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E. Feinstein, H. Ashush, M. E. Kleinman, M. Nozaki, H. Kalinski, I. Mett, J. Ambati; PF-04523655 (REDD14), an siRNA Compound Targeting RTP801, Penetrates Retinal Cells Producing Target Gene Knockdown and Avoiding TLR3 Activation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5693.
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PF-04523655 (REDD14), a synthetic chemically modified 19-mer siRNA molecule targeting RTP801, significantly inhibited laser-induced CNV in mice compared to control siRNA. This compound is currently in clinical trials of DME and AMD under the sponsorship of Pfizer, Inc. Our previous studies have shown that unmodified minimum 21-mer siRNA molecules do not enter retinal and choroid cells but rather exert an anti-angiogenic effect via stimulation of TLR3 (Kleinman et al., Nature 452:591-7, 2008). The objective of this study was to experimentally demonstrate that PF-04523655 enters its target cells in the back of the eye and does not activate TLR3 receptor.
: Fluorescent or non-labeled PF-04523655 was injected into mouse vitreous. Fluorescent siRNA in retinal and choroid cells was detected by FACS analysis using co-staining with cell-specific antibodies. Non-labeled siRNA was detected by in situ hybridization on eye sections. Knockdown of RTP801 mRNA was analyzed separately in neuronal retina and choroid using qPCR. Mouse and human TLR activation was analyzed in vitro in the reporter cells.
Fluorescent PF-04523655 was detected in RGC, RPE and retinal and choroid endothelial cells. In situ hybridization showed guide-strand-specific signal throughout the retina over numerous cell types with the most concentration in RGC. qPCR analysis revealed RTP801 KD in PF-04523655-injected eyes. Treatment of mouse and human TLR3 reporter cells with 1nM to 10 uM concentrations of PF-04523655, either naked or lipofectamine formulated, did not elicit reporter activation. Similar results were obtained in mouse and human TLR7 and TLR9 reporter systems.
We concluded that PF-04523655, unlike some other siRNA molecules, enters target cells in the back of the eye and elicits its pharmacological effect via target gene knock-down (KD) and not via TLR3 activation.
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