Efficient gene transfer and expression of biologically active glial cell line-derived neurotrophic factor in rat motoneurons transduced wit lentiviral vectors

Several studies have shown the ability of human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors to infect nondividing brain and retinal neurons with high efficiency and long-term expression of the transduced gene. We show that purified embryonic motoneurons can be efficiently (>95%) transduced in culture using an HIV1-based lentiviral vector encoding LacZ. Expression of beta-galactosidase was observed for at least 9 days in these conditions. Furthermore, motoneurons transduced with a lentiviral vector expressing glial cell line-derived neurotrophic factor survived in the absence of additional trophic support, showing that the overexpressed protein was biologically active. Our results demonstrate the potential of lentiviral vectors in studying the biological effects of proteins expressed in motoneurons and in the development of future gene therapy for motoneuron diseases.     

In vivo manipulation of gene expression in non-human primates using lentiviral vectors as delivery vehicles

Non-human primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.     

Real-time in vivo bioluminescence imaging of lentiviral vector-mediated gene transfer in mouse testis

Although much research has focused on transferring exogenous genes into living mouse testis to investigate specific gene functions in spermatogenic, Sertoli, and Leydig cells, relatively little is known regarding real-time gene expression in vivo. In this study, we constructed a bicistronic lentiviral vector (LV) encoding firefly luciferase and enhanced green fluorescence protein (EGFP); this was a highly efficient in vivo gene transfer tool. After microinjecting LV into the seminiferous tubules the ICR mouse testis, we detected luciferase and EGFP expression in vivo and ex vivo in the injected tubules using bioluminescence imaging (BLI) with the IVIS-200 system and fibered confocal fluorescence microscopy (CellViZio), respectively. In addition, with an in vivo BLI system, luciferase expression in the testis was detected for ∼3 mo. Furthermore, EGFP expression in seminiferous tubules was confirmed in excised testes via three-dimensional fluorescent imaging with a confocal laser-scanning microscope. With immunostaining, EGFP expression was confirmed in several male germ cell types in the seminiferous tubules, as well as in Sertoli and Leydig cells. In conclusion, we demonstrated that real-time in vivo BLI analysis can be used to noninvasively (in vivo) monitor long-term luciferase expression in mouse testis, and we verified that EGFP expression is localized in seminiferous tubules after bicistronic LV-mediated gene transfer into mouse testes. Furthermore, we anticipate the future use of in vivo BLI technology for real-time study of specific genes involved in spermatogenesis.     

Supramolecular assemblies of zwitterionic nanoliposome-polynucleotide complexes as gene transfer vectors: Nanolipoplex formulation and in vitro characterisation

Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg²⁺ on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg²⁺ ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs.     

Functional in vivo gene transfer into the myofibers of adult skeletal muscle

The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.     

Inhibitory effect of locally produced and exogenous interleukin-6 on tumor growth in vivo

In order to define the potential antitumor activity of the multifunctional cytokine interleukin-6 (IL-6), retrovirus-mediated gene transfer was used to introduce and express a cDNA encoding human IL-6 in the murine fibrosarcoma cell line Fsa-R. Although these genetically modified tumor cells appeared morphologically and phenotypically identical to control Fsa-R cells and had a similar plating efficiency in vitro, they were found to exhibit greatly reduced tumorigenicity in vivo following intravenous injection into syngeneic recipients. Exogenous IL-6 was shown to produce a similar inhibition of tumor growth in the lung if administered intraperitoneally. In contrast, tumor growth in subcutaneous sites was inhibited only if the tumor cells were engineered to express IL-6 locally, or if IL-6 was administered intratumorally. Intraperitoneal injection of IL-6 had no inhibitory effect. Tumors that did grow from IL-6-producing tumor cell inocula in subcutaneous sites were found to contain large numbers of macrophages. These results demonstrate that the antitumor activity of systemically administered IL-6 varies depending on the site of tumor growth and suggest an important role for IL-6 in the recruitment, proliferation and/or survival of tumor-associated macrophages.Supported by grants from the B. C. Health Care Research Foundation (BCHRF), the National Cancer Institute (NCI) and the National Cancer Institute of Canada (NCIC). G.J.D. is a Research Scientist of the National Cancer Institute of Canada (NCIC). R.S.L. is a RSNA Research and Education Fund Scholar     

