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Brian P Rossmiller, Haoyu Mao, Alfred S Lewin; Gene therapy With Self-complementary Recombinant Adeno-associated Virus in Models of Autosomal Dominant Retinitis Pigmentosa Caused by RHO Mutations. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5410.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa is the leading hereditary cause of blindness with 30-40% of cases attributable to autosomal dominant retinitis pigmentosa (ADRP). ADRP arises from mutations in at least 24 known genes with 30% arising in the rhodopsin gene (RHO). Given the large heterogeneity of mutations in RHO leading to ADRP, we propose knocking down of endogenous RHO and replacing it with a “hardened” copy, or a RHO with nucleotide changes that preserve the amino acid sequence but decrease the efficiency of knock-down. Here we report the use of a self-complementary Adeno-associated virus (scAAV) serotype 8 (Y733F) to express a hardened human rhodopsin (hRHO) under the control of the human opsin proximal promoter (HOPS) and an H1 promoter driven shRNA.
Four different knock-down methods were tested, ribozyme (Rz) 407, Rz525, miRNA 301 and shRNA 301 against both the wild-type and hardened RHO target regions in HEK293 cells. The reduction in expression of luciferase was measured at 24 and 48 hours post transfection.<br /> <br /> Mice were treated at postnatal day 5 or 15 using scAAV to deliver these RNA knockdown agents to mouse models of ADRP: Rho I307N and human RHO transgenic T17M and P23H. The I307N mouse model exhibits very slow degeneration under ambient light but is reduced in visual response to light by 50% in one week post exposure to 10,000 lux. Intravitreal injections were done using two constructs, hRHO+shRNA301 or hRHO+shRNA750 in one eye and a control AAV-HOPS-mCherry or sham injection in the other. The mice were followed using electroretinogram and optical coherence tomography.<br />
The knock-down results show shRNA301 and ribozyme 525 to cause the largest reduction of RHO mRNA. At one month post injection there was no statistically significant difference between T17M or P23H RHO eyes injected with AAV-hRHO-shRNA750 and the sham injected eyes.
We have generated a series of combination RNA knockdown and replacement AAV vectors that may be useful for the treatment of ADRP. At early time points, our tests of these specific vectors have not been conclusive. The injected mice will be followed for longer intervals and additional mice will be added to the study to determine if the difference in visual function of the experimentally treated eyes versus the control is statistically significant.<br />
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