Background We are testing several approaches, including gene therapy, small molecule screening for drug discovery, and retinal reconstruction. All strategies are guided by the knowledge of molecular mechanisms of photoreceptor development, homeostasis, and disease acquired through other projects (see EY000450-451-473-475).
 Results 1. Gene therapy In collaboration with Drs. P. Colosi and T. Li, we are focusing on the development of gene therapy strategies for the treatment of Leber congenital amaurosis (LCA) caused by CEP290 mutations and of retinitis pigmentosa (RP) caused by mutations in the RPGR, RP2 or other related genes (see EY000PETERS REPORT#). As a novel tool for gene therapy, we are testing AAV vectors containing 0.3 kb of Nrl promoter/enhancer that can specifically direct the target gene to both developing and mature rod photoreceptors (see EY000450-04) 2. Drug discovery We are developing reagents and protocols for high throughput screens of small molecules in collaboration with the NIH Chemical Genomics Center (NCGC), to discover new drugs to prevent photoreceptor degeneration. We are using zebrafish as animal model and developing in vitro biomaterial-based cell cultures. We also plan to use iPS cells to investigate the molecular mechanisms of retinal disease and for small molecule testing. Candidate molecules will be further investigated in preclinical studies using our well-characterized mouse models of retinal degeneration. 2.1. Small molecule screening in zebrafish A pilot screen in zebrafish is under way to identify small molecules that can affect cone photoreceptor development. We are assaying the NCI Diversity Set II library, consisting of about 2000 compounds, on zebrafish expressing GFP under the cone specific transducin a promoter (TαCP-GFP).
Our aim i s to screen the library on several retinal degeneration transgenic lines to identify small molecules that can halt or reverse degeneration. To this end, we are developing a transgenic line carrying a truncation in the cone specific cGMP phosphodiesterase gene in the TaCP-GFP background. In this line, cone degeneration is evident by 3 days post fertilization (dpf) and is complete by 6 pdf. By TILLING (Targeting Induced Local Lesions in Genomes) we have also generated mutations in zebrafish orthologs of human retinal diseased genes, including cep290, rp2, and rpgr. 2.2. Biomaterial-based in vitro cultures We have successfully encapsulated primary photoreceptor precursors from postnatal day (P)4 Nrl-GFP mice into the photopolymerizable hydrogel, poly ethylene glycol PEG. In addition, we have evaluated the effect of co-culture with RPE cells and of culturing the photoreceptor gel constructs in RPE conditioned medium. Viability as well as GFP expression within the 3-D matrix have been evaluated with the aid of 3-D confocal microscopy in cells maintained in 3-D matrices for up to 8 days. Improved viability and GFP expression have been noted in the presence of conditioned medium and in co-cultures with human fetal RPE. Subsequent studies will address the incorporation of an RPE-laden film into the hydrogel matrix, alongside the encapsulated photoreceptor precursors. These cultures will be used to develop assays for small molecule screening. 3. Cell-based therapies for retinal degenerative diseases 3.1 Use of iPS cells to investigate the molecular basis of disease and for developing therapies This year we have received support through the NIH Center for Regenerative Medicine (NCRM) Awards Program for a project on Therapies for early onset retinal degeneration using iPS cell technology.
Our aims are to generate iPS cell lines from rd16 mice and rdAc cats, two established animal models of CEP290-LCA, and from five patients with distinct CEP290 mutations. We will identify functional parameters of both the animal and human cell lines (including biomarkers, gene expression profiles, growth on biomaterials, differentiation to neurons, survival) that can be exploited for high-throughput screening of small molecules and for drug discovery. For this project, we have recruited two new postdocs with expertise in stem cell biology and in deriving retinal photoreceptors from pluripotent stem cells: Dr. K. Homma, from Dr. Takahashis laboratory in Japan and Dr. J. Cooke, from Dr. Dicks laboratory in the UK. We are using iPS control cells available from the NCRM to test their differentiation into photoreceptors in the presence of multiple fluorescent photoreceptor-specific gene reporters. Since the BAC system is not applicable to human iPS cells for stable gene expression, we have adopted the piggyback transposon methodology. We have generated several DNA constructs for multiple fluorescent labeling, which will allow us to tag newly formed retinal photoreceptors at specific stages of differentiation. We have also initiated the collection of fibroblasts from retinal degeneration patients to generate iPS cells to be used for disease modeling. 3.2 Retinal reconstruction We hypothesize that due to the unique hierarchical, layered structure of the mammalian retina, the position and orientation of the transplanted cells both during and after delivery is critical to optimal cell integration. Furthermore, in view of the distinct polarity that exists between photoreceptors and RPE, it is desirable to develop a biomaterials-based in vitro model that approximates the native anatomy and function. Finally, injectable biomaterials may provide structural support to the degenerating retina and therefore aid in vision restoration, particularly in patients with rapid rod degeneration. We are focusing on: assessing biomaterial candidates for their suitability as scaffolds for in vitro cell culture of photoreceptor progenitors and cell transplantation;and evaluating the use of injectable biomaterials for their ability to support the degenerating retina and contribute to vision restoration. Primary retinal cells isolated from Nrl-GFP mice at stages ranging from E11 to P10 have been cultured on biomaterial substrates including collagen vitrigel (from Dr. T. Takezawa) and silk fibroin films (from Dr. D. Kaplan) and evaluated for cell growth, viability, GFP and rhodopsin expression. Cultures of P4-derived cells demonstrate clustering behavior on collagen vitrigel, not observed in poly-D-lysine control plates. Furthermore, cells grown on collagen vitrigel exhibit greater cell viability and GFP expression compared to poly-D-lysine controls, while silk only and silk-RGD films do not support retinal cell growth and appear to promote cell death. Employing rd16 mice as a model system, we have injected liquid, photopolymerizable PEG into the subretinal space at various stages of development in order to assess the ability of the PEG to provide physical support to the degenerating retina. We have optimized surgical technique for the injection of a PEG solution, followed by sub-retinal in situ photopolymerization of the injected PEG. Furthermore, we have shown PEG to be histologically biocompatible within the subretinal space and have demonstrated that polymerized PEG persists within the retina for several days without toxic effects. Future experiments will focus on verification of biocompatibility, morphological assessments of retinas post-injection and electroretinography (ERG) to determine functional outcome.

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National Eye Institute (NEI)
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Swaroop, Manju; Brooks, Matthew J; Gieser, Linn et al. (2018) Patient iPSC-derived neural stem cells exhibit phenotypes in concordance with the clinical severity of Mucopolysaccharidosis I. Hum Mol Genet :
DiStefano, Tyler; Chen, Holly Yu; Panebianco, Christopher et al. (2018) Accelerated and Improved Differentiation of Retinal Organoids from Pluripotent Stem Cells in Rotating-Wall Vessel Bioreactors. Stem Cell Reports 10:300-313
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Shimada, Hiroko; Lu, Quanlong; Insinna-Kettenhofen, Christine et al. (2017) In Vitro Modeling Using Ciliopathy-Patient-Derived Cells Reveals Distinct Cilia Dysfunctions Caused by CEP290 Mutations. Cell Rep 20:384-396
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Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei et al. (2016) Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines. Invest Ophthalmol Vis Sci 57:ORSFl1-ORSFl11
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