Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo Nonsense Suppression

<div><p>Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the <i>Idua</i> locus to test whether NMD attenuation can enhance PTC suppression <i>in vivo</i>. <i>Idua</i> encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing <i>Idua</i> transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy <i>in vivo</i>.</p></div

Caffeine and NMDI-1 attenuate NMD.

<p>A) HeLa cells expressing luciferase-based NMD reporters <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060478#pone.0060478-Boelz1" target="_blank">[32]</a> were used to monitor the effect of various drugs on NMD efficiency, which was expressed as the normalized N39X <i>Renilla</i> expression relative to WT ×100 (% WT <i>Renilla</i> activity). B) The effect of cycloheximide (CHX) (open circles, dashed line) and caffeine (closed circles, solid line) on NMD. C) The effect of ellipticine (open circles, dashed line) and NMDI-1 (closed circles, solid line) on NMD. The data shown are expressed as the mean +/− sd of a representative assay performed in triplicate.</p

Comparison of 2-week and 9-week co-administration of gentamicin with NMDI-1 on MPS I-H biochemical endpoints in <i>Idua</i>^W392X mouse brain.

<p>Comparison of 2-week and 9-week co-administration of gentamicin with NMDI-1 on MPS I-H biochemical endpoints in <i>Idua</i>^W392X mouse brain.</p

Improvements in MPS I-H biochemical markers are sustained in <i>Idua</i>^W392X mice co-administered gentamicin and NMDI-1 for 9-weeks.

<p><i>Idua</i>^W392X mice were administered gentamicin 3-times weekly for 9 weeks without (−) or with (+) co-administration of NMDI-1 or vehicle alone (v) two times weekly. MPS I-H biochemical markers were analyzed in brain tissue from treated <i>Idua</i>^W392X mice and controls. A) α-L-iduronidase (Idua) specific activity was determined in brain protein lysates. The data are expressed as the specific activity in the mutant mouse tissues relative to WT controls × 100 (% WT Idua Activity). Data are the mean +/− sd of values obtained from 5 mice per group, performed using ≥4 replicates (n = 5). <i>p</i> values above the columns compare treated mice to untreated controls, while the <i>p</i> values above the brackets compare mice treated with NMDI-1 to those without NMDI-1 treatment. B) Sulfated GAGs were quantitated in defatted, dried brain homogenates from WT mice (light gray bars) and from untreated and treated <i>Idua</i>^W392X mice (dark gray bars). GAG levels were quantitated as micrograms GAGs per milligram protein. The dashed line represents the WT GAG level as a reference. Data are expressed as mean +/− sd of 15–18 assays from 5 mice for each experimental group (n = 5). <i>p</i> values above the columns compare treated versus untreated W392X mice. <i>p</i> values above the brackets compare mice co-treated with both gentamicin and NMDI-1 compared to mice treated with gentamicin alone. C) β-hexosaminidase (β-hex) and β-glucuronidase (β-gluc) enzyme activities were determined in brain protein lysates. The data are expressed as the fold-elevation in enzyme specific activity measured in treated and untreated <i>Idua</i>^W392X mice relative to the level in WT mice, which was normalized to 1. The data are the mean +/− sd of the values obtained from 5 mice per treatment group performed with ≥4 replicates (n = 5). <i>p</i> values above the brackets compare mice administered NMDI-1 versus those without NMDI-1 administration within each treatment group.</p

NMDI-1 co-administration with gentamicin, but not NB84, enhances PTC suppression in <i>Idua</i>^W392X mice.

<p>Homozygous WT and <i>Idua</i>^W392X mice were administered readthrough (RT) drugs gentamicin (GENT) or NB84 for 14 days without (−) or with (+) NMDI-1 administration during the final 3 days of treatment. α-L-iduronidase (Idua) specific activity was determined in A) brain and D) spleen. The data are expressed as the specific activity in the mutant mouse tissues relative to WT controls × 100 (% WT Idua Activity). Data in A & D are the mean +/− sd of values obtained from 3–4 mice per group, performed using ≥8 replicates (n = 3 or 4). <i>p</i> values above the brackets compare mice treated with NMDI-1 to those without NMDI-1 treatment. B, C, E, F) Sulfated GAG levels were quantitated in brain and spleen from WT mice (white bars) and from untreated and treated <i>Idua</i>^W392X mice (gray bars). GAG levels were quantitated as micrograms GAGs per mg protein in B) brain and E) spleen from the gentamicin treatment group, or in C) brain and F) spleen from the NB84 treatment group. The dashed line represents the WT GAG level as a reference. Data in B, C, E, F are expressed as mean +/− sd of 15–18 assays from 5–6 mice for each experimental group (n =  5 or 6). <i>p</i> values above the columns compare treated versus untreated W392X mice. <i>p</i> values above the brackets compare mice co-treated with both RT drug and NMDI-1 compared to mice treated with RT drug alone.</p

NMD attenuators reduce NMD efficiency and enhance PTC suppression in <i>Idua</i>^W392X MEFs.

