June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The human retinoblastoma WERI-Rb1 cell line as an in vitro model for photoreceptor-specific AAV transgene expression
Author Affiliations & Notes
  • Cristina Martinez-Fernandez de la Camar
    Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxfordshire, United Kingdom
  • Maria In�s Patrício
    Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxfordshire, United Kingdom
    John Radcliffe Hospital, Oxford Eye Hospital, Oxford University Hospitals NHS Trust, Oxford, Oxfordshire, United Kingdom
  • Michelle E. McClements
    Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxfordshire, United Kingdom
  • Alun R Barnard
    Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxfordshire, United Kingdom
    John Radcliffe Hospital, Oxford Eye Hospital, Oxford University Hospitals NHS Trust, Oxford, Oxfordshire, United Kingdom
  • Robert E MacLaren
    Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, Oxfordshire, United Kingdom
    John Radcliffe Hospital, Oxford Eye Hospital, Oxford University Hospitals NHS Trust, Oxford, Oxfordshire, United Kingdom
  • Footnotes
    Commercial Relationships   Cristina Martinez-Fernandez de la Camar, None; Maria Patrício, None; Michelle McClements, None; Alun Barnard, Nightstarx Ltd (C); Robert MacLaren, Nightstarx Ltd (C)
  • Footnotes
    Support  Nightstarx Ltd and the Royal College of Surgeons of Edinburgh
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1773. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Cristina Martinez-Fernandez de la Camar, Maria In�s Patrício, Michelle E. McClements, Alun R Barnard, Robert E MacLaren; The human retinoblastoma WERI-Rb1 cell line as an in vitro model for photoreceptor-specific AAV transgene expression. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1773.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : The development of vectors encoding therapeutic genes driven by human photoreceptor-specific promoters is of special interest to increase the efficacy and the safety of gene therapy treatment in photoreceptor degenerations, such as retinitis pigmentosa. At present, however, there is no regulatory approved in vitro model to confirm photoreceptor-specific transgene expression with adeno-associated viral (AAV) vectors. Hence the development of a robust and reproducible in vitro system would facilitate progress towards clinical trials.

Methods : In this study, we used the retinoblastoma-derived WERI-Rb1 cell line to establish an in vitro model for the expression of a green fluorescent protein (GFP) reporter gene driven by the human rhodopsin kinase (GRK1) or chicken β-actin (CAG) promoter. Recombinant AAV2/8 vectors were prepared using iodixanol gradients (rAAV2/8.CAG.GFP and rAAV2/8.GRK1.GFP). Since several proteins activated during cellular DNA replication and transcription have been reported to interfere with viral DNA replication, we used different enzymatic inhibitors to improve AAV transduction. GFP expression levels were monitored overtime using epifluorescence microscopy.

Results : Drug toxicity was first assessed using a MTT cell viability assay to find the highest non-toxic concentration. A range of multiplicities of infection (MOI) from 2,000 to 50,000 was tested to give the best transduction efficiency, having used rAAV2/8.CAG.GFP as a positive control. Epifluorescence microscopy analysis revealed that the inhibition of some of these proteins involved in DNA metabolism such as topoisomerases increases the GFP expression under the human photoreceptor-specific promoter GRK1.

Conclusions : The human GRK1 promoter delivered by the AAV2/8 vector can be assessed in human WERI cells. This study provides a helpful human in vitro model to test the expression of therapeutic genes specifically in photoreceptors using the human GRK1 promoter delivered by the AAV serotype 8 vector, thus making available a reliable and specie-specific preclinical model to validate therapeutic vectors.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×