Poly-l-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model

Abstract

Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20 -40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr ؊/؊ hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.Previous studies have shown that aminoglycosides such as gentamicin and amikacin can suppress translation termination at disease-causing premature stop (nonsense) mutations and partially restore the expression of functional proteins in mammalian cells (for a review, see Ref. 1). In particular, gentamicin has been shown to suppress nonsense mutations and partially restore protein expression in mouse models of Duchenne muscular dystrophy (2) and cystic fibrosis (CF) 2 (3, 4). However, the use of aminoglycosides is commonly associated with serious side effects, including nephrotoxicity and ototoxicity (5, 6). The high dose of gentamicin (34 mg/kg) initially used to demonstrate readthrough in mouse models also resulted in serum concentrations that were far in excess of their maximum clinically recommended levels (2-4). More recently, we demonstrated that a lower dose of gentamicin (5 mg/kg) produced peak serum concentrations in a CF mouse model that were within the accepted clinical range for these compounds (4). Functional CFTR protein was produced under those conditions, as shown by immunofluorescence and short circuit current measurements. However, the level of suppression obtained was significantly less than was observed with higher doses. Consistent with the efficacy of these clinically relevant doses in mice, small clinical trials have suggested that g...

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

“…Other approaches to augment rescue of CFTR nonsense mutations, such as the coadministration of polyanions, such as poly-L-aspartic acid, or NMD inhibitors, such as NMDI-1, are other options that could be tested in the future (13,55). Furthermore, time-dependent increases in activity have been observed with prolonged administration of NB84 in a mouse model of Hurler's syndrome (38), a finding also observed in patients with CF administered ataluren in an open label trial (16), suggesting that enhanced readthrough with these agents may be possible with chronic administration in vivo.…”

“…For example, poly-L-aspartic acid, a compound previously shown to significantly reduce aminoglycoside toxicity, was found to increase both the level and duration of readthrough in a CFTR-G542X transgenic mouse (13). In a second approach, PTC Therapeutics, Inc. (South Plainfield, NJ) used high-throughput screening to identify the nonaminoglycoside readthrough compound, ataluren (also known as PTC124) (14).…”

“…Cftr knockout mice expressing a human CFTR-G542X transgene under intestine-specific rat fatty acid binding protein were treated by subcutaneous injection once daily for 14 days (6,13,15). Freshly mounted intestinal slices were stimulated with forskolin (10 mM) and IBMX (100 mM) in Ussing chambers using a partial chloride secretory gradient; glybenclamide was used to confirm specificity.…”

Renoprotective effects of the direct renin inhibitor aliskiren on gentamicin-induced nephrotoxicity in rats

“…GM concentrations were determined using a fluorescence polarization immunoassay. 13,14 Cell culture and application of GM and aliskiren to HK-2 cells An immortalized proximal tubule epithelial cell line from HK-2 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured. Briefly, cells were passaged every 3-4 days in 100-mm dishes (Falcon, Bedford, MA, USA) using Dulbecco's modified Eagle's medium-F12 (Sigma Chemical Co., St Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies Inc., Gaithersburg, MD, USA), insulin-transferrin-sodium selenite media supplement (Sigma Chemical Co., St Louis, MO, USA), 100 U/ml penicillin and 100 mg/ ml streptomycin (Sigma Chemical Co., St Louis, MO, USA).…”

Thérapie spécifique d’allèle

“…Le problème de la toxicité est d'autant plus aigu que les patients concernés sont en majorité des enfants dont le traitement devra être prolongé. Plusieurs approches sont actuellement envisagées afin de contrer ces effets secondaires, telles que l'administration d'antioxydants, qui permettent de réduire le niveau de radicaux libres [24], ou de polyanions, comme le poly-L-aspartate, qui semblent réduire les effets toxiques de la gentamicine [25]. [26] et les composés NB54, NB74 et NB84 [27,28].…”