Effects Of Adenoviral Gene Transfer Research Articles

HIV-infection resistance in PMBC-derived dendritic cells modified with recombinant virus

This study aimed to identify the characteristics of recombinant-adenovirus-modified PBMC-derived dendritic cells and their resistance to HIV-1 infection by integrating the CCR5∆32, CCR5siRNA, HIV-1 pol and HIV-1 int genes into a recombinant adenovirus vector using the AdEasy system. Dendritic cells (DCs) were isolated from human PBMCs from blood of healthy donors. The expression of CCR5∆32, CCR5, CXCR4 and HIV-1 p24 in PBMCs or modified cells was measured by western blot, p24 expression in cell lysates was measured by ELISA, and HIV-1 entry was measured by β-galactosidase assay. Furthermore, T-cell immunity induced by the recombinant adenovirus was measured by ELISPOT assay. After the cells were modified by Ad-R5∆32siRNA, the expression of CCR5∆32 increased, while the expression of CCR5 and CXCR4 decreased. There was no adverse effect of adenoviral gene transfer on DC development. CD83 expression on the surface of mature DCs did not change after gene transfer. The expression of p24 remained at low levels in modified cells when challenged by HIV-1. The modified cells showed resistance to HIV-1 infection. Results indicated that recombinant-adenovirus-modified cells demonstrated good resistance to HIV-1 infection. Modification of HSC-derived immune cells, such as DCs, may be a potent strategy to resist HIV-1 infection.

Abstract 4944: The impact of adenoviral gene transfer of microRNA-34b/c on malignant pleural mesotheliomas

Abstract Background: Altered expression of microRNA (miRNA) is strongly implicated in human malignancies. miRNAs are small non-coding RNAs that regulate translations of messenger RNAs of various genes. Similar to encoding genes, tumor suppressive miRNAs are silenced with DNA methylation. The miR-34b/c is direct transcriptional targets of p53 and has been reported to be down-regulated in human malignancies. Unlike other malignant tumors, mutation of TP53 gene is rare in malignant pleural mesotheliomas (MPMs). In our previous study, aberrant methylations of miR-34b/c were frequent in MPMs (−100%), resulting in the slicing of their expression. Ectopic expression of miR-34b/c by transit or stable plasmid transfection, that promoted physiological expression level of miR-34b/c, could inhibit the proliferation and invasion of MPM cells. In this study, we investigate the anti proliferative effect of adenoviral gene transfer of miR-34b/c on MPM cells to aim development of miR-34b/c gene therapy. Methods: We examined three MPM cell lines (H28, H290, and H2052) and a lung cancer cell line (H125). Three adenovirus vectors expressing miR-34b/c (Ad-miR-34b/c), p53 (Ad-p53) and luciferase (Ad-Luc) driven by CMV promoter were generated by homologous recombination and were infected to cell lines above. Expression of miR-34b/c was determined by quantitative PCR. Protein expression, cell proliferation, and cell cycle were analyzed by western blotting, MTS assay, and flow cytometry, respectively. Results: TP53 was wild-type and miR-34b/c was methylated in all three MPM cell lines, while TP53 was mutated and miR-34b/c was unmethylated in H125. After Ad-p53 transfer, p53 and p21 were up-regulated in all four cell lines. Of note, Ad-p53 transfer increased expression of miR-34b/c in H125 but not in all three MPM cell lines, resulting in a decrease of the cell viability in H125 but not in MPM cell lines. The expression of miR-34b and 34c were highly un-regulated after Ad-miR-34b/c transfer in all three MPM cell lines (100 to 2000 fold), that were much higher than physiological level. In addition, the cell viability was also decreased after Ad-miR-34b/c transfer compared with that after Ad-Luc transfer (P < 0.01) in MPM cell lines. Ad-miR-34b/c transfer promoted the strong down-regulation of Bcl-2 protein expression in MPM cell lines and caused an increase in the sub-G0-G1 DNA content, compared with that in cell lines infected with Ad-Luc, indicating the induction of apoptosis. In contrast, Ad-p53 transfer did not induce a drastic change in the sub-G0-G1 DNA content in MPM cell lines. Conclusion: Current study suggests that the adenoviral gene therapy of miR-34b/c, but not TP53, inhibits the cell proliferation of MPM cells by induction of apoptosis, indicating the possibility of miR-34b/c gene therapy in MPMs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4944. doi:10.1158/1538-7445.AM2011-4944

