Systemic aminoglycoside treatment in rodent models of retinitis pigmentosa
Introduction
Molecular genetic studies in patients with inherited retinal disease have identified a large number of the causative genes. Interestingly, a significant fraction is caused by premature termination codon (PTC) mutation (Daiger et al., 2007). In many instances these PTC mutations disrupt photoreceptor- or retina-specific genes (Sénéchal et al., 2006, Hong et al., 2004). However, there are also numerous examples of syndromic retinal degeneration (Bardet–Biedl syndrome, Usher syndrome, choroideremia) caused by PTC mutations in ubiquitously expressed genes (Leroy et al., 2001, Adato et al., 1997). Biochemical studies show that some PTC mutations generate a truncated protein (Hong et al., 2004, Friedman et al., 2006), whereas other PTC mutations result in a complete loss of the protein with a concomitant reduction in mRNA levels due to nonsense-mediated mRNA decay (Zhang et al., 2002, Frischmeyer and Dietz, 1999).
A large number of systemic diseases including cystic fibrosis, Duchenne muscular dystrophy, and β-thalassemia are also known to be caused by PTC. It is estimated that 12% of all mutations are single point mutations that result in a premature termination codon (Human Gene Mutation Database) (Krawczak et al., 2000, Cooper et al., 2007 in http://www.hgmd.cf.ac.uk/ac/index.php). A specific tabulation of PTC mutations for the identified retinal degeneration genes (RetNet, http://www.sph.uth.tmc.edu/Retnet/) have not been conducted, but it may be assumed that the proportion of retinal diseases caused by PTCs are similar to other systemic disease (12%). A small molecule therapy that targets these premature stop codons and converts these truncated proteins into full-length proteins could therefore treat a substantial portion of patients, making the approach practical and economical.
There are two classes of treatment under study for diseases resulting from PTC mutations: homologous replacement of the premature stop mutation with a wildtype sequence and delivery of pharmacological agents or suppressor tRNAs to reduce the efficiency of premature translation termination. Clearly, homologous replacement of the defective gene with a normal copy is the most elegant strategy to treat monogenic disease. However, to date the efficiency of homologous recombination methods in tissues has been quite low (Urnov et al., 2005, Moehle et al., 2007) with typical targeted integration frequencies of ∼15%. At these efficiencies, targeted gene addition is currently only appropriate for ex vivo gene therapy in isolated cells where selection of cells with successful homologous recombination can be performed. Homologous recombination at these low efficiencies is not currently efficacious as a therapy for complex tissues such as retina.
Pharmacologic treatments for PTCs are designed to reduce the premature translation termination and generate “read-through” to the genuine termination codon, thereby converting a potentially toxic, truncated protein into a full-length, functional protein. Aminoglycosides therapy often produces an increased amount of full-length functional protein, although generally a low percentage (Howard et al., 2000, Howard et al., 2004, Barton-Davis et al., 1999, Hein et al., 2004). This effect can result in a significant improvement in the disease phenotype, particularly in recessive disorders resulting from nonsense mutations in genes encoding an enzyme, where protein expression and activity is very low. Restoration of a small percentage of normal protein function may result in a clinically less severe or apparently normal phenotype (Wagner et al., 2001). Indeed, it has been primarily recessive disorders where cell culture and clinical experiments with aminoglycosides have given the most promising results.
Retinitis pigmentosa (RP) is a debilitating disease affecting significant numbers of patients worldwide. Currently, there is no cure and the few approved treatments (vitamin A supplements, cataract extraction, and acetazolamide for macular edema) are of very limited efficacy. Within the last decade a great deal of molecular genetic study has been undertaken and over 32 genes (http://www.sph.uth.tmc.edu/Retnet/) have now been identified to cause RP, making it the most genetically heterogeneous disease known in humans. Gene identification has allowed the subsequent development of a number of very useful transgenic and knockout animal models of RP. Unfortunately, the large degree of genetic heterogeneity raises significant problems with regard to developing new treatments. With a frequency in the population of 1:10,000, each identified gene corresponds to a relatively small number of affected patients relative to the cost of treatment development and initiation of clinical trials. Mutation-specific techniques, such as viral mediated gene augmentation therapy for recessive disease, and ribozyme or siRNA mediated knockdown for dominant diseases may fractionate the patient population to the point that they are financially unviable. Currently, techniques that are broadly applicable and to some extent mutation-independent, may be much more feasible in treating large numbers of patients.
