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Chris Reid, William Hauswirth, Daniel M Lipinski; Micro-RNA induced silencing of cytotoxic transgenes leads to increased recombinant adeno-associated virus (AAV) titers. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
The production of high titer recombinant adeno-associated virus (AAV) vector is essential for the successful treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may substantially limit injectable volumes. Problematically, cytotoxicity arising from over-expression of the transgene during vector production frequently leads to a reduction in vector yield. This is particularly true of transgenes intended for ‘suicide’ gene therapy, such as herpes simplex virus thymidine kinase (HSVtk) or Bcl-2 associated X protein (BAX), which may be useful for the treatment of retinoblastoma. Herein, we evaluate the use of micro-RNA (miRNA) mediated silencing to effectively limit over-expression of cytotoxic transgenes during packaging as a method of increasing vector yield for ocular gene therapy applications.
Production of AAV serotype 2, 5 and 8 vectors packaging either a non-toxic fluorescent reporter (mCherry) or cytotoxic transgene (HSV-tk, ND4 or Bax) upstream of a quadruple repeated miRNA recognition site (mmu-miR-122-5p, mmu-miR-367-3p or hsa-miR-373-5p) was carried out in HEK293T cells by standard plasmid co-transfection. Transgene silencing was achieved during production by over-expression of the paired mature miRNA from either a separate expression plasmid or the adenoviral helper plasmid (pHelper-MIR). The titer (vg/ml) was compared to concurrently produced vector preparations without silencing by a Picogreen DNA assay.
Over-expression of paired mature miRNA during vector production led to a significant (p<0.01) increase in viral titer for all serotypes when packaging the cytotoxic HSV-tk, ND4 and BAX transgenes. Vector yield was not substantially increased when packaging the mCherry construct. Importantly, the addition of the miRNA recognition site to the transgene cassette did not affect the ability of the packaged transgene to be expressed in the rodent eye.
The significant increase in vector yield observed following over-expression of miRNA indicates that transgene silencing during production is an effective method of increasing vector yields for ocular gene therapy applications. This finding may have significant economic and logistical implications for the production of AAV vectors for research and clinical use.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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