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William A Beltran, Artur V Cideciyan, Raghavi Sudharsan, Michael Massengill, Brian Rossmiller, Valerie Liliane Dufour, felipe Pompeo Marinho, Tatyana Appelbaum, Malgorzata Swider, Mychajlo S. Kosyk, Simone Iwabe, William W Hauswirth, Samuel G Jacobson, Alfred S Lewin, Gustavo D Aguirre; AAV-mediated Knockdown and Replacement of Rhodopsin Protects Rods in a Canine Model of RHO-ADRP. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4483. doi: https://doi.org/.
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To develop an allele-independent corrective gene therapy approach for RHO-ADRP that combines the knockdown (KD) of both the mutant and WT RHO alleles and their replacement with a “resistant” codon-modified human RHO cDNA, and to test its efficiency in preventing rod loss in the light-sensitive canine T4R RHO mutant dog.
An shRNA (shRNA820) designed to target a homologous region of both human and dog RHO, and shown to efficiently KD RHO in 293T cells expressing GFP-tagged human RHO was packaged in an AAV2/5 vector under the H1 promoter (cloned as a self-complementary cassette). Four adult WT (RHO+/+) and 4 mutant (RHOT4R/+) dogs were each injected subretinally (volume: ~ 150 ml) with one of four titers ranging from 1 x 1011 to 1 x 1012 vg/ml. Seven to eight weeks post-injection, retinal integrity was assessed by cSLO/OCT imaging, and by histology /IHC on cryosections. Efficiency of RHO KD was measured both at the RNA and protein levels from retinal tissue punches collected both in the treated (vector bleb) and untreated areas. Following identification of the optimal therapeutic titer, an AAV2/5 construct containing both shRNA820 (under H1 promoter control), and a resistant codon-modified human RHO cDNA (RHO820) under control of a human opsin (HOP) promoter was tested in RHOT4R/+ dogs. Testing employed an acute light damage paradigm that rapidly assesses the effect of therapeutic intervention in preventing light-induced loss of rods in the T4R RHO mutant dog.
scAAV2/5-H1-shRNA820 achieved a KD efficiency that ranged between 5% and 100 % at the protein level and between 26% and 100 % at the RNA level with titers ranging between 1x1011 and 1x1012 vg/ml. Efficient (85-100 %) KD of RHO protein was achieved at 5 x 1011 vg/ml. Light-induced retinal degeneration in RHOT4R/+ dogs was prevented, yet was associated with a loss of rod OS and reduced RHO immunolabeling. However, treatment with the combination virus (scAAV2/5-HOP-RHO820-H1-shRNA820) at 5x1011 vg/ml preserved ONL thickness and rod OS integrity.
We have identified a very efficient KD reagent (shRNA820) able to suppress canine RHO expression when delivered with viral titers that appear to be safe. Its combination with the replacement (resistant human RHO cDNA) component results in protection from light-induced retinal degeneration and paves the way for a novel allele-independent gene therapy for RHO-ADRP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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