The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22–23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3′-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.
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The authors thank Dr Q Li and Dr A Verma for providing access to the fluorescent plate reader. Authors would also like to thank the Retinal Gene Therapy Vector Laboratory (University of Florida) for large-scale vector production. DML was funded through a Fulbright-Fight for Sight UK Research Scholarship (#1396) and is supported by intra-mural funding from the Medical College of Wisconsin. Additional funding was provided by NIH grant P30EY021721 and an unrestricted grant to the University of Florida department of Ophthalmology from Research to Prevent Blindness.
CAR, SLB, WWH and DML designed the study. CAR carried out all in vitro experiments and produced the figures. CAR and DML carried out in vivo experiments. CAR and DML prepared the manuscript with input from co-authors.
WWH and the University of Florida have a financial interest in the use of AAV therapies, and own equity in a company (AGTC) that might, in the future, commercialize some aspects of this work. The remaining authors declare no conflict of interest.
Supplementary Information accompanies this paper on Gene Therapy website
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Reid, C., Boye, S., Hauswirth, W. et al. miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity. Gene Ther 24, 462–469 (2017). https://doi.org/10.1038/gt.2017.50
Applied Microbiology and Biotechnology (2018)