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Chris Reid, Daniel M Lipinski; Riboswitch-mediated modulation of transgene expression following rAAV delivery to the mouse retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4088.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant adeno-associated virus (rAAV) vectors have emerged as promising tools for mediating gene therapy for diseases of the retina. Effectively controlling gene expression levels following vector delivery is paramount to the success of potential gene therapies, where uncontrolled over-expression of the therapeutic transgenes can lead to toxicity. Traditionally, inducible promoter systems have been employed. Unfortunately, due to the limited coding capacity of AAV, and the large size of elements required to make such systems work effectively, their inclusion is frequently not practical. Here, we evaluate the use of small (~100bp) ligand responsive self-cleavable riboswitches to modulate gene expression in the mouse retina.
Six riboswitches were evaluated in HEK293T cells to determine optimal copy number (largest dynamic range) and dose-responsiveness to its activating ligand using a dual luciferase assay. Next, the optimal copy number of each riboswitch was cloned into a rAAV GFP reporter cassette and packaged in an AAV2 capsid. Each GFP-riboswitch cassette was intravitreally injected into C57Bl/6j mice in combination with an AAV2 control vector harboring a non-inducible mCherry reporter gene. Four weeks post-injection, mCherry and GFP fluorescence levels were quantified in vivo using a custom confocal scanning laser ophthalmoscope (cSLO). Mice subsequently received a 1000mg/kg dose of its activating ligand, and fluorescence levels were quantified 2 and 24 hours post-gavage.
Cell culture experiments revealed significant changes in luminescence in response to dosing of the appropriate ligand (p<0.01). In vivo results demonstrated dosing mice with the activating ligand of each riboswitch could achieve a highly significant change in GFP fluorescence at 2 hours post-gavage compared to pre-treatment levels (p<0.01). Importantly, GFP fluorescence was recovered to pre-treatment levels 24 hours after dosing
All riboswitch constructs were capable of modulating transgene expression in vivo. Concurrent dual color cSLO imaging allowed riboswitch-mediated changes in GFP fluorescence to be normalized (to mCherry), effectively standardizing our in vivo measurements. Due to their small size and lack of immunogenicity, incorporation of riboswitches in rAAV constructs could have potential clinical applications where tightly controlled gene expression is paramount.
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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