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Cristina Martinez-Fernandez dela Camara, Christopher I Cox, Pamela Whalley, Clare Blue, Robert E MacLaren; The accurate quantification of AAV genomic titre depends on the conformation of the plasmid reference. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4542. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant adeno-associated viral (AAV) vectors have shown safety and efficacy in delivering small transgenes to cells of all retinal layers and area currently being assessed in phase III clinical trials. The determination of the physical viral genome titer by qPCR is a critical step to ensure the product potency and safety. The aim of this study is to analyse the influence of the plasmid conformation used as standard and the enzymatic digestion during the sample preparation on the physical genome titration of a research grade (non-clinical) AAV8.RPGR vector.
To analyse the influence of the DNA standard conformation, two standard curves were prepared using the supercoiled plasmid and the linearized form. Seven serial dilutions of each plasmid standard (109 – 103 copies of plasmid DNA) were used to generate the standard curves. To extract the DNA from purified AAV vectors, two enzymatic methods were used: single digestion with DNase I and double digestion with an additional proteinase K treatment. QPCR was performed using primers and Taqman probe specific to the transgene sequence.
The use of supercoiled plasmid as a standard resulted in a higher apparent titre of AAV8.RPGR vector, compared to the use of linearized plasmid (p < 0.0001, Paired t-test). Based on the data generated, the absolute difference in the titre values was approximately one half log unit higher using supercoiled standard compared to the linearized standard. This is due to the difference in QPCR values of these control plasmids since the real titre of AAV.RPGR was identical for both samples. No significant difference (p=0.075, Paired t- test) in the titre was found between samples treated with DNase I and the same samples with the additional Proteinase K treatment.
The structure of the DNA reference plasmid influences absolute quantification by qPCR of the control sample, thereby generating different AAV8.RPGR titres. One possible reason for this discrepancy is that linearized DNA may be easier for primers to bind to compared with supercoiled plasmids. Since the DNA in AAV is also linear after extraction, the linearized plasmid reference may provide a more accurate titre of AAV. Ideally, all AAV publications should state which form of reference plasmid has been used to calculate titres, so that appropriate comparisons relating to dose toxicity and transduction efficacy can be made.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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