PRESERVATION OF MECHANICAL AND ENERGETIC FUNCTION AFTER ADENOVIRAL GENE TRANSFER IN NORMAL RAT HEARTS

1. The aim of the present study was to examine the acute and chronic effects of adenoviral gene transfer on cardiac function in terms of left ventricular (LV) mechanoenergetic function. Recombinant adenoviral vector carrying beta-galactosidase and green fluorescent protein genes (Ad.betagal-GFP) was used. Cardiac function was examined in cross-circulated rat heart preparations, where end-systolic/diastolic pressure-volume relationships (ESPVR/EDPVR), systolic pressure-volume area (PVA), LV relaxation rate, equivalent maximal elastance at mid-range LV volume (eE(max) at mLVV), coronary blood flow, coronary vascular resistance and myocardial oxygen consumption (VO(2)) were also measured. 2. To examine the ex vivo acute effects of the adenoviral vector, data were obtained before and 30-90 min after intracoronary infusion of Ad.betagal-GFP in the excised, cross-circulated hearts that underwent serotonin pretreatment. To examine the in vivo chronic effects of adenoviral gene transfer, normal rat hearts received Ad.betagal-GFP or saline by a catheter-based technique and data were obtained 3 days after the injection of Ad.betagal-GFP or saline. 3. The ESPVR, EDPVR, LV relaxation rate, eE(max) at mLVV, coronary blood flow and coronary vascular resistance remained unchanged in Ad.betagal-GFP-transfected hearts in both ex vivo acute and in vivo chronic experiments. Moreover, the ex vivo and in vivo transfection caused no change in the slope and VO(2) intercept of the VO(2)-PVA relationship, VO(2) for basal metabolism and for Ca(2+) handling in excitation-contraction coupling and O(2) costs of LV contractility. 4. These results indicate that adenoviral gene transfer has neither acute nor chronic toxic effects on LV mechanical and energetic function. A special combination of in vivo adenoviral gene transfer and a cross-circulation experimental system may provide a useful novel strategy to explore the functional and mechanoenergetic role of specifically targeted genes in the diseased heart.

Dysregulation of cGMP‐dependent protein kinase 1 (PKG‐1) impairs erectile function in diabetic rats: influence of in vivo gene therapy of PKG1α

To investigate the expression of cGMP-dependent protein kinase 1 (PKG1)alpha and PKG1beta in the corpus cavernosum, and to evaluate the effect of adenoviral gene transfer of PKG1alpha to the erectile compartment on erectile function in a rat model of diabetes. Diabetic (DM; induced by streptozotocin) male Sprague Dawley rats were transfected with adenoviruses (AdCMVbetagal or AdCMVPKG1alpha, in 10 rats each) 2 months after the induction of DM. Intracavernosal pressure (ICP) during stimulation of the cavernosal nerve (CN) was assessed, and compared with mean arterial pressure (MAP). Erectile tissue was harvested for Western blot analysis, immunohistochemistry and total PKG activity. Ten age-matched rats without DM served as the control. Compared to controls, AdCMVbetagal-transfected DM rats had significantly lower peak ICP responses, ICP/MAP ratios, and filling rates during CN stimulation. In DM rats transfected with AdCMVPKG1alpha, peak ICP, ICP/MAP ratios and filling rates were significantly better than in DM rats transfected with the reporter gene. As assessed by Western blot and immunohistochemistry, expression of PKG1alpha and PKG1beta was lower in corporal tissue from DM AdCMVbetagal-transfected rats than in controls. PKG1alpha expression was improved after AdCMVPKG1alpha gene therapy. Total PKG activity was lower in DM rat corporal tissue than in controls, and PKG1alpha gene transfer significantly improved DM corporal PKG activity to a value greater than in the control. PKG1alpha and PKG1beta activities are reduced in the erectile tissue of the diabetic rat, and gene transfer of PKG1alpha to the penis restored PKG activity and erectile function in vivo in diabetic rats. Gene therapy procedures targeting PKG1alpha might be an interesting future therapeutic approach to overcome diabetic erectile dysfunction resistant to oral pharmacotherapy.

