Abstract
BackgroundLentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression.ResultsWe compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes.ConclusionThe different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others.
Highlights
Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and nondividing cells
Primers and probe are directed against the U5 region of the 5' LTR and the 5' end of the gag gene, sequences that are present in all HXB2-derived lentiviral vector constructs [18] (Figure 1A)
For basic studies and eventually clinical trials, it is imperative that the performance characteristics and the variability inherent with these titration methods are known
Summary
Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and nondividing cells. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression. Since lentiviral vectors (LV) integrate stably into the host-cell genome of non-dividing cells such as neurons and in haematopoietic stem cells [1,2,3], they offer great potential for gene therapeutic applications [4]. Production of lentiviral vectors is routinely achieved by transient transfection of human embryonic kidney (293T) cells using high concentrations of the different plasmids, implicating the presence of residual plasmid DNA in the vector preparation, even after concentration. Transduction by lentiviral vectors (page number not for citation purposes)
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