title: Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.
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.
authors.affiliation: National Measurement Institute, Lindfield, New South Wales, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Baoutina
authors.affiliation: National Measurement Institute, Lindfield, New South Wales, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Bhat
authors.affiliation: Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, New South Wales, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Zheng
authors.affiliation: National Measurement Institute, Lindfield, New South Wales, Australia.
authors.forename: L
authors.initials: L
authors.lastname: Partis
authors.affiliation: National Measurement Institute, Lindfield, New South Wales, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Dobeson
authors.affiliation: Gene Therapy Research Unit, The Children's Hospital at Westmead and Children's Medical Research Institute, Westmead, New South Wales, Australia.|Discipline of Child and Adolescent Health, The University of Sydney, New South Wales, Australia.
authors.forename: I E
authors.initials: IE
authors.lastname: Alexander
authors.affiliation: National Measurement Institute, Lindfield, New South Wales, Australia.
authors.forename: K R
authors.initials: KR
authors.lastname: Emslie
mesh_terms: D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy / Q000592:standards*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism / Q000592:standards*; D012015:Reference Standards; D019076:Transgenes
chemical_list: D004274:DNA, Recombinant; D012333:RNA, Messenger; D004921:Erythropoietin