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ZZ Chen, S Wen, D Maneval, M Hess, J Nery, P Kaufman, R Nickells, B Faha; Biodistribution of an Adenovirus Encoding Human p21WAF1/Cip-1 (rAd-p21) Following Subconjunctival Injection in Rabbits . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3334.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Human p21WAF1/Cip-1 is a cell cycle inhibitor, investigated as a potential gene therapy approach to prevent wound healing after glaucoma surgery. rAd-p21 inhibits the proliferation of Tenon's fibroblasts in vitro and in vivo post subconjunctival injection and subsequent unguarded sclerectomy in rabbits. This study was to assess the systemic exposure and extent of transgene expression of rAd-p21 after subconjunctival injection in rabbits. Methods: 6 New Zealand white rabbits were injected unilaterally with 100µl of rAd-p21 (9.5x 1010 particles [p]) in the subconjunctival space. Animals were sacrificed d1 and d4 post injection (3/timepoint). Controls included 1 rabbit injected intravenously with 1ml of rAd-p21 (9.5 x 1011 p) and 1 rabbit injected in the subconjunctival space with excipient. Organs sampled for PCR and RT-PCR included the liver, kidney, spleen, heart, lung and ovary. Eyes from all animals were enucleated for PCR and RT-PCR. Total RNA and DNA were extracted from 100mg of each tissue using Tri-Reagent. rAd-p21 RNA and DNA were quantified using quantitative RT-PCR and PCR. Blood samples were collected from all animals before injection, and at 5m, 30m, 2h and 1d post injection. DNA was extracted from whole blood and analyzed by PCR for rAd-p21 DNA. Results: After subconjunctival injection, rAd-p21 DNA and RNA were detected in all rAd-p21 injected eyes (3/3 at d1 and 3/3 at d4) with similar levels (DNA ranging 1.6 x 107 ∼ 8.4 x 106 genome equivalents (GE)/mg-tissue, and RNA 1.8 x 107 ∼ 1.1 x 106 copies/mg), but not detected in any other organ except 1/3 spleen samples showing 1.6x 101 GE/mg DNA at d1. Similarly, rAd-p21 DNA was not detected in any blood samples except one at 30m. All tissues from the excipient-injected animal were negative. Interestingly, rAd-p21 DNA was detected in the contralateral eyes in 3/3 animals one day after injection at very low levels (only 0.002% of that from the injected eye). After intravenous injection, rAd-p21 DNA was present in all blood samples at all timepoints, ranging from 7.5 x 108 GE/ml at 5m to 1.0 x 106 GE/ml at 24h. At d1, samples from the liver, spleen, kidney, ovary, lung, heart and eye were tested positive for rAd-p21 RNA and DNA. Conclusion: Systemic exposure following subconjunctival injection of rAd-p21 is minimal and rAd-p21 transgene expression is stable in the eye. Thus, subconjunctival injection of adenovirus vectors may be a suitable delivery mode for ocular gene therapy.
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