June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Improved packaging efficiency for ocular applications of AAV vectors through inclusion of miRNA recognition sequences
Author Affiliations & Notes
  • Chris Andrew Reid
    Ophthalmology, University of Florida, Gainesville, FL
  • William W Hauswirth
    Ophthalmology, University of Florida, Gainesville, FL
  • Daniel M Lipinski
    Ophthalmology, University of Florida, Gainesville, FL
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships Chris Reid, None; William Hauswirth, AGTC (C), AGTC (P), Bionic (I); Daniel Lipinski, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 3635. doi:
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      Chris Andrew Reid, William W Hauswirth, Daniel M Lipinski; Improved packaging efficiency for ocular applications of AAV vectors through inclusion of miRNA recognition sequences . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3635.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Adeno-associated virus (AAV) mediated ocular gene therapy is a promising therapeutic option for the treatment of degenerative retinal diseases. Currently, the inability to efficiently package some transgenes that are toxic to host cells during viral production, such as aquaporin and NADPH dehydrogenase subunit 4 (ND4), represents a significant barrier to the clinical translation of therapies for Leber’s hereditary optic atrophy and glaucoma. Here, we evaluate the ability to package toxic transgenes through exploitation of miRNA-based gene silencing during production.

Methods: Selected miRNAs (mmu-MiR122, mmu-MiR216, mmu-MiR367 and hsa-MiR373) with flanking genomic sequence (600bp max) were cloned into a ubiquitous expression plasmid containing an EGFP reporter (pCMV); miRNA expression levels for each construct were confirmed by qPCR/miRNA capture following transient transfection in HEK293T cells. Quadruplet repeat target sequences for each miRNA were cloned into an AAV2.mCherry expression vector (pTR-UF11) and silencing efficiency of each miRNA pair was quantified both by flow cytometry (mCherry reporter) and dual-luciferase reporter assay. Stable cell lines were generated for each miRNA expression plasmid to enable gene silencing during AAV packaging; multiple batches of ND4 and aquaporin vectors containing the most effectively silenced miRNA target sequence were produced to confirm efficient packaging using the miRNA system.

Results: High-level expression of miRNAs in HEK293T stable packaging cell lines was not observed to have any adverse impact on cell survival or growth. The efficiency with which transgenes with known toxicity (ND4/aquaporin) could be packaged using stable miRNA expressing packaging cell lines was observed to be consistently higher than when packaged using unmodified HEK293T.

Conclusions: Transgene silencing during AAV vector production may allow the packaging of transgenes that display substantial toxicity in host HEK293T cells more effectively than currently available packaging technologies, potentially allowing more efficient production of AAV vector for future clinical applications.


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