Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy

Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy / A. Baoutina, S. Bhat, M. Zheng, L. Partis, M. Dobeson, I.E. Alexander, K.R. Emslie. - (Gene Therapy 23 (2016) 10 (October; p. 708-717)

  • PMID: 27439362
  • DOI: 10.1038/gt.2016.47


Abstract

There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.

Original document

Parameters

Science
Study
Date
7 June 2016
People
Alexander, I.E.
Baoutina, A.
Bhat, S.
Dobeson, M.
Emslie, K.R.
Partis, L.
Zheng, M.
Country
Australia
Language
English
Other organisations
Children's Hospital at Westmead
Children's Medical Research Institute (CMRI)
National Measurement Institute, Australia
University of Sydney (USyd)
Analytical aspects
Testing method development
Doping classes
M3. Gene And Cell Doping
S2. Peptide Hormones, Growth Factors
Substances
Erythropoietin (EPO)
Medical terms
Gene Therapy
Document category
Scientific article
Document type
Pdf file
Date generated
14 January 2021
Category
  • Legal Source
  • Education
  • Science
  • Statistics
  • History
Country & language
  • Country
  • Language
Other filters
  • ADRV
  • Legal Terms
  • Sport/IFs
  • Other organisations
  • Laboratories
  • Analytical aspects
  • Doping classes
  • Substances
  • Medical terms
  • Various
  • Version
  • Document category
  • Document type
Publication period
Origin