Significant advances are being made in the study of many other inherited diseases and this can be extrapolated to retinitis pigmentosa research’. For instance, recently, aminoglycosides such as gentamicin have shown therapeutic benefit in nonsense mutation animal models of cystic fibrosis and Duchenne muscular dystrophy by causing read-through of premature stop codons (Du et al., 2002, Barton-Davis et al., 1999). This is most likely due to aminoglycoside interaction with ribosomes, reducing the stringency of codon–anticodon pairing (Mankin and Liebman, 1999). Successful in vitro proof of principle has been demonstrated in cell lines of Hurler's syndrome (Keeling et al., 2001), late-infantile neuronal ceroid lipofuscinosis (Sleat et al., 2001) and coagulation factor VII deficiency (Pinotti et al., 2006). Therapeutic efficacy has also been achieved in animal models of cystic fibrosis (Du et al., 2002), Duchenne muscular dystrophy (Barton-Davis et al., 1999) and nephrogenic diabetes insipidus (Sangkuhl et al., 2004). Other groups have failed to reproduce this success (Dunant et al., 2003), but nonetheless, the positive results have lead to human trials in cystic fibrosis (Linde et al., 2007, Wilschanski et al., 2003) and Duchenne muscular dystrophy (Wagner et al., 2001, Politano et al., 2003), which have shown therapeutic benefit in patients.
Systemic aminoglycoside therapy has been previously attempted in models of RP. Gentamicin treatment was tried in a human cell line (Grayson et al., 2002) and an animal model of RP (Pittler et al., 2001) with limited effectiveness. Despite limitations in these studies, the fact that the approach could be applicable to large numbers of RP patients with different stop mutations in different genes, prompted us to take up a more systematic assessment using disease models closer to human disease.
Section snippets
Rodent models of retinal degeneration
Two models of monogenic retinal disease caused by PTC mutations were tested, a rat model of dominant disease and a mouse recessive model. These animal models were selected because they exhibit a relatively slow rate of degeneration, which allows sufficient time for the effect of a systemic treatment to be assessed. Unlike many other rodent models, these slowly progressing degeneration models are more representative of the slow, progressive degeneration seen in human disease.
The S334ter
In vitro luciferase reporter assays
The efficacy of gentamicin-induced translational read-through (the ability of the drug to promote gene translation past a termination sequence or nonsense mutation) was tested on the mutant Rho S334ter construct in COS-7 cells. In an initial dose response experiment the highest level of read-through (6.1%, n = 6) was with a 50 μg/ml gentamicin dose (Fig. 1A). At higher doses, gentamicin was toxic to the cells. Using a 50 μg/ml dose we repeated the luciferase assays in three independent experiments
Discussion
Using systemically administered aminoglycosides, we have found a significant slowing of retinal degeneration in an animal model of retinitis pigmentosa caused by a premature stop mutation in rhodopsin. This finding is surprising given results in other studies (Grayson et al., 2002, Pittler et al., 2001) and may reflect our choice of model. The S334ter-4 rat exhibits a relatively slow progression of retinal degeneration in comparison to many other rodent models of disease, particularly the rd
Acknowledgements
This work was supported by the National Eye Institute Grant R01-EY013533; the Foundation Fighting Blindness, USA; and the British Retinitis Pigmentosa Society, Project Number GR550.
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The authors contributed equally to this work.