Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system

Keeling, Kim M.; Bedwell, David M.

doi:10.1007/s00109-001-0317-z

Cited by 128 publications

(98 citation statements)

References 53 publications

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“…1B), as previously reported using various cell-based systems (23)(24)(25)(26). In preliminary experiments, the optimal gentamicin to PAA ratio was determined (data not shown).…”

Section: Treatment Of Cultured Cells With Paa and Gentamicinsupporting

confidence: 73%

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

Keeling

Fan

et al. 2009

Journal of Biological Chemistry

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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...

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“…1B), as previously reported using various cell-based systems (23)(24)(25)(26). In preliminary experiments, the optimal gentamicin to PAA ratio was determined (data not shown).…”

Section: Treatment Of Cultured Cells With Paa and Gentamicinsupporting

confidence: 73%

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

Keeling

Fan

et al. 2009

Journal of Biological Chemistry

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“…CFTR is a large, hydrophobic protein of generally low abundance, the expression of which is normally limited to epithelial cells. Because previous studies have shown that the local sequence context around a PTC acts as a primary determinant of readthrough efficiency (29,30), we inserted the CFTR-G542X mutation (TGA) and its local sequence context (three codons on either side) into a construct amenable to protein purification from HEK293 cells and mass spectrometry. This construct contained TGFP, the CFTR-G542X mutation and context, and C-terminal HA and 6× His tags (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Roy

Friesen

Tomizawa

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren’s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren’s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression.

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“…However, in several studies, no expression of full-length functional proteins was actually observed, suggesting no response to the aminoglycoside treatment (15,16,(21)(22)(23)(24)(25). This variability in response was proposed to be associated with the identity of the PTC and its sequence context (26)(27)(28), as well as with the chemical composition of the aminoglycoside, the duration of treatment, and the method of application.…”

Section: Introductionmentioning

confidence: 99%

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin

Linde¹,

Boelz²,

et al. 2007

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Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of fulllength proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

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Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system

Cited by 128 publications

References 53 publications

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

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

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin

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