Vivo Gene Research Articles

STAT3 Targets the Regulatory Regions of Gluconeogenic Genes in Vivo

The regulation of expression of gluconeogenic genes including glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) in the liver plays an important role in glucose homeostasis, because aberrant expression of these genes contributes to the development of type 2 diabetes. Previous reports demonstrate that signal transducer and activator of transcription 3 (STAT3) plays a key role in regulating gluconeogenic gene expression, but the mechanism remains unclear. Herein we demonstrate that phosphorylated STAT3 is required for repression of G6Pase expression by IL-6 in both HepG2 cells and mouse liver. Interestingly, PEPCK expression is regulated by STAT3 independent of IL-6 activation. Using in vivo chromatin immunoprecipitation, we demonstrate that STAT3 binds to the promoters of the G6Pase, PEPCK, and suppressor of cytokine signaling (SOCS)3 genes, and its recruitment increases at the G6Pase and SOCS3 promoters with IL-6 treatment. Whereas persistent recruitment of RNA polymerase II is seen on the SOCS3 promoter, consistent with its induction by IL-6, a decrease in polymerase II recruitment and histone H4 acetylation is seen at the G6Pase promoter with IL-6 treatment. Thus STAT3 mediates negative regulation of hepatic gluconeogenic gene expression in vivo by interacting with regulatory regions of these genes.

Endothelin-1 Inhibits Thick Ascending Limb Transport via Akt-stimulated Nitric Oxide Production

Endothelin-1 inhibits sodium reabsorption in the thick ascending limb (THAL) via stimulation of nitric oxide (NO) production. The mechanism whereby endothelin-1 stimulates THAL NO is unknown. We hypothesized that endothelin-1 stimulates THAL NO production by activating phosphatidylinositol 3-kinase (PI3K), stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport. We measured 1) NO production by fluorescence microscopy using DAF2-DA, 2) Akt activity using a fluorescence resonance energy transfer-based Akt reporter, 3) phosphorylated NOS3 and Akt by Western blotting, and 4) NKCC2 activity by fluorescence microscopy. In isolated THAL, endothelin-1 (1 nmol/liter) increased NO production from 0.23 +/- 0.24 to 2.81 +/- 0.32 fluorescence units/min (p < 0.001; n = 5) but failed to stimulate NO production in THALs isolated from NOS3-/- mice. Wortmannin (150 nmol/liter), a PI3K inhibitor, reduced endothelin-1-stimulated NO by 83% (0.49 +/- 0.13 versus 3.31 +/- 0.49 fluorescence units/min for endothelin-1 alone; p < 0.006; n = 5). Endothelin-1 stimulated Akt activity by 0.16 +/- 0.02 arbitrary units as measured by fluorescence resonance energy transfer (p < 0.001; n = 5) and increased phosphorylation of Akt at Ser473 by 56 +/- 11% (p < 0.002; n = 7). Dominant-negative Akt blocked endothelin-1-induced NO by 60 +/- 8% (p < 0.001 versus control; n = 6), and an Akt inhibitor had a similar effect. Endothelin-1 increased phosphorylation of NOS3 at Ser1177 by 89 +/- 24% (p < 0.01; n = 7) but had no effect on Ser633. Endothelin-1 inhibited NKCC2 activity, an effect that was blocked by dominant-negative Akt and NOS inhibition. We conclude that endothelin-1 stimulates THAL NO production by activating PI3K, stimulating Akt activity, and phosphorylating NOS3 at Ser1177. This enhances NO production and inhibits sodium transport.

Retroviral-Based BMP-4 In Vivo Gene Transfer Strategy with Intramedullary Viral Delivery Optimizes Transgene Expression in Rat Femur Fractures

