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Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina
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AAV Serotype Testing on Cultured Human Donor Retinal Explants.

Thilo M Buck1, Lucie P Pellissier2,3, Rogier M Vos2,4

  • 1Department of Ophthalmology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

Methods in Molecular Biology (Clifton, N.J.)
|December 1, 2017
PubMed
Summary

This study presents a new method to test adeno-associated virus (AAV) infectivity in human retinal explants. The cost-effective protocol allows for detailed analysis of AAV tropism and gene expression in retinal cells ex vivo.

Keywords:
Adeno-Associated Virus (AAV)ExplantHumanNeural retina tissue cultureOrganotypicPhotoreceptorsPostmortemPotency assaysRetinal Müller glial cell

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Area of Science:

  • Ophthalmology
  • Virology
  • Molecular Biology

Background:

  • Adeno-associated viruses (AAVs) are promising vectors for gene therapy.
  • Understanding AAV tropism and infectivity in human retinal cells is crucial for developing effective ocular gene therapies.
  • Existing methods may not fully capture the complexity of AAV interactions within the human retina.

Purpose of the Study:

  • To establish and validate a cost-effective protocol for screening AAV serotype infectivity and tropism in human retinal explants.
  • To enable cell-type specific gene expression analysis using green fluorescent protein (GFP) vectors.
  • To facilitate ex vivo studies of AAV-mediated gene delivery and functional assays in human retinal tissue.

Main Methods:

  • Human donor eyes were enucleated, and retinas were isolated and sectioned into 6-mm explants.
  • Retinal explants were cultured ex vivo on specialized inserts, with photoreceptors oriented towards a membrane.
  • Adeno-associated viruses (AAVs) were applied to explants for transduction, followed by culturing in serum-free medium for up to 21 days.
  • Quality control measurements and histological staining were performed to assess transduction and cell viability.

Main Results:

  • The protocol successfully allowed for the transduction of human retinal explants by AAVs.
  • Cell-type specific or ubiquitous GFP expression was observed, indicating successful gene delivery.
  • The ex vivo culture system supported retinal explant viability and function for 21 days, enabling extended observation periods.
  • The method proved to be cost-effective and time-efficient for AAV screening.

Conclusions:

  • This protocol provides a robust and accessible method for evaluating AAV infectivity and tropism in human retinal explants.
  • The ex vivo system supports long-term culture, enabling comprehensive analysis of AAV-mediated gene expression and functional studies.
  • This approach can accelerate the development of AAV-based gene therapies for retinal diseases by allowing detailed preclinical assessment.