SNPs (-log10p) for ASO efficacy on chromosome 8. Red arrow indicates position of gene (B) Plot of hepatic Adi1 expression and hepatic potency of 556089 shows differential Adi1 expression among the HMDP strains. Low expressing strains are indicated in grey. (C) HMDP strains with lower hepatic Adi1 expression show higher potency compared to strains with higher hepatic Adi1 expression. **** p ≤ 0.0001, unpaired t-test (D) Adi1 expression in MHT cells treated with either control siRNA or Adi1 siRNA. Data shows mean mean ± S.E.M *** p < 0.001, unpaired t-test (E) Plot of Malat ASO efficacy across indicated doses in MHT cells treated with Control siRNA or Adi1 siRNA. Data shows significant differences in ASO activity at 25 and 50nM Malat1 ASO, multiple t-tests using the Holm-Sidak method, with alpha = 0.05. * p ≤ 0.05.