Aminoglycoside antibiotics remain first line therapy for gram negative infections and are used in conjunction with other antibiotics for certain gram positive organisms. Aminoglycosides, especially gentamicin, have also been used to suppress premature stop codon termination of protein synthesis leading to monogenic diseases like cystic fibrosis and Duchene's Muscular Dystrophy. Unfortunately, they still have an unacceptably high rate of dose and duration-dependent nephrotoxicity, especially in the elderly and in patients with chronic kidney, heart and liver disease. We have previously identified non-nephrotoxic, yet bactericidal, congeners of gentamicin that we now propose to use to determine their efficacy in suppressing premature termination codons (PTC). To do this we will use a human proximal tubule cell line with a novel FRET probe allowing for ratiometric quantitation of readthrough of different embedded stop codons. This will be followed by in vivo studies, again using the FRET probe, but now with 2-photon microscopy in kidney proximal tubule cells labeled by subcapsular delivery of specifically designed plasmids based on the cell culture data. To test the clinical potential of this approach we will study the mdx mouse and quantify dystropin synthesis in response to dose and duration dependent therapy. Finally, we will develop a transgenic mouse for the most appropriate stop codon, determined by the rat studies, and quantify readthrough in multiple tissues throughout the body. This is particularly important as many solid tumor cancers have monogenic premature termination codons that lead to malignant transformation of the cell type and these are also responsive to suppression therapy by aminoglycosides. We hypothesize that gentamicin congeners, with markedly reduced nephrotoxicity, will provide enhanced suppression of PTC leading to synthesis of functional proteins needed to reduce and/or prevent multiple PTC diseases. The nontoxic congeners will allow us to directly compare and contrast differences between toxic and nontoxic forms thereby leading to greater understanding of the factors mediating premature termination codon suppression in multiple cell types. These studies will provide the information necessary to enhance translation into clinical studies of multiple diseases.

Public Health Relevance

Aminoglycoside antibiotics remain the main line therapy for gram negative infections and are used in conjunction with other antibiotics for certain gram positive organisms. Aminoglycosides, especially gentamicin can help patients with monogenic diseases like Cystic Fibrosis and Duchene's Muscular Dystrophy (DMD) by allowing production of the necessary protein to reduce or prevent the disease. Unfortunately, aminoglycosides have an unacceptably high rate of causing acute and chronic kidney disease especially in the elderly and in patients with pre-existing kidney, heart and liver disorders. The present studies use a unique approach to determine the efficacy of nephrotoxic and non-nephrotoxic forms of gentamicin in cultured cells, rats and a transgenic mice form of a human disease (DMD). A specific imaging probe will allow for quantitative results that will enable and direct future therapeutic approaches in patients.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Nephrology (NEPH)
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Rlr VA Medical Center
Independent Hospitals
United States
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