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J. Liu, S.-H. Min, S. L. Boye, J. J. Alexander, M. Ding, S. Mani, V. A. Chiodo, A. S. Lewin, W. W. Hauswirth; Combined Nanoparticle DNA-rAAV Vector Gene Therapy for Treatment of Retinal Diseases. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4610.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a rapid onset but long-lasting gene therapy technique for the retina, we examined the time course of expression of subretinally delivered DNA nanoparticles (NP) and then tested them for therapy in the rapidly degenerating rd1 mouse either alone or combined with an rAAV vector.
Serotype 5 rAAV was prepared according to standard procedures in our lab. Two plasmids (pTR-CBA-GFP and pTR-CBA-mPDEß) were used for making two linear polyethylenimine-based NP DNA complexes, each at an N/P ratio of 6. To examine the onset of expression, a NP and or rAAV5 vector containing GFP cDNA was injected into eyes of adult C57BL/6 mice. At 2, 4, 7, 14, 21, 30, 60 and 90 days post-injection, eyes examined by GFP antibody staining of retinal whole mounts. In order to examine the therapeutic potential of NP-DNA either alone or combined with a rAAV5 vector, rd1 mice were intravitreally injected at P3-P7. ERGs were recorded and histology analysed at 3, 4, 5, and 6 weeks post-injection.
The effect of combined NP-DNA plus vector rAAV5 treatment was first examined by GFP expression in wild-type mouse retinas. By whole-mounted immunohistochemistry, combined NP-DNA plus rAAV5 GFP expression initiated at 2 days post-injection and persisted for longer than 90 days. GFP levels were significantly higher after 14 days than for treatment with NP-DNA alone. Before 14 days, GFP signal was very weak with rAAV5 alone. Therefore, combined NP-DNA plus rAAV5 vector gene delivery efficiently expresses passenger genes in the mouse retina from day 2 to day 90 or more. To further assess its therapeutic potential, the combined NP-DNA plus rAAV5 vector system was tested in the rd1 mouse after intravitreally injection at P3-P7. A CBA promoter regulating mouse PDEß was used for both the NP and vector. With combined treatment there was a 10%~50% rescue of the scotopic ERG at 3 and 4 weeks post-injection, but only a 5% ~ 15% of rescue with the NP DNA alone and no rescue for the rAAV5 vector alone. Histologic results showed 2~5 layers of outer nuclear layer preservation in treated eyes compared to one row in untreated partner control eyes.
Combined linear polyethylenimine-based nanoparticle DNA plus rAAV5 vector-mediated gene therapy for treatment of early onset retinal diseases was demonstrated to be effective in the rapidly degenerating rd1 mouse.
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