We have developed an intramedullary delivery strategy to administer retroviral vectors expressing a therapeutic gene to promote healing of a closed rat femur fracture. This strategy involves implantation of an indwelling catheter with the stabilizing Kirschner (K)-wire during the surgery prior to fracture of the femur by the three-point bending technique. It uses the openings in the bone that were already created for the stabilizing K-wire and the catheter insertion. In this study, transgene expression and callus bone formation induced by intramedullary delivery of MLV-based vectors expressing the bone morphogenetic protein-2/4 (BMP-2/4) hybrid gene or -galactosidase (-gal) gene were compared with those pro- duced by percutaneous injections of the same vectors at the periosteum of the fracture site. The percutaneous injections of MLV-BMP-2/4 vector led to massive but asymmetric transgene expression in surrounding tissues within the fracture cal- lus and large amounts of supraperiosteal as well as asymmetric callus bone formation. In contrast, the intramedullary ad- ministration produced a robust and symmetric pattern of transgene expression at the fracture site with very minimal trans- duction at cells of surrounding tissues, resulting in normal subperiosteal bone development around the entire fracture cal- lus without supraperiosteal bone formation. In summary, we have developed an intramedullary retroviral vector delivery strategy with a rat femur fracture model that led to uniform transgene expression around the entire fracture site, which op- timizes the gene therapy-enhanced fracture repair. This strategy should readily be adapted to administer large dosages of any therapeutic vehicle (therapeutic molecules, peptides, or proteins, as well as viral or non-viral vectors) throughout much of early fracture repair, and thus it would be an ideal rat model for in vivo testing of various therapeutic agents to promote fracture repair.

Modification of Adenoviral Vectors With Polyethylene Glycol Modulates In Vivo Tissue Tropism and Gene Expression

Polyethylene glycol (PEG) is a hydrophilic polymer that has been used to coat adenoviral (Ad) vectors to improve their pharmacology. To analyze the effects of PEG on Ad5 tropism, Ad5 was covalently modified with different sizes of PEG and in vitro and in vivo transduction was analyzed. All tested PEGs ablated in vitro transduction. When protein C (PC) and factors VII, IX, and X were added, only factors IX and X increased transduction by the PEGylated vectors with the largest effect by X. Inactivation of these factors with warfarin drastically reduced liver transduction in mice by the PEGylated vectors after intravenous (i.v.) injection. Ad5 conjugated with 5 kd PEG maintained normal liver transduction while conjugation with larger 20 and 35 kd PEGs significantly reduced liver transduction. When intraperitoneal (i.p.) injection was tested, Ad transduced the peritoneum efficiently with only low level liver transduction. When Ad5 was modified with 5 kd PEG, peritoneal transduction was reduced and the virus preferentially transduced the liver. These data demonstrate the effects of different sizes of PEG on in vivo Ad tropism and suggest that this approach may be useful in retargeting and detargeting Ad in vivo.

320. Modification of Adenovirus with Polyethlyene Glycol Modulates In Vivo Tissue Transduction and Gene Expression

320. Modification of Adenovirus with Polyethlyene Glycol Modulates In Vivo Tissue Transduction and Gene Expression

106. Transduction of Human Fibroblast-Like Synoviocytes with Adeno-Associated Virus Type 5 Is Persistently Increased after Proteasome Inhibition: A Possible Application for Rheumatoid Arthritis In Vivo Gene Therapy

106. Transduction of Human Fibroblast-Like Synoviocytes with Adeno-Associated Virus Type 5 Is Persistently Increased after Proteasome Inhibition: A Possible Application for Rheumatoid Arthritis In Vivo Gene Therapy

978. Preparation of Fine DNA Particles and High Level Tumor-Targeted In Vivo Gene Expression after Intravenous Injection

978. Preparation of Fine DNA Particles and High Level Tumor-Targeted In Vivo Gene Expression after Intravenous Injection

825. In Vivo Gene Delivery and Long-Term Expression in Mouse by a Helper Dependent Adenovirus-Epstein-Barr Virus Hybrid Vector

825. In Vivo Gene Delivery and Long-Term Expression in Mouse by a Helper Dependent Adenovirus-Epstein-Barr Virus Hybrid Vector

Role of Biophysical Parameters on ex Vivo and in Vivo Gene Transfer to the Airway Epithelium by Polyethylenimine/Albumin Complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.

Protein Trans -Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

Protein Trans -Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

In Vivo Gene Transfer of PPAR.GAMMA. Is Insulfficient to Induce Adipogenesis in Skeletal Muscle

Skeletal muscle contains several progenitor/stem cells with myogenicity as well as adipogenicity such as satellite cells. Our previous study demonstrated that forced expression of PPAR gamma is sufficient to induce transdifferentiation of predetermined myoblasts in vitro. In the present study, we examined whether introduction of PPAR gamma gene could induce adipogenesis of satellite cells in vivo. A plasmid vector containing enhanced green fluorescent protein (EGFP) or PPAR gamma gene was introduced into rat tibialis anterior muscle by electroporation. Histological analyses revealed that electroporation induces degenerative/regenerative response in skeletal muscle, including activation of satellite cells. When EGFP gene was introduced, newly formed myotubes resulted from fusion of activated satellite cells, showed EGFP expression, indicating that electroporation could transfect satellite cells with exogenously introduced gene. Gene transfer of PPAR gamma resulted in an increase of PPAR gamma-positive mononucleated cells on day 3 after electroporation but failed to induce adipogenesis thereafter. These results suggested that, in addition to an expression of PPAR gamma, niches that support adipogenesis are required for satellite cells to enter adipogenesis in vivo.

