Retina expression and cross-species validation of gene silencing by PF-655, a small interfering RNA against RTP801 for the treatment of ocular disease.
OBJECTIVE
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.


CONCLUSIONS
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.
Drug Safety, Pfizer Global Research and Development, San Diego, California 92121, USA. dong.lee2@pfizer.com
Dong U
DU
Lee
Wenhu
W
Huang
Kay D
KD
Rittenhouse
Bart
B
Jessen
D000818:Animals; D002461:Cell Line, Transformed; D003315:Cornea / Q000378:metabolism; D005128:Eye Diseases / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D015870:Gene Expression; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D011817:Rabbits; D012160:Retina / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D014162:Transfection
C469991:DDIT4 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors