A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1Citation formats

  • External authors:
  • Michelle E. McClements
  • Cristina Martinez-Fernandez de la Camara
  • Maria M Patricio
  • Carolina Uggenti
  • Sumathi Sekaran
  • Alun R. Barnard
  • Robert E MacLaren

Standard

A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1. / Wood, Shaun; McClements, Michelle E.; Martinez-Fernandez de la Camara, Cristina; Patricio, Maria M; Uggenti, Carolina; Sekaran, Sumathi; Barnard, Alun R.; Manson, Forbes; MacLaren, Robert E.

In: Human Gene Therapy Methods, 2019.

Research output: Contribution to journalArticlepeer-review

Harvard

Wood, S, McClements, ME, Martinez-Fernandez de la Camara, C, Patricio, MM, Uggenti, C, Sekaran, S, Barnard, AR, Manson, F & MacLaren, RE 2019, 'A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1', Human Gene Therapy Methods. https://doi.org/10.1089/hgtb.2018.267

APA

Wood, S., McClements, M. E., Martinez-Fernandez de la Camara, C., Patricio, M. M., Uggenti, C., Sekaran, S., Barnard, A. R., Manson, F., & MacLaren, R. E. (2019). A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1. Human Gene Therapy Methods. https://doi.org/10.1089/hgtb.2018.267

Vancouver

Wood S, McClements ME, Martinez-Fernandez de la Camara C, Patricio MM, Uggenti C, Sekaran S et al. A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1. Human Gene Therapy Methods. 2019. https://doi.org/10.1089/hgtb.2018.267

Author

Wood, Shaun ; McClements, Michelle E. ; Martinez-Fernandez de la Camara, Cristina ; Patricio, Maria M ; Uggenti, Carolina ; Sekaran, Sumathi ; Barnard, Alun R. ; Manson, Forbes ; MacLaren, Robert E. / A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1. In: Human Gene Therapy Methods. 2019.

Bibtex

@article{d2e492d120ae4dedbc5f79e694e15a4d,
title = "A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1",
abstract = "Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as 'bestrophinopathies', for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localises to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, we present an assay that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, western blot and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increased in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product which will support a future application for regulatory approval.",
keywords = "Gene Therapy, Ophthalmology, AAV, Bestrophin-1, BEST1",
author = "Shaun Wood and McClements, {Michelle E.} and {Martinez-Fernandez de la Camara}, Cristina and Patricio, {Maria M} and Carolina Uggenti and Sumathi Sekaran and Barnard, {Alun R.} and Forbes Manson and MacLaren, {Robert E}",
year = "2019",
doi = "10.1089/hgtb.2018.267",
language = "English",
journal = "Human Gene Therapy. Methods",
issn = "1946-6536",
publisher = "Mary Ann Liebert Incorporated",

}

RIS

TY - JOUR

T1 - A quantitative chloride channel conductance assay for efficacy testing of AAV.BEST1

AU - Wood, Shaun

AU - McClements, Michelle E.

AU - Martinez-Fernandez de la Camara, Cristina

AU - Patricio, Maria M

AU - Uggenti, Carolina

AU - Sekaran, Sumathi

AU - Barnard, Alun R.

AU - Manson, Forbes

AU - MacLaren, Robert E

PY - 2019

Y1 - 2019

N2 - Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as 'bestrophinopathies', for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localises to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, we present an assay that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, western blot and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increased in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product which will support a future application for regulatory approval.

AB - Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as 'bestrophinopathies', for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localises to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, we present an assay that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, western blot and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increased in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product which will support a future application for regulatory approval.

KW - Gene Therapy

KW - Ophthalmology

KW - AAV

KW - Bestrophin-1

KW - BEST1

U2 - 10.1089/hgtb.2018.267

DO - 10.1089/hgtb.2018.267

M3 - Article

JO - Human Gene Therapy. Methods

JF - Human Gene Therapy. Methods

SN - 1946-6536

ER -