raltegravir potassium Induced pluripotent stem cell iPSC tec

Induced pluripotent stem cell (iPSC) technology (Jin et al., 2012; Singh et al., 2013; Takahashi and Yamanaka, 2006; Tucker et al., 2013a,b, 2014a, 2015) makes it possible to derive cell types in vitro that would otherwise be inaccessible in living patients. This in turn allows one to evaluate the pathophysiology of novel genetic mutations in patients with rare inherited eye diseases (Sohn et al., 2015; Songstad et al., 2015; Tucker et al., 2013a, 2015). To study the disease mechanisms associated with hypomorphic genotypes of TRNT1, we used retinal organoids derived from the iPSCs of patients with TRNT1-associated RP. When compared to cells derived from an age matched individual with no history of ocular disease, the patient derived cells displayed a significant reduction in total TRNT1 protein, a truncated protein isoform, an autophagy defect and elevated levels of oxidative stress.
Materials and methods
Results
Discussion Although treatments capable of arresting RP progression are not currently available, gene augmentation strategies, such as those being developed for the treatment of RPE65-associated LCA (Maguire et al., 2008; Simonelli et al., 2010), (Maguire et al., 2009; Testa et al., 2013), hold great promise. However, more than 3000 different RP causing raltegravir potassium have been reported to date (Cooper and Krawczak, 1996; Daiger et al., 2013; Ferrari et al., 2011; Hamel, 2006, 2014). This extreme genetic heterogeneity, and the historical reliance on animal models to demonstrate a treatment effect, have both hindered the development of effective gene therapies. For many disease causing genes, especially those as rare as TRNT1, mammalian models suitable for testing novel therapeutics do not exist. Although, one could generate and evaluate a new rodent model for a single human retinal disease gene with a year or two of effort, there is no guarantee that the newly developed model would accurately recapitulate the human disease phenotype. For example, deletion of Ush2A, the gene most commonly associated with recessive RP in humans (Baux et al., 2007; McGee et al., 2010), does not cause photoreceptor cell death in mouse (Jacobson et al., 2008). If one could devise a system that could augment or replace animals in the therapeutic testing pipeline, one could dramatically reduce the cost and increase the speed with which new therapies could be evaluated. The advent of the iPSC makes it possible to generate retinal tissue from individual patients diagnosed with an inherited retinal degenerative disorder. As we and others have demonstrated, these cells can be used to determine if and how newly identified genetic mutations cause photoreceptor cell death (Jin et al., 2012; Tucker et al., 2015; Tucker et al., 2013b, 2011; Zhong et al., 2014). Disease-specific phenotypes identified during this process can in turn be used to test the efficacy of novel therapeutics (Giacalone et al., 2016; Parfitt et al., 2016; Wiley et al., 2016b; Wiley et al., 2015a). For instance, in a recent study, Parfitt and colleagues used iPSC-derived retinal organoids, generated from a patient homozygous for the common CEP290 IVS26 mutation, to demonstrate the ability of a novel antisense oligonucleotide to mitigate aberrant splicing and correct the associated disease phenotype (Parfitt et al., 2016). The goal of this study was to determine how recently identified mutations in TRNT1 (DeLuca et al., 2016), a gene previously implicated in the severe syndromic disorder SIFD (Chakraborty et al., 2014), cause photoreceptor cell death (Hull et al., 2016). To do so, iPSCs and retinal organoids were generated from 3 patients with molecularly confirmed TRNT1-associated RP. Compared to controls, patient-specific cells were found to have increased lipidation of LC3-1, production of LC3-II, and decreased LAMP-1 expression, which collectively suggest a defect in autophagy. The patient-specific cells were also found to have increased levels of oxidative stress, which we hypothesize to be the ultimate cause of photoreceptor cell injury and death in these patients. Under normal physiological conditions, autophagy is a cell protective pathway that plays an important role in degradation of dysfunctional proteins and discarded organelles (Boya et al., 2013; Ward et al., 2016). The autophagy pathway can function as a cellular defense mechanism, protecting cells from stress induced apoptosis (Li et al., 2015). Thus, it is not surprising that defects in the autophagy system have been linked to a number of neurodegenerative disorders including Alzheimer Disease, Huntington\'s Disease, Parkinson\'s Disease and glaucoma (Li et al., 2015; Metcalf et al., 2012; Navarro-Yepes et al., 2014; Tucker et al., 2014b; Wong and Cuervo, 2010). Age-dependent decreases in beclin-1, a critical regulator of autophagy and apoptosis, have been observed in affected brain regions of patients with Alzheimer and Huntington Disease (Pickford et al., 2008; Shibata et al., 2006). This reduction has been shown to cause decreased autophagic activity, aggregation of mutant proteins and neurodegeneration (Pickford et al., 2008; Shibata et al., 2006). In normal tension glaucoma, aberrant autophagy has been implicated as an important pathway in neural degeneration, with mutations in crucial regulators of autophagy associated with disease (Tucker et al., 2014b). For instance, duplication of TBK1 has been shown to cause over-activation of autophagy and increased expression of LC3-II, which ultimately induces apoptotic death of retinal ganglion cells (Tucker et al., 2014b).