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D. L. Simons, M. Abd-El-Barr, B. Appukuttan, E. Simmons, T. McFarland, W. W. Hauswirth, J. R. Lupski, P. J. Francis, S. M. Wu; Characterization of Retinal Degeneration in a Bardet-Biedl Syndrome Mouse Model and Development of a Gene Therapy Strategy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1745.
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We have previously shown that Bardet-Biedl Syndrome 4 (BBS4) knockout (-/-) mice experience severe retinal degeneration starting in the first month of life. Our current objectives are to characterize retinal function in +/- animals and develop viral gene therapy constructs for the prevention of photoreceptor death in this mouse model.
Dark-adapted mice underwent electroretinograms (ERGs) to assess rod and cone function. Retinal immunohistochemistry was performed to evaluate the integrity of intraflagellar transport in the photoreceptors. Two classes of gene therapy vectors were developed: a lentivirus (HIV-derived) and a pseudotyped adeno-associated virus (AAV2/5). Each class of virus contained one construct expressing only BBS4 and one construct expressing GFP. The GFP constructs of each class were either injected subretinally into wild-type mice or delivered into cultures of HEK293 cells as an initial validation of transgene expression. The BBS4 constructs were injected subretinally into P14 -/- mice and retinal function in these mice was followed longitudinally by ERG.
We found that the rod a-wave ERG response was much lower in -/- mice than in +/+ controls as early as 4 weeks of age. Additionally, the rod a-wave was lower in +/- mice compared to +/+ controls at all time points examined. This difference was relatively small at 4 and 8 weeks of age, but became more pronounced at 14 weeks of age (21% decrease, p < .05) and persisted at 28 weeks. The cone b-wave followed a similar course, with the +/- amplitude 44% lower than the +/+ controls at 14 weeks (p < .01). We also successfully developed two classes of viral vectors: the first class consists of HIV-CMV-mBBS4 and HIV-CMV-mBBS4-IRES-GFP constructs, while the second class consists of AAV2/5-MOP-hBBS4 and AAV2/5-MOP-GFP. Transgene expression was verified in vivo from AAV2/5-MOP-GFP and in vitro from HIV-CMV-mBBS4-IRES-GFP. Photoreceptor response to treatment with the BBS4-expressing vectors is currently under investigation.
Our results indicate that loss of just one BBS4 allele in mice causes a statistically significant decrease in both rod and cone function by 14 weeks of age. This raises the possibility that human carriers of BBS mutations may be predisposed to age-related macular degeneration. Our successful development of both lentiviral and AAV vectors carrying the BBS4 gene demonstrate the feasibility of rescuing photoreceptors from degeneration in this model of Bardet-Biedl Syndrome.
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