Alleviation of Trigeminal Nociception Using p75 Neurotrophin Receptor Targeted Lentiviral Interference Therapy.

Acute and chronic trigeminal (TG) neuropathies are the cause of considerable distress, with limited treatments available at present. Nociceptive neurons enriched with the vanilloid type 1 receptor (VR1) partake in pain sensation and sensitization in the TG system. While VR1 blockers with anti-nociceptive potential are of substantial medical interest, their use remains limited due to poor selectivity and lack of cell-targeting capabilities. This study describes a methodology for the alleviation of nociception via targeted depletion of VR1 in TG sensory neurons in rats. In cultured TG ganglion neurons, VR1 expression was virtually abolished by lentiviral short hairpin RNA (LV-VR1). By decorating GFP encoding LV (LV-GFP) and LV-VR1 with IgG192 for targeting TG sensory neurons enriched with the p75 neurotrophin receptor (p75NTR), transduction of a reporter GFP and VR1 depletion was achieved after injection of targeted vectors into the whisker pad. In IgG192/LV-VR1-injected rats, the behavioral response to capsaicin exposure as well as Erk 1/2 phosphorylation and VR1 current activation by capsaicin were significantly reduced. This pioneering investigation, thus, provides a proof of principle for a means of attenuating TG nociception, revealing therapeutic potential.

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses.

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

Natural stilbenoids isolated from grapevine exhibiting inhibitory effects against HIV-1 integrase and eukaryote MOS1 transposase in vitro activities.

Polynucleotidyl transferases are enzymes involved in several DNA mobility mechanisms in prokaryotes and eukaryotes. Some of them such as retroviral integrases are crucial for pathogenous processes and are therefore good candidates for therapeutic approaches. To identify new therapeutic compounds and new tools for investigating the common functional features of these proteins, we addressed the inhibition properties of natural stilbenoids deriving from resveratrol on two models: the HIV-1 integrase and the eukaryote MOS-1 transposase. Two resveratrol dimers, leachianol F and G, were isolated for the first time in Vitis along with fourteen known stilbenoids: E-resveratrol, E-piceid, E-pterostilbene, E-piceatannol, (+)-E-ε-viniferin, E-ε-viniferinglucoside, E-scirpusin A, quadragularin A, ampelopsin A, pallidol, E-miyabenol C, E-vitisin B, hopeaphenol, and isohopeaphenol and were purified from stalks of Vitis vinifera (Vitaceae), and moracin M from stem bark of Milliciaexelsa (Moraceae). These compounds were tested in in vitro and in vivo assays reproducing the activity of both enzymes. Several molecules presented significant inhibition on both systems. Some of the molecules were found to be active against both proteins while others were specific for one of the two models. Comparison of the differential effects of the molecules suggested that the compounds could target specific intermediate nucleocomplexes of the reactions. Additionally E-pterostilbene was found active on the early lentiviral replication steps in lentiviruses transduced cells. Consequently, in addition to representing new original lead compounds for further modelling of new active agents against HIV-1 integrase, these molecules could be good tools for identifying such reaction intermediates in DNA mobility processes.

Lentivirus-mediated α-melanocyte-stimulating hormone overexpression in the hypothalamus decreases diet induced obesity in mice.

Melanocyte stimulating hormone (MSH) derived from the pro-hormone pro-opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long-term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long-term anti-obesity effects of hypothalamic α-MSH overexpression in mice. An α-MSH lentivirus (LVi-α-MSH-EGFP) vector carrying the N'-terminal part of POMC and the α-MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi-α-MSH-EGFP or control LVi-EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high-fat diet. The effects of 6-week-treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14-day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi-α-MSH-EGFP treatment is dependent on decreased food intake. The 6-week LVi-α-MSH-EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi-EGFP treated mice. Food intake was decreased during the first week in the LVi-α-MSH-EGFP treated mice but subsequently increased to the level of LVi-EGFP treated mice. The LVi-EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi-α-MSH-EGFP treated mice. We demonstrate that gene transfer of α-MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α-MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site-specific effects of POMC peptides.

Retronectin enhances lentivirus-mediated gene delivery into hematopoietic progenitor cells.

Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.

Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia.

DYT1 is the most common inherited dystonia. Currently, there are no preventive or curative therapies for this dominantly inherited disease. DYT1 dystonia is caused by a common three-nucleotide deletion in the TOR1A gene that eliminates a glutamic acid residue from the protein torsinA. Recent studies suggest that torsinA carrying the disease-linked mutation, torsinA(DeltaE) acts through a dominant-negative effect by recruiting wild-type torsinA [torsinA(wt)] into oligomeric structures in the nuclear envelope. Therefore, suppressing torsinA(DeltaE) expression through RNA interference (RNAi) could restore the normal function of torsinA(wt), representing a potentially effective therapy regardless of the biological role of torsinA. Here, we have generated short hairpin RNAs (shRNAs) that mediate allele-specific suppression of torsinA(DeltaE) and rescue cells from its dominant-negative effect, restoring the normal distribution of torsinA(wt). In addition, delivery of this shRNA by a recombinant feline immunodeficiency virus effectively silenced torsinA(DeltaE) in a neural model of the disease. We further establish the feasibility of this viral-mediated RNAi approach by demonstrating significant suppression of endogenous torsinA in mammalian neurons. Finally, this silencing of torsinA is achieved without triggering an interferon response. These results support the potential use of viral-mediated RNAi as a therapy for DYT1 dystonia and establish the basis for preclinical testing in animal models of the disease.

Effects of recombinant interferon-tau on ovine lentivirus replication.

As a pregnancy recognition signal, sheep trophoblast cells secrete a type I interferon, ovine interferon-tau (OvIFN-tau), which has potent antiviral activity. We studied the effects of a recombinant protein (rOv-IFN-tau) on the replication of ovine lentivirus (OvLV) in goat synovial membrane cells. The amount of provirus DNA, as measured by polymerase chain reaction (PCR), the virus titers, and the number of OvLV-induced syncytia were 76.5%, 82%, and 95%, respectively, lower in cultures treated with rOv-IFN-tau than in placebo-treated controls (p < 0.01). rOv-IFN-tau also reduced OvLV reverse transcriptase activity and protected cells from OvLV-induced cell lysis, but the effect was less dramatic. The antiviral activity increased with the concentration up to a maximum with 256 antiviral units of rOv-IFN-tau per ml.