Tissue-Targeted In Vivo Gene Transfer Coupled with Histone Deacetylase Inhibitor Depsipeptide (FK228) Enhances Adenoviral Infection in Rat Renal Cancer Allograft Model Systems

Although the adenoviral vector represents an efficient delivery system, hepatotropic accumulation often has detrimental effects on adenoviral vector-mediated cancer therapy. To overcome this disadvantage, we performed in vivo local gene transfer, in combination with the histone deacetylase inhibitor, depsipeptide (FK228), in a rat renal cancer model. Renal cancer cells induced by ferric nitrilotriacetate in ACI rats were used in this study. Adenoviral vectors containing luciferase cDNA were introduced into the tumor-burdened kidney by way of a catheter placed in the renal artery. Subcutaneous tumors were treated by herpes simplex virus thymidine kinase cDNA followed by intraperitoneal ganciclovir. The levels of Coxsackie-adenovirus receptor in various tissue were determined by quantitative reverse transcriptase-polymerase chain reaction. Depsipeptide (1 mg/kg) was intravenously administered 24 hours before adenoviral vector transduction. The catheter-based adenoviral vector delivery enabled strong gene transduction of the tumor-burdened kidney. Moreover, depsipeptide treatment before adenoviral vector injection significantly improved transgene expression at tumor sites. Quantitative reverse transcriptase-polymerase chain reaction analysis showed that depsipeptide increased the expression levels of the Coxsackie-adenovirus receptor in the renal tumor (13-fold), but not in other normal tissues. Furthermore, the use of herpes simplex virus thymidine kinase cDNA-expressing adenoviral vector followed by ganciclovir markedly inhibited the established tumor growth in combination with depsipeptide compared with herpes simplex virus thymidine kinase cDNA alone. The tissue-targeted in vivo gene transfer coupled with depsipeptide significantly enhanced adenoviral infection at tumor sites. Sensitization of tumor cells with depsipeptide can improve the efficacy of adenoviral vector-mediated suicide gene therapy. Thus, application of depsipeptide could be one of the beneficial adjunct for adenoviral vector-mediated cancer gene therapy.

Abstract 1744: Adenylate-Cyclase VI Transforms Ventricular Cardiomyocytes Into Biological Pacemaker Cells

Background- Genetic therapy of arrhythmic disorders of the heart has been subject of extensive studies. Cyclic AMP (cAMP) is generated in response to β-adrenergic receptor stimulation and also binds to HCN channels, where it regulates spontaneous rhythmic activity in the heart. Aim of this study was to demonstrate whether in-vivo adenoviral gene transfer of Adenylate-Cyclase type VI (AC VI ) can create biological pacemaker activity in a porcine atrioventricular node (AV-node) block model. Methods and Results- Adenovirus encoding either AC VI or β-Galactosidase (lacZ) gene were injected into the lateral wall of the left ventricle of adult pigs via anterolateral thoracotomy at a dose of 10 10 virus particles each. After 12 days, the AV-node was ablated and three-dimensional electrophysiological cardiac mapping was performed using the Ensite™ electro-anatomical system. After rapid ventricular pacing and administration of Isoprenalin all animals of the AC-VI group exhibited an escape rhythm originating from the area of the left ventricular injection site at a rate of 100 ± 7 beats/minute (n = 5), while the escape rhythms in the control group (n = 4) originated from the right ventricle. Western blot analysis of the injection sites revealed a significantly higher expression of AC-VI in the respective group as compared to the control group. Conclusions- Our study demonstrates that AC-VI gene transfer may be a novel method to create a biological pacemaker

Sine-wave Current for Efficient and Safe In Vivo Gene Transfer

Recently, electro-gene transfer has become a powerful tool to enhance the efficiency of gene expression in different organs and particularly in muscle, which provides efficient secretion of therapeutic proteins into the circulation. However, its toxicity, owing to the high field strengths of conventional direct current (DC) square-waves, should be taken into account and should be minimized if electroporation is to be used routinely for the treatment of human diseases. In this study, we demonstrate that efficient in vivo gene transfer could be safely achieved using pulses of alternating current sine-waves (ACSWs) with a frequency of 60 Hz. Importantly, the field strength was decreased to as low as 20 V/cm and the efficiency of muscle gene transfer increased more than tenfold and showed less toxicity than with conventional DC square-wave pulses. Using ACSW pulses to transfer human clotting factor IX (hFIX) plasmid into muscle, we observed significant phenotypic correction in mice with hemophilia B.

