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L. K. Buie, C. A. Rasmussen, R. J. Samulski, P. L. Kaufman, T. Borras; Self-Complementary Adeno-Associated Viruses (scAAV) Transduce Anterior Segment Tissues of Living Rats and Monkeys Without Detrimental Secondary Effects. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2050. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant AAV viruses produce stable expression and elicit low immune response. scAAV are modified AAV which bypass the required second strand DNA synthesis to achieve transcription of the transgene. In contrast to conventional AAV, scAAV are able to transduce human trabecular meshwork (TM) in cultured cells and perfused organ cultures. Our goal is to obtain optimal, non immunogenic delivery of transgenes to the TM in a living system. Our purpose was to investigate scAAV transgene transduction in living rodents and non human primates.
scAAV.GFP vector was prepared at the UNC vector core facility. Wistar rats received single dose intracamerally injection (6-8X109 viral particles, VP) of scAAV or vehicle. Intraocular pressures (IOP) (calibrated Tonometer and Tonopen) were taken at baseline and once a week thereafter. Animals were euthanized at 2, 5-7, and 9-10.5 wks post injection and their anterior segments processed for fluorescence histochemistry. Two cynomolgus monkeys received transcorneal injection of a 30 µl single dose (3X1010 VP). Follow-up examinations included IOP (Goldmann), slit lamp biomicroscopy, gonioscopy, fundus camera and microscope/cooled-CCD camera photography to detect GFP fluorescence.
Rats: scAAV injected eyes showed no clinical signs of inflammation or cataract formation. Whole mounts and cryosections showed intense GFP transduction to the iris, TM, and some corneal endothelium. Successful, GFP positive injections occurred in all eyes at 2 wks, in 13 out of 14 at 7 wks, and in 17 out of 18 at 2.5 months (last point tried). Delta-IOP values were not significantly different from baseline and/or from eyes injected with vehicle. Monkeys: At abstract submission, 2 wk post-injection, in vivo gonioscopic examination revealed strong fluorescence in the anterior ciliary muscle / iris processes / uveal meshwork, and in the pupillary iris sphincter in both animals. Slit lamp biomicroscopy revealed no visible inflammatory reaction in the scAAV treated eyes. IOP measurements remained similar between the eyes. Further follow-up over the next few months will be presented.
scAAV is the first adeno-associated vector shown to transduce the living animal iris and trabecular meshwork. Because of the long term, non immunogenic transgene expression history of AAV vectors in eye diseases of the retina, scAAV holds good promise for developing gene transfer/gene therapy regimens to the TM.
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