Abstract
BackgroundProviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. Here, we present a method that can achieve this.ResultsProviral gene expression was detected by the activity of the puromycin resistance gene encoded in the viral vector, and quantified by comparing the growth curve of the sample under puromycin selection to that of a series of calibration cultures. Reproducible estimates of the efficiency of proviral gene expression could be derived. We confirm that contamination from unstable species such as passively transferred plasmid used in viral vector production and unintegrated viral DNA can seriously confound estimates of the efficiency of transduction. This can be overcome using a PCR based on limiting dilution analysis.ConclusionA simple, low cost method was developed that should be useful in studying the biology of retroviruses and for the development of expression systems for retrovirus based gene therapy.
Highlights
Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors
An human immunodeficiency virus (HIV)-1 vector (HVP) with a long terminal repeats (LTR) driven puromycin resistance reporter was utilised in this study
The provirus in HVP was inactivated by number of deletions: the viral gene pol was truncated, nef and part of env was deleted; nef being replaced by the puromycin resistance gene
Summary
Proviral gene expression is a critical step in the retroviral life cycle and an important determinant in the efficiency of retrovirus based gene therapy vectors. There is as yet no method described that can assess the efficiency of proviral gene expression while vigorously excluding the contribution from unstable species such as passively transferred plasmid and LTR circles. One of the features of the retroviral life cycle is integration, where the viral genome is incorporated into that of the host. A retrovirus can complete its life cycle only if gene expression from the provirus occurs. Gene therapy associated insertional oncogenesis in a clinical trial [2] and in experimental models of fetal gene transfer [3] highlighted the importance of assessing the expression efficiency of the therapeutic vectors employed. Information on the efficiency of retroviral vector expression can aid in determining the number of integrations necessary to produce a therapeutic effect, improving the accuracy of the risk assessment [4,5], and limiting the dosage of vector used [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
