Abstract
Purpose: :
Mutations in the RP1 gene are a common cause of dominant retinitis pigmentosa. All pathologic mutations in RP1 found to date are nonsense or frameshift mutations, and are predicted to result in premature termination of translation in the RP1 protein. However, the pathogenesis of the RP1 form of RP is not yet understood. We have generated Rp1h-Q662X knockin mice to study the mechanism by which mutations in RP1 lead to photoreceptor cell death, ant to test potential therapies for RP1 disease using viral delivery systems.
Methods: :
The Rp1h-Q662X mice were generated using standard gene targeting techniques. The mouse Rp1h cDNA was cloned into adeno-associated virus (AAV) and trans-splicing AAV vectors, and AAV viruses generated using standard techniques. The full length mouse Rp1h gene was cloned into a helper dependent adenovirus (HD-Ad) vector, and adenoviruses generated using standard techniques. The viral vectors were delivered to photoreceptor cells in Rp1h-Q662X knockin mice by sub-retinal injection and the expression of full-length Rp1h protein was detected by anti-C-Rp1h antibody. The morphology of transduced photoreceptor cells was evaluated by confocal microscope and 3D imaging reconstruction.
Results: :
The homozygous Rp1hQ662X/Q662X mice develop a rapid retinal degeneration phenotype, with disorganized outer segments. In addition, a 661aa truncated Rp1h protein is detected in the retina of the knockin mice. Sub-retinal injection of the AAV 2/5-Rp1h and the trans-splicing AAV-Rp1h vectors in neonatal Rp1hQ662X/Q662X mice results in expression of full-length Rp1h protein, but in only rare photoreceptor cells. The transduction efficiency following injection of the HD-Ad-Rp1h vector is notably better, with many photoreceptor cells in the injected portions of the retina showing expression of the full-length Rp1h protein in their axonemes. Analysis of the outer segments of photoreceptor cells transduced with these vectors demonstrates that expression of the full-length Rp1h protein does not correct the defects in outer segment structure caused by the mutant Rp1h-Q662X protein.
Conclusions: :
These results indicate that haploinsufficiency is not the cause of RP1 disease. Rather, the truncated Rp1h protein produced in the retinas of the Rp1h-Q662X mice appears to have a toxic effect on photoreceptor OS structure, via either a gain-of-function or dominant-negative activity. Additional studies to confirm these results, and distinguish between the two dominant mechanisms of disease are in progress.
Keywords: retinal degenerations: hereditary • gene transfer/gene therapy • retinal degenerations: cell biology