<p>MEFs were cultured +/− 7.5 mM caffeine for 4 hr or 5 μM NMDI-1 for 24 hr prior to RNA isolation. RT-qPCR was performed to quantitate NMD substrate steady state levels. A) Quantitation of <i>Idua, Atf4</i>, and <i>Gas5</i> mRNAs in treated relative to untreated <i>Idua</i>^W392X MEFs (indicated by the dashed line  = 1). B) Quantitation of <i>Idua</i>, <i>Atf4</i>, and <i>Gas5</i> mRNAs in treated relative to untreated WT MEFs (indicated by dashed line = 1). For A & B, results were normalized to 5S rRNA. Similar results were obtained when NMD substrates were normalized to 18S rRNA or <i>Rpl13a</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060478#pone.0060478.s003" target="_blank">Figure S3</a>). All data are the mean +/− sd of at least 3 independent experiments performed with ≥6 replicates (n = 3). <i>p</i> values above the columns compare treated to untreated MEFs. C) α-L-iduronidase activity in <i>Idua</i>^W392X MEFs cultured without readthrough (RT) drugs, with gentamicin (GENT), or with NB84 in the absence (−) or presence (+) of NMDI-1. The data shown are the mean +/− sd of 3 independent experiments performed in triplicate (n = 3). D) Sulfated GAGs were quantitated in <i>Idua</i>^W392X MEFs cultured without readthrough drugs, with gentamicin (GENT), or with NB84 in the absence (−) or presence (+) of NMDI-1. The dashed line represents the WT GAG level. The data shown are expressed as the mean +/− sd of 3 independent experiments performed at least in quadruplicate (n = 3). For C & D, <i>p</i> values above the brackets compare NMDI-1 treated cells versus untreated controls.</p

NMDI-1 increases the abundance of endogenous NMD substrates in <i>Idua</i>^W392X mice.

<p>Homozygous <i>Idua</i>^W392X mice were administered 5 mg/kg NMDI-1 for 3 days via once daily subcutaneous injections. After treatment, RNA was isolated from the brain, heart, and spleen and analyzed by RT-qPCR to determine NMD substrate steady state levels in NMDI-1 treated mice relative to untreated controls. The data shown are quantitation of the A) <i>Idua</i>, B) <i>Atf4</i>, and C) <i>Gas5</i> mRNAs normalized to 5S rRNA. Similar results were obtained when NMD substrates were normalized to 18S rRNA or <i>Rpl13a</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060478#pone.0060478.s004" target="_blank">Figure S4</a>). The data are expressed as the fold-change in RNA levels in <i>Idua</i>^W392X mice treated with NMDI-1 relative to untreated <i>Idua</i>^W392X mice (indicated by dashed line  = 1). All data are the mean +/− sd of values obtained from 3 mice per group, performed with ≥6 replicates (n = 3). <i>p</i> values above the columns compare treated to untreated mice.</p

NMDI-1 co-administration with gentamicin alleviates secondary MPS I-H lysosomal markers.

<p><i>Idua</i>^W392X mice were administered gentamicin (g) or NB84 (n) for 14 days without (−) or with (+) co-administration of NMDI-1 during the final 3 days of treatment. β-hexosaminidase (β-hex) and β-glucuronidase (β-gluc) enzyme activities were determined in tissue lysates from A) brain and B) spleen. The data are expressed as the fold-elevation in enzyme specific activity measured in treated and untreated <i>Idua</i>^W392X mice relative to the level in WT mice, which was normalized to 1. The data are the mean +/− sd of the values obtained from 3–4 mice per treatment group performed with ≥8 replicates (n =  3 or 4). <i>p</i> values above the brackets compare mice administered NMDI-1 versus those without NMDI-1 administration within each treatment group.</p