Direct chitosan-mediated gene delivery to the rabbit knee joints in vitro and in vivo

Chitosan vector system is expected to be useful for direct gene therapy for joint disease. This study first sought to confirm that foreign genes can be transferred to articular chondrocytes in primary culture. Next, chitosan-DNA nanoparticles containing IL-1Ra or IL-10 gene were injected directly into the knee joint cavities of osteoarthritis rabbits to clarify the in vivo transfer availability of the chitosan vectors. Clear expression of IL-1Ra was detected in the knee joint synovial fluid of the chitosan IL-1Ra-injected group. While no expression was detected in the chitosan IL-10-injected group, this demonstrates that the transfection efficiency of chitosan-DNA nanoparticles was closely related to the type of the gene product. A significant reduction was also noted in the severity of histologic cartilage lesions in the group that received the chitosan IL-1Ra injection. This avenue may therefore represent a promising future treatment for osteoarthritis.     

Adeno-associated viral gene transfer of dominant negative RhoA enhances erectile function in rats

We previously reported the inhibition of Rho-kinase to result in increased intracavernosal pressure (ICP) in an in vivo rat model of erection. Expression of an upstream activator of Rho-kinase, RhoA, has been demonstrated in the penile vasculature; however, the functional role of RhoA in the regulation of erection remains unknown. We used adeno-associated viral gene transfer of a dominant negative RhoA mutant (T19NRhoA) into rat cavernosum to test the hypothesis that RhoA activation is physiologically important for maintenance of the non-erect state and inhibition of this pathway leads to erection. Anesthetized, male, Sprague-Dawley rats transfected with the T19NRhoA mutant exhibited an elevated baseline ICP/mean arterial pressure (MAP) and nerve stimulation-induced ICP/MAP as compared with beta-galactosidase-transfected controls. The novel findings of this study demonstrate a functional role of RhoA in maintaining the flaccid penis and provide support for the inhibition of RhoA as a potential therapy for the enhancement of erectile function.     

Active component of Fatsia japonica enhances the transduction efficiency of Tat-SOD fusion protein both in vitro and in vivo

It has been reported that Tat-SOD can be directly transduced into mammalian cells and skin and acts as a potential therapeutic protein in various diseases. To isolate the compound that can enhance the transduction efficiency of Tat-SOD, we screened a number of natural products. 3-O-[beta-D-Glucopyranosyl(1-->4)-alpha-L-arabinopyranosyl]- hederagenin (OGAH) was identified as an active component of Fatsia japonica and is known as triterpenoid glycosides (hederagenin saponins). OGAH enhanced the transduction efficiencies of Tat-SOD into HeLa cells and mice skin. The enzymatic activities in the presence of OGAH were markedly increased in vitro and in vivo when compared with the controls. Although the mechanism is not fully understood, we suggest that OGAH, the active component of Fatsia japonica, might change the conformation of the membrane structure and it may be useful as an ingredient in antiaging cosmetics or as a stimulator of therapeutic proteins that can be used in various disorders related to reactive oxygen species (ROS).     

Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT

Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 ± 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was ∼10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.     

Differential effects of selegiline on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

The action of selegiline, a selective and irreversible inhibitor of monoamine oxidase B, commonly applied in the therapy of Parkinson's disease, on glucose formation was investigated in isolated rabbit hepatocytes and kidney-cortex tubules, maintaining the whole body glucose homeostasis via gluconeogenic pathway activity. An intensive hepatic metabolism of selegiline resulted in formation of selegiline-N-oxide, desmethylselegiline, methamphetamine and amphetamine, whereas during slow degradation of the drug in freshly isolated renal tubules selegiline-N-oxide was mainly produced. At 100 μM concentration selegiline markedly diminished glucose synthesis in isolated renal tubules incubated with dihydroxyacetone or alanine + glycerol + octanoate (by about 60 and 30%, respectively), while at 5 μM concentration a similar degree of inhibition was achieved in renal tubules grown in primary culture under the same conditions (about 40 and 60%, respectively). Moreover, desmethylselegiline and selegiline-N-oxide considerably diminished glucose production in renal tubules whereas selegiline and its metabolites did not affect gluconeogenesis in hepatocytes. Contrary to control animals, following selegiline administration to alloxan-diabetic rabbits for 8 days (10 mg kg⁻¹ body wt. daily) the blood glucose and serum creatinine levels were significantly diminished, suggesting a decrease in renal gluconeogenesis and improvement of kidney functions.

