1. Retina expression and cross-species validation of gene silencing by PF-655, a small interfering RNA against RTP801 for the treatment of ocular disease.
- Author
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Lee DU, Huang W, Rittenhouse KD, and Jessen B
- Subjects
- Animals, Cell Line, Transformed, Cornea metabolism, Eye Diseases metabolism, Gene Expression, Gene Silencing, Genetic Therapy methods, HEK293 Cells, Humans, RNA, Messenger genetics, RNA, Small Interfering administration & dosage, Rabbits, Transcription Factors biosynthesis, Transcription Factors deficiency, Transfection, Eye Diseases genetics, Eye Diseases therapy, RNA, Small Interfering genetics, Retina metabolism, Transcription Factors genetics
- Abstract
Purpose: PF-655, a synthetic 19-mer siRNA, targeting the RTP801 gene is currently in clinical trials for the treatment of wet age-related macular degeneration and diabetic macular edema. Preclinical studies have shown a dose-related suppression of RTP801 expression in rat disease models. Investigative studies were conducted with PF-655 to validate the Dutch-Belted rabbit as a biologically relevant species for gene silencing to support nonclinical ocular toxicity and continual dosing studies., Methods: Cross-species comparison and DNA sequencing was done to determine the level of homology between PF-655 and rabbit RTP801. Human (HEK 293) and rabbit (SIRC cornea) cell lines were stimulated with CoCl(2) to mimic hypoxic stress (an inducer of RTP801 expression) and treated with PF-655. Taqman-polymerase chain reaction and immunoblot analysis were performed to gauge RTP801 expression in cell culture and rabbit retinas., Results: Sequence analysis showed a 1-base mismatch in the PF-655 targeting site from genomic DNA of Dutch-Belted rabbit and the SIRC cell line, a cornea cell derived from the New Zealand White rabbit. HEK and SIRC CoCl(2)-stressed cells induced RTP801 expression 10-20-fold above control conditions. Treatment with 20 or 100 nM PF-655 showed a decrease in gene expression, 40%-50% relative to appropriate controls. RTP801 mRNA was detectable in primary rabbit retina tissues, with cycle threshold values showing a large linear range for the assay., Conclusion: These results support our investigation into cross-species validation of gene suppression by a therapeutic siRNA designed to a human gene. The SIRC cell line was utilized as a surrogate to test the degree of RTP801 gene silencing induced by PF-655 in vitro. With a 1-base mismatch, the level of silencing in a rabbit ocular cell line was comparable to that of a human cell line. Sequence analysis and expression data confirmed the relevance of the RTP801 target gene in rabbits and the utility of this species as a relevant animal model. Additionally, our work outlines a tractable method that validates relevant larger non-rodent species for ophthalmic drug testing.
- Published
- 2012
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