Browsing Department of Animal Biology by Supervisor "Indumathi Mariappan"
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ItemDeveloping in vitro and in vivo models of retinal degeneration(University of Hyderabad, 2019) Praveen Joseph, S ; Indumathi MariappanRetinal degeneration (RD) is a progressive disorder wherein, there is a gradual loss of rod and cone cells leading to disease manifestations like night blindness, which progresses to peripheral vision loss and finally leading to loss of central vision and complete blindness. Pathogenic mutations in over 260 genes associated with retinal development, maintenance and function are linked to the disease. However, the underlying molecular mechanisms that result in retinal cell loss remain elusive for most of the reported gene mutations. Studies based on limited patient samples, retinal cell lines and knock out animal models has led to our current understanding of the disease. However, the diverse disease phenotypes and severities observed among patients are attributed to hundreds of different mutations reported within the same gene involved. Whole gene knockout animal models are therefore insufficient to understand such diverse retinal phenotypes. This study is aimed to establish relevant developmental model systems to evaluate the effect of patient-specific mutations in two of the RD conditions – Autosomal recessive retinal degeneration, ARRD and Leber congenital amaurosis, LCA12. The ARRD patientspecific iPSC line (KR) carrying mutation in ABCA4, showed abnormal eye field commitment with defective optic cup formation and the retinal progenitors exhibited preferential fate commitment towards RPE lineage. Also, the RPE cells derived from KR, displayed abnormal tight junctions and microvilli projections that resulted in severely altered epithelial barrier functions and ion transport functions. The LCA12 patient-specific iPSC line (VS) carrying a mutation in RD3, showed normal eye field commitment and gave rise to three-dimensional retinal organoids and RPE, upon retinal differentiation. While the RPE cells derived from VS showed no significant difference in their morphology and function when compared to the healthy control cells (F2), the retinal organoids indicated possible lamination defects. Similar lamination defects was also observed in the homozygous zebrafish mutant model of rd3 (rd3-/-), created in this study. The patient specific iPSC models thus generated can serve as useful tools, to understand the molecular mechanisms behind inherited retinal degenerative conditions; for potential drug screening applications and to carry out proof-of-concept studies on mutation correction. The zebrafish mutant models are ideal to understand disease manifestations at various time points, right from early stages of retinal degeneration to its progression into late and severe form of the disease, leading to complete blindness. Finally, a lentivirus-based vector system, encoding the RD3 promoter driven RD3 transgene cassette with a GFP reporter, was designed and constructed to evaluate its applications in future gene supplementation studies