Although harnessing essential steps in the viral life cycle, these viral vectors separate the regulatory elements needed for transduction and transgene expression from the structural and enzymatic proteins needed for viral particle production. Although a range of platforms have been developed to deliver such genetic material to target cell populations, viral vectors are a particularly effective and versatile tool. The core tenet of gene therapy as a treatment modality is the ability to deliver and express a transgene capable of imparting therapeutic benefit. We examine the function of each region in a lentiviral vector, the molecular differences between vectors, and where optimization may guide development of the lentiviral delivery systems. Phylogenetic analysis of the cPPT/CTS indicated multiple sources, perhaps because of its later inclusion into lentiviral vector systems, whereas other regions revealed node clusters around the HIV-1 reference genomes HXB2 and NL4-3. The 3′ LTR was the most divergent sequence with a range of deletions. The Ψ signaling sequence demonstrated the greatest similarity between all vectors with only minor changes. All vectors included required elements: 5′ long terminal repeat (LTR) through the Ψ packaging signal, central polypurine tract/chain termination sequence (cPPT/CTS), Rev responsive element (RRE), and 3′ LTR, including a poly(A) signal.
With the use of representative vectors developed for clinical/commercial applications, we compared the vector backbone sequences to the initial sources of the HIV-1. Importantly, the coding regions of viral proteins were deleted, and the cis-acting regulatory elements were retained. These vectors incorporate features to provide long-term gene transfer and expression while minimizing generation of a replication-competent virus or pathogenicity. Three gene therapy strategies have received US Food and Drug Administration (FDA) approval one includes HIV-1-based lentiviral vectors. Gene Editing: Technology & Applications.This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS).
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