Since in renal tubules selegiline induced a decline in the intracellular levels of gluconeogenic intermediates and ATP content accompanied by a decrease in oxygen consumption in both kidney-cortex and hepatic mitochondria it seems possible that its inhibitory action on renal gluconeogenesis might result from an impairment of mitochondrial function, while an intensive selegiline metabolism in hepatocytes causes decrease of its concentration and in consequence no inhibition of gluconeogenesis. In view of these observations it is likely that an increased risk of selegiline-induced hypoglycemia might be expected particularly in patients exhibiting an impairment of liver function and following transdermal administration of this drug, i.e. under conditions of increased serum selegiline concentrations.     

Gene expression-based in vivo and in vitro prediction of liver toxicity allows compound selection at an early stage of drug development

We have analyzed gene expression and histopathology of rat liver treated with a histamine-3 receptor inverse agonist under development for the treatment of obesity 24 h after a single acute administration. While histopathology did not identify a clear liver toxicity, analysis of gene changes strongly suggested the development of toxicity. This prediction was confirmed in a 2-week repeat-dose rat study where prominent liver pathology occurred, while gene changes that lead to the prediction persisted. A subset of these genes was analyzed in vitro in both rat and human hepatocytes to reveal the potential relevancy of the findings for the situation in humans. This comprehensive analysis of the development compound at the gene expression level allowed interpretation of findings of the follow-up compound in a frontloaded 24-h single-dose acute study that was initiated before regular 2-week repeat-dose studies started. The high similarity of the follow-up compound to the lead compound based on gene expression lead to the immediate termination of the development program for this compound series. Our data demonstrate the value of genomics-based early toxicity prediction in short-term in vivo studies for the characterization of compounds to allow prioritization and selection of suited candidates before compound-, animal-, and cost-intensive longer term studies are undertaken.     

The biosynthesis of taurine fromN-acetyl-l-cysteine and other precursorsin vivo and in rat hepatocytes

Summary The synthesis of taurine fromN-acetylcysteine has been examined in ratsin vivo and in rat hepatocyte suspensionsin vitro. In ratsin vivo, administration ofN-acetylcysteine significantly increased urinary taurine (3 fold) 24h after dosing and liver glutathione levels. Liver taurine was not increased significantly. In hepatocytes incubated in the presence ofN-acetylcysteine, glutathione concentration increased to a maximum after 1 hour but the increase was not dependent on the concentration ofN-acetylcysteine. In contrast, after an initial lag phase, taurine synthesis increased in relation to the concentration ofN-acetylcysteine and continued for 3 hours. Glutathione synthesis seems to be preferential to taurine synthesis. Taurine synthesis from cysteine sulphinate was greater and from hypotaurine was greatest and maximal after 1 hour. Implications for the mechanism of protection byN-acetylcysteine are discussed.     

Enhanced gene delivery in vitro and in vivo by improved transferrin-lipoplexes

Cationic liposomes and the complexes they form with DNA (lipoplexes) constitute the most promising alternative to the use of viral vectors for gene therapy. One of the limitations to their application in vivo, however, is the inhibition of gene delivery by serum. In a previous study, we demonstrated that transferrin (Tf)-lipoplexes were superior to plain lipoplexes in transfecting HeLa cells in the presence of high concentrations of serum. With the goal of obtaining efficient gene expression in vivo, we evaluated the efficacy of Tf-lipoplexes (containing DOTAP and cholesterol) in transfecting primary hepatocytes and adipocytes in the presence of high serum concentrations. The association of transferrin with cationic liposomes increased luciferase expression compared to plain lipoplexes in primary cells as well as in HepG2 and 3T3-L1 differentiated adipocytes. The complexes were not cytotoxic and were highly effective in protecting DNA from attack by DNase I. An efficient and reliable method was developed to prepare lipoplexes containing both Tf and protamine sulfate, where the latter was mixed with transferrin, followed by the addition of cationic liposomes and DNA. The resulting protamine-Tf-lipoplexes increased significantly the levels of gene expression in cultured cells and in various tissues in mice following i.v. administration.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.