July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
In vitro expression model for AAV2.VMD2.BEST1 gene therapy vectors
Author Affiliations & Notes
  • Cristina Martinez-Fernandez dela Camara
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom
  • Richard Truran
    Nightstar Therapeutics, United Kingdom
  • Gregory Robinson
    Nightstar Therapeutics, United Kingdom
  • Robert E MacLaren
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford University Hospitals NHS Trust, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   Cristina Martinez-Fernandez dela Camara, University of Oxford (E); Richard Truran, Nightstar Therapeutics (E); Gregory Robinson, Nightstar Therapeutics (E); Robert MacLaren, Nightstar Therapeutics (P), Nightstar Therapeutics (I), Nightstar Therapeutics (C), Nightstar Therapeutics (F), Spark Therapeutics Inc (C), University of Oxford (E), University of Oxford (P)
  • Footnotes
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Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3416. doi:
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      Cristina Martinez-Fernandez dela Camara, Richard Truran, Gregory Robinson, Robert E MacLaren; In vitro expression model for AAV2.VMD2.BEST1 gene therapy vectors. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3416.

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

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Purpose : Mutations in BEST1 have been found in association with clinically different retinal degenerations, termed bestrophinopathies. Autosomal recessive bestrophinopathy (ARB) is a good candidate for treatment using gene replacement therapy. To ensure the efficacy and the safety of the therapy, the development of adeno-associated viral (AAV) vectors comprising the normal BEST1 gene driven by a human retinal pigment epithelium (RPE)-specific promoter is of special interest. The purpose of this study was to develop an in vitro assay to analyse the expression of an AAV2.VMD2.BEST1 gene therapy vector as part of a potency assay required for clinical trials and the final product release.

Methods : Postconfluent cultures of ARPE19 were maintained in complete media for up to 4 months for epithelial differentiation. Recombinant AAV2/2 vectors containing BEST1 gene or the green fluorescent protein (GFP) reporter gene under the control of the VMD2 promoter, as well as the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) were prepared using iodixanol gradients. Differentiated ARPE19 cells were pre-treated with doxorubicin to improve AAV transduction with a range of multiplicities of infection (MOI) from 10,000 to 40,000. GFP expression levels were monitored overtime using epifluorescence microscopy. BEST1 expression was analysed by immunocytochemistry and western blot.

Results : Successful BEST1 expression was detected by immunocytochemistry in ARPE19 cells differentiated for just 1 week, and transfected or transduced with VMD2.BEST1 vectors. In plasmid-transfected cells, expression was detected as early as 2 days post-transfection; whilst in AAV-transduced cells, the peak of protein expression was observed at day 10 post-transduction. Doxorubicin significantly improved the AAV transduction efficiency in this cellular model. Correct molecular weight of BEST1 protein was confirmed by western blot in ARPE19 cells lysates previously transduced with AAV2.VMD2.BEST1.WPRE at 20,000 gp/cell.

Conclusions : The development and validation of AAV vector potency assay is critical for the translation from the research bench to clinical practice. In this study, for the first time, we demonstrate a suitable and biological relevant human in vitro model to test AAV2.VMD2.BEST1 gene therapy vectors, providing a useful tool to ensure the safety and quality of AAV-based biologic product for ARB.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.


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