For dominantly inherited disorders development of gene therapies, targeting the primary genetic lesion has been impeded by mutational heterogeneity. An example is rhodopsin-linked autosomal dominant retinitis pigmentosa with over 150 mutations in the rhodopsin gene. Validation of a mutation-independent suppression and replacement gene therapy for this disorder has been undertaken. The therapy provides a means of correcting the genetic defect in a mutation-independent manner thereby circumventing the mutational diversity. Separate adeno-associated virus (AAV) vectors were used to deliver an RNA interference (RNAi)-based rhodopsin suppressor and a codon-modified rhodopsin replacement gene resistant to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. Viruses were subretinally coinjected into P347S mice, a model of dominant rhodopsin-linked retinitis pigmentosa. Benefit in retinal function and structure detected by electroretinography (ERG) and histology, respectively, was observed for at least 5 months. Notably, the photoreceptor cell layer, absent in 5-month-old untreated retinas, contained 3–4 layers of nuclei, whereas photoreceptor ultrastructure, assessed by transmission electron microscopy (TEM) improved significantly. The study provides compelling evidence that codelivered suppression and replacement is beneficial, representing a significant step toward the clinic. Additionally, dual-vector delivery of combined therapeutics represents an exciting approach, which is potentially applicable to other inherited disorders.
Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset
Chemically-induced vascular toxicity during embryonic development may
cause a wide range of adverse effects. To identify putative vascular disrupting
chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA
ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals
for their potential to disrupt vascular development. Thirty-seven compounds were
selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP)
zebrafish embryos to identify chemicals that impair developmental angiogenesis.
We hypothesized that zebrafish angiogenesis toxicity data would correlate with
human cell-based and cell-free in vitro HTS ToxCast data.
Univariate statistical associations used to filter HTS data based on
correlations with zebrafish angiogenic inhibition in vivo
revealed 132 total significant associations, 33 of which were already captured
in the pVDC signature, and 689 non-significant assay associations. Correlated
assays were enriched in cytokine and extracellular matrix pathways. Taken
together, the findings indicate the utility of zebrafish assays to evaluate an
HTS-based predictive toxicity signature and also provide an experimental basis
for expansion of the pVDC signature with novel HTS assays.
Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3–5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.
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