Faculty Collaboration Database

miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity. Gene Ther 2017 Aug;24(8):462-469

Date

06/16/2017

Pubmed ID

28617420

DOI

10.1038/gt.2017.50

Scopus ID

2-s2.0-85030779767 (requires institutional sign-in at Scopus site)   10 Citations

Abstract

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.

Author List

Reid CA, Boye SL, Hauswirth WW, Lipinski DM

Author

Daniel M. Lipinski PhD Associate Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Dependovirus
Gene Silencing
Gene Targeting
HEK293 Cells
Humans
Mice
Mice, Inbred C57BL
RNA, Small Interfering
RNAi Therapeutics
Transgenes
Virus Replication