Engineering Baculo- and Lentiviral Vectors for Enhanced and Targeted Gene Delivery

One of the major goals of gene therapy is the development of vectors able to precisely deliver a gene of interest to specific cells or organs in vivo. In this study we aimed at introducing more efficient and targetable baculoand lentiviral vectors to the field of gene therapy. In addition, we studied the effects of baculovirus nuclear entry and viral gene transcription in human cells. In the first article we show that a 21amino acid EctoDomain in conjunction with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED), deprived of its tropism-mediating epitope, augments baculovirus-mediated gene-delivery to vertebrate cells in vitro and in vivo. We suggest that VSV-GED enhances baculovirus transduction by potentiating the membrane fusion activity of baculovirus envelope protein gp64. However, VSV-GED does not provide cell specificity and this is why other targeting strategies were sought. An ideal targeting strategy would use a general system eliminating the need to engineer new vectors for each new ligand. The use of (strept)avidin and its extraordinary tight interaction with biotin (Kd ~ 10-10M) could offer an effective approach. In the second article we developed a targeting strategy based on metabolical biotinylation of baculovirus vectors. This was achieved by displaying a small biotin acceptor peptide, BAP, fused either to different sites in the baculovirus glycoprotein gp64 or to VSV-GED. Transduction efficiencies of different contructs showed significant differences highlighting the importance in choosing the peptide insertion site. Only vectors displaying BAP inserted at amino acid position 283 of the gp64 protein showed improved transduction when targeted to cancer cell lines with biotinylated ligands or antibodies. These vectors could also be concentrated by streptavidin conjugated paramagnetic particles to reach titers up to 10 pfu/ml. For applications requiring long-term transgene expression development of targeted lentiviral vectors is of great importance. In the third article we constructed lentiviral vectors displaying avidin and streptavidin fused to VSV-GED, codisplayed with gp64. We present data on targeting of these lentivirus vectors to transferrin, epidermal growth factor and CD46 receptors overexpressed on tumor cells in vitro. Further, we demonstrate the capability of avidin-display in non-invasive imaging in vivo. The insect baculoviruses have the ability to transduce mammalian cell lines without replication. However, the baculovirus transduction can lead to the expression of some baculoviral immediate early genes in mammalian cells. In the last article we further studied the transcription and expression of viral immediate-early genes in human cells and examined the interactions between viral components and subnuclear structures. In conclusion, this work presents a simple means to enhance baculoviral gene transfer by VSVGED pseudotyping and gives the proof of principle of the utility of avidin-biotin display as a versatile tool for targeting baculoand lentivirus transduction. This conjugate-based strategy is readily adaptable for different targets in order to increase the gene delivery for ex vivo and in vivo applications. Finally, we elucidated the intranuclear events followed by baculovirus transduction in human cells. Together these results provide new insights into the future design of safer and more specific gene therapy vectors. National Library of Medicine Classification: QU 195, QU 350, QU 470, QU 475, QU 55.7, QW 162, QW 168.5. H6, QZ 52, WN 185, WN 203 Medical Subject Headings: Avidin; Baculoviridae; Biotin; Biotinylation; Cell Nucleus Structures; Cell Line; Cells, Cultured; Choroid Plexus; Gene Expression; Gene Targeting; Gene Therapy; Gene Transfer Techniques; Genetic Vectors; HIV-1; Lentivirus; Membrane Glycoproteins; Magnetic Resonance Imaging; Muscle, Skeletal; Rabbit; Rats; Receptor; Brain; Tomography, Emission-Computed, SinglePhoton; Transcription, Genetic; Vertebrates; Viral Envelope Proteins; Viral Fusion Proteins