Bone Regeneration by Regulated In Vivo Gene Transfer Using Biocompatible Polyplex Nanomicelles

Gene therapy is a promising strategy for bone regenerative medicine. Although viral vectors have been intensively studied for delivery of osteogenic factors, the immune response inevitably inhibits bone formation. Thus, safe and efficient non-viral gene delivery systems are in high demand. Toward this end, we developed a polyplex nanomicelle system composed of poly(ethyleneglycol) (PEG)-block-catiomer (PEG-b-P[Asp-(DET)]) and plasmid DNA (pDNA). This system showed little cytotoxicity and excellent transfection efficiency to primary cells. By the transfection of constitutively active form of activin receptor-like kinase 6 (caALK6) and runt-related transcription factor 2 (Runx2), the osteogenic differentiation was induced on mouse calvarial cells to a greater extent than when poly(ethylenimine) (PEI) or FuGENE6 were used; this result was due to low cytotoxicity and a sustained gene expression profile. After incorporation into the calcium phosphate cement scaffold, the polyplex nanomicelles were successfully released from the scaffold and transfected surrounding cells. Finally, this system was applied to in vivo gene transfer for a bone defect model in a mouse skull bone. By delivering caALK6 and Runx2 genes from nanomicelles incorporated into the scaffold, substantial bone formation covering the entire lower surface of the implant was induced with no sign of inflammation at 4 weeks. These results demonstrate the first success in in vivo gene transfer with therapeutic potential using polyplex nanomicelles.

C/EBPβ Activates E2F-regulated Genes in Vivo via Recruitment of the Coactivator CREB-binding Protein/P300

The E2F transcription factors play an essential role in regulating the G(1)- to S-phase transition of the cell cycle. Previous studies have identified the importance of interactions between E2Fs and other transcription factors as a mechanism for transcriptional control of a subset of E2F regulated target genes. However, the mechanisms responsible for E2F target gene specificity remain incompletely understood. Here we report that in a mammalian in vivo model of synchronized proliferation, C/EBPbeta occupancy on the promoters of E2F-regulated growth-related genes increases as a function of cell cycle progression. C/EPBbeta binding to these promoters is associated with recruitment of the coactivator CBP/p300, histone H4 acetylation, and maximal activation of E2F target genes. Moreover, binding of CBP/p300 to E2F targets is markedly reduced in C/EBPbeta null mice, resulting in reduced expression of E2F regulated genes. These findings identify C/EBPbeta as a direct activator of E2F target genes in mammalian cell cycle progression through a mechanism that involves recruitment of CBP/p300. The demonstration of a functional link between C/EBPbeta and CBP/p300 for E2F target gene activation provides a potential mechanism for how coactivators such as CBP/p300 can be selectively recruited to E2F target genes in response to tissue-specific growth stimuli.

