ABSTRACT
Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes without inducing chronic toxicity. However, vector therapeutic index is narrow because of a toxic acute response with potentially lethal consequences elicited by high vector doses. Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) are major barriers to efficient hepatocyte transduction. We investigated two small peptides (PP1 and PP2) developed by phage display to block scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I), respectively, for enhancement of HDAd-mediated hepatocyte transduction efficiency. Pre-incubation of J774A.1 macrophages with either PP1 or PP2 prior to HDAd infection significantly reduced viral vector uptake. In vivo, fluorochrome-conjugated PP1 and PP2 injected intravenously into mice co-localized with both CD68 and CD31 on KCs and LSECs, respectively. Compared with saline pre-treated animals, intravenous injections of both peptides prior to the injection of an HDAd resulted in up to 3.7- and 2.9-fold increase of hepatic transgene expression with PP1 and PP2, respectively. In addition to greater hepatocyte transduction, compared with control saline injected mice, pre-treatment with either peptide resulted in no increased levels of serum interleukin-6, the major marker of adenoviral vector acute toxicity. In summary, we developed small peptides that significantly increase hepatocyte transduction efficacy and improve HDAd therapeutic index with potential for clinical applications.
Subject(s)
Adenoviridae/genetics , Hepatocytes/metabolism , Peptides/pharmacology , Scavenger Receptors, Class A/antagonists & inhibitors , Scavenger Receptors, Class F/antagonists & inhibitors , Transduction, Genetic , Amino Acid Sequence , Animals , Genetic Vectors , Helper Viruses/genetics , Hepatocytes/drug effects , Humans , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Peptide Library , Peptides/geneticsSubject(s)
Gene Expression Regulation, Developmental/genetics , Serotonin Plasma Membrane Transport Proteins/genetics , Animals , Brain/embryology , Brain/growth & development , Brain/metabolism , Embryonic Development , Heart/embryology , Heart/growth & development , Homeostasis , Homozygote , Mice , Mice, Knockout , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins/deficiencyABSTRACT
The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSHTSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pit(dw)pit(dw)) or of a functional TSHR (tshr(hyt)tshr(hyt) and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.