September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Potency Assay for AAV Vector Encoding Retinal Pigment Epithelial 65 Protein
Author Affiliations & Notes
  • Linda B Couto
    Spark Therapeutics, Philadelphia, Pennsylvania, United States
  • George Buchlis
    Spark Therapeutics, Philadelphia, Pennsylvania, United States
  • Rafal Farjo
    EyeCRO, Oklahoma City, Oklahoma, United States
  • Katherine High
    Spark Therapeutics, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Linda Couto, Spark Therapeutics (E); George Buchlis, Spark Therapeutics (E); Rafal Farjo, EyeCRO (E); Katherine High, Spark Therapeutics (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 759. doi:
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      Linda B Couto, George Buchlis, Rafal Farjo, Katherine High; Potency Assay for AAV Vector Encoding Retinal Pigment Epithelial 65 Protein. Invest. Ophthalmol. Vis. Sci. 2016;57(12):759.

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

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Purpose : A validated potency assay for release of a medicinal product is required by the FDA prior to commercialization. SPK-RPE65 is a gene transfer vector being developed for the treatment of inherited retinal degeneration due to mutations in the retinal pigment epithelial 65 (RPE65) gene. In a Phase 3 trial, it demonstrated restoration of functional vision in subjects progressing toward blindness. We developed a non-radioactive in vitro assay to measure the activity of SPK-RPE65, in anticipation of potential commercialization of this product.

Methods : The RPE65 gene encodes an isomerohydrolase that converts all-trans-retinol to 11-cis-retinol, and is critical to the visual cycle. The potency assay is a modification of a radioactive assay (Moiseyev et al PNAS, 2005), and includes three components: (1) cell transduction by SPK-RPE65; (2) enzymatic assay measuring RPE65 activity; and (3) detection of the reaction product. In the first step, HEK293 cells constitutively expressing lecithin:retinol acyltransferase (LRAT) were infected with SPK-RPE65 and cell lysates were prepared. Cell lysates were then incubated with all-trans-retinol and a cofactor of the reaction, cellular retinaldehyde binding protein (CRALBP). In the third step, the product of the reaction, 11-cis-retinol, was detected by liquid chromatography/mass spectrometry (LC-MS/MS).

Results : The assay was analyzed to meet the following criteria: sensitivity, accuracy, and reproducibility. The biochemical assay performance was evaluated using a range of substrate and cofactor concentrations, different volumes, and in different solvents. The limit of quantitation of the assay is 1 nM 11-cis-retinol, and detection of 11-cis-retinol is linear over a range of 1-25 nM. Detection of 11-cis-retinol following transduction of cells with SPK-RPE65 at a multiplicity of infection (MOI) of 1x105 vector genomes/cell is reproducible.

Conclusions : A non-radioactive potency assay has been developed for SPK-RPE65. The assay is useful for release and stability testing of the vector. In addition, it is being used to test the activity of many mutant RPE65 genes in order to confirm and uncover disease-causing variants.

Reference: Moiseyev et al 102:12413-12418 (2005)

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.


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