Gene therapy for Parkinson's disease: early data

Parkinson's disease is the second most common neurodegenerative disorder, affecting at least 1% of people above the age of 65 years, a proportion that increases with age. The introduction of replacement therapy with levodopa and, more recently, various adjunct drugs has substantially improved survival and quality of life. However, these therapies are associated with several long-term complications that can limit the tolerated dose. None of the available drugs has a convincing effect on the inexorable progression of the disease. Surgical approaches, such as deep-brain stimulation of the subthalamic nucleus, are effective 1 Deuschl G Schade-Brittinger C Krack P et al. A randomized trial of deep-brain stimulation for Parkinson's disease. N Engl J Med. 2006; 355: 896-908 Crossref PubMed Scopus (2018) Google Scholar but technically complex, and continuing care from an expert team is needed. Although there is interest in cell-based therapies, double-blind controlled trials of human fetal mesencephalic transplantation were disappointing. 2 Freed CR Greene PE Breeze RE et al. Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N Engl J Med. 2001; 344: 710-719 Crossref PubMed Scopus (1968) Google Scholar , 3 Olanow CW Goetz CG Kordower JH et al. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson's disease. Ann Neurol. 2003; 54: 403-414 Crossref PubMed Scopus (1244) Google Scholar Several gene-therapy approaches have been suggested or are in development, ranging from those that attempt to restore dopamine synthetic capacity 4 Bankiewicz KS Forsayeth J Eberling JL et al. Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC. Mol Ther. 2006; 14: 564-570 Summary Full Text Full Text PDF PubMed Scopus (235) Google Scholar , 5 Pardridge WM Tyrosine hydroxylase replacement in experimental Parkinson's disease with transvascular gene therapy. NeuroRx. 2005; 2: 129-138 Crossref PubMed Scopus (83) Google Scholar to those that protect against cell death with trophic factors, 6 Kordower JH Herzog CD Dass B et al. Delivery of neurturin by AAV2 (CERE-120)-mediated gene transfer provides structural and functional neuroprotection and neurorestoration in MPTP-treated monkeys. Ann Neurol. 2006; 60: 706-715 Crossref PubMed Scopus (216) Google Scholar to molecules that interfere with the aberrant protein aggregation in Parkinson's disease. 7 Lo Bianco C Schneider BL Bauer M et al. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an α-synuclein rat model of Parkinson's disease. Proc Natl Acad Sci USA. 2004; 101: 17510-17515 Crossref PubMed Scopus (270) Google Scholar , 8 Mochizuki H Yamada M Mizuno Y Parkin gene therapy for α-synucleinopathy: a rat model of Parkinson's disease. Parkinsonism Relat Disord. 2006; 12: S107-S109 Summary Full Text Full Text PDF Scopus (6) Google Scholar Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trialAAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases. Full-Text PDF

Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial

Dopaminergic neuronal loss in Parkinson's disease leads to changes in the circuitry of the basal ganglia, such as decreased inhibitory GABAergic input to the subthalamic nucleus. We aimed to measure the safety, tolerability, and potential efficacy of transfer of glutamic acid decarboxylase (GAD) gene with adeno-associated virus (AAV) into the subthalamic nucleus of patients with Parkinson's disease. We did an open label, safety and tolerability trial of unilateral subthalamic viral vector (AAV-GAD) injection in 11 men and 1 woman with Parkinson's disease (mean age 58.2, SD=5.7 years). Four patients received low-dose, four medium-dose, and four high-dose AAV-GAD at New York Presbyterian Hospital. Inclusion criteria consisted of Hoehn and Yahr stage 3 or greater, motor fluctuations with substantial off time, and age 70 years or less. Patients were assessed clinically both off and on medication at baseline and after 1, 3, 6, and 12 months at North Shore Hospital. Efficacy measures included the Unified Parkinson's Disease Rating Scale (UPDRS), scales of activities of daily living (ADL), neuropsychological testing, and PET imaging with 18F-fluorodeoxyglucose. The trial is registered with the ClinicalTrials.gov registry, number NCT00195143. All patients who enrolled had surgery, and there were no dropouts or patients lost to follow-up. There were no adverse events related to gene therapy. Significant improvements in motor UPDRS scores (p=0.0015), predominantly on the side of the body that was contralateral to surgery, were seen 3 months after gene therapy and persisted up to 12 months. PET scans revealed a substantial reduction in thalamic metabolism that was restricted to the treated hemisphere, and a correlation between clinical motor scores and brain metabolism in the supplementary motor area. AAV-GAD gene therapy of the subthalamic nucleus is safe and well tolerated by patients with advanced Parkinson's disease, suggesting that in-vivo gene therapy in the adult brain might be safe for various neurodegenerative diseases.

In-vivo gene transfer into newly hatched chick brain by electroporation

Newly hatched domestic chicks serve as ideal models for studies of the neural basis of behavioral plasticity, particularly for understanding the mechanisms of learning such as filial imprinting. To elucidate the molecular basis and gene functions involved in learning, we developed an in-vivo gene-transfer system in the brain of a living chick using electroporation. When green fluorescent protein-encoding plasmids were transfected to a chick brain, green fluorescence was clearly observed, and expression at the protein level was confirmed by immunoblotting. Most of the transfected brain cells were neuronal cells with dendrites. This neuron-selective electroporation system will facilitate the analysis of gene functions in the living chick brain and provide further clues as to the molecular mechanisms of avian learning.

973. Protective Effects of Limited In Vivo Gene Transfer of Phospholamban Antisense in a Catecholamine Induced Hypertrophic Rat Heart Model

973. Protective Effects of Limited In Vivo Gene Transfer of Phospholamban Antisense in a Catecholamine Induced Hypertrophic Rat Heart Model