(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

SPECT/CT imaging of baculovirus biodistribution in rat.

Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.

Magnetic resonance imaging of viral particle biodistribution in vivo.

We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ventricles. No specific signal loss was detected when wild-type baculoviruses or intact biotinylated USPIO particles were injected into the lateral ventricles. Cryosectioned brains were stained for nuclear-targeted beta-galactosidase gene expression, which was found to colocalize with MRI contrast. This study provides the first proof of principle for robust and non-invasive viral vector MRI by using avidin-displaying viruses in vivo. Considering the widespread use of MRI in current medical imaging, the approach is likely to provide numerous future applications in imaging of therapeutic gene transfer.

Water deprivation and rat adrenal mRNAs for tyrosine hydroxylase and the norepinephrine transporter.

Experiments were performed in rats to test the hypothesis that adrenal mRNA levels of tyrosine hydroxylase (TH) and the norepinephrine transporter (NET) would be modified by water deprivation via activation of the sympathetic nervous system. TH and NET mRNA levels were measured using the ribonuclease protection assay. Adrenal TH mRNA was higher (P < 0.001) in water-deprived (921 +/- 39 fg/microgram total RNA) compared with the water-replete rats (657 +/- 45 fg/microgram total RNA). In contrast, water deprivation decreased (P < 0.01) adrenal NET mRNA levels (275 +/- 66 vs. 433 +/- 63 fg/microgram total RNA). The dehydration-induced increase in TH mRNA was prevented by prior splanchnicectomy, but the decrease in NET mRNA was produced even in the absence of adrenal nerves. Water deprivation also increased (P < 0.05) plasma adrenocorticotropic hormone (84 +/- 16 vs. 42 +/- 14 pg/ml) and corticosterone (358 +/- 87 vs. 44 +/- 15 ng/ml) levels. Interestingly, the corticosterone response was reduced (P < 0.05) by unilateral adrenal denervation. These results suggest that water deprivation increases both adrenal medullary and adrenocortical activity at least in part by stimulation of sympathetic nerve activity.