Effects of adenoviral gene transfer of mutated IkappaBalpha, a novel inhibitor of NF-kappaB, on human monocyte-derived dendritic cells1

To investigate the effects of adenoviral gene transfer of IkappaBalpha mutant (IkappaBalphaM), a novel inhibitor of nuclear factor kappaB (NF-kappaB), on apoptosis, phenotype and function of human monocyte-derived dendritic cells (DC). Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with AdIkappaBalphaM or AdLacZ. The transcription and expression of the IkappaBalphaM gene, and the inhibitory effect of IkappaBalphaM on tumor necrosis factor (TNF)-alpha-induced NF-kappaB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction. PCR and RT-PCR were used to detect a unique 801 bp band in AdIkappaBalphaM-transfected mature DC, and also a dose- and time-dependent expression of the IkappaBalphaM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIkappaBalphaM significantly suppressed the TNF-alpha-induced NF-kappaB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC. AdIkappaBalphaM effectively transfected and potently inhibited NF-kappaB activation in monocyte-derived mature DC. Overexpression of the IkappaBalphaM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.

Regulation of vascular smooth muscle cell function by adenylyl cyclase isoforms

Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. Regulation patterns differ between the AC isoforms, however the consequences of activation of any individual AC isoform (e.g., to catalyze the synthesis of cAMP) were thought to be common to all isoforms. To examine this hypothesis we studied the effect of adenoviral gene transfer of representative AC isoforms AC I, II, V and VI on whole cell cAMP activity, regulation of vascular smooth muscle cell proliferation, isoproterenol (ISO)-mediated arborization responses (an index of cytoskeletal re-organization) and protein kinase A (PKA) activity Although all AC isoforms when expressed in smooth muscle cells comparably enhanced forskolin (FSK)-stimulated cAMP synthesis (186–208% of the AC activity in GFP-infected cells), only AC I-infected cells demonstrated an enhancement of FSK-stimulated inhibition of proliferation (19±3% of GFP, p<0.01). Only gene transfer of AC VI enhanced ISO-mediated arborization (155±16% of GFP, p<0.05). To further examine the contribution of AC VI to the arborization response, we utilized a “knockdown” approach based on expression of a truncated-AC VI dominant negative mutant (TM1T). TM1T expression significantly reduced FSK-stimulated AC activation (79±8% of GFP alone) and ISO-mediated arborization (54±6% of GFP alone). This reduction in arborization was recovered by the co-expression of AC isoform VI (84±10%). Lastly, FSK-stimulated PKA activity was significantly reduced (61±5% of GFP) by AC I expression and significantly enhanced (132±8% of GFP) by AC VI expression. In aggregate these data support a previously unappreciated role for specific AC isoforms in directing the effect of elevation of intracellular cAMP in the regulation of vascular function. Supported by CIHR.

Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus

We have previously shown that neuroprotective effects of an adenoviral glial cell line-derived neurotrophic factor (GDNF) gene transfer on the lesioned adult rat motoneurons in the nucleus ambiguus. In the present study, we examined neuroprotective effects of adenoviral gene transfer of brain-derived neurotrophic factor (BDNF) or/and GDNF to motoneurons in nucleus ambiguus using an adult rat vagal nerve avulsion model. The animals avulsed and inoculated with adenoviral vectors encoding BDNF (AxCAmBDNFME) or/and GDNF (AxCAhGDNF) showed immunolabeling for BDNF or/and GDNF in the nucleus ambiguus on the treated side, respectively, and expression of virus-induced BDNF or/and GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF or AxCAmBDNFME significantly prevented the loss of vagal motoneurons in comparison to the control; the protective effect of AxCAmBDNFME was greater than that of AxCAhGDNF. The combined treatment with AxCAmBDNFME and AxCAhGDNF acted synergistically and significantly larger number of vagal motoneurons was preserved as compared to either AxCAmBDNFME treatment or AxCAhGDNF treatment. The treatment with AxCAmBDNFME or/and AxCAhGDNF after avulsion also suppressed the activity of nitric oxide synthase in lesioned motoneurons in the nucleus ambiguus. These results indicate that adenovirus-mediated BDNF and GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.

Therapeutic effects of adenoviral gene transfer of bone morphogenic protein-7 on a corneal alkali injury model in mice

An alkali burn in the cornea is a common serious clinical problem often leading to permanent visual impairment. Since transforming growth factor-β (TGF-β) is involved in the response to corneal injury, we evaluated the therapeutic effects of adenoviral gene transfer of mouse bone morphogenic proten-7 (BMP-7), which has antagonistic effects on TGF-β in tissue fibrosis. Burned cornea did not express endogenous BMP-7 mRNA and protein. Resurfacing of the burned cornea by invading conjunctival epithelium was accelerated by adenoviral introduction of BMP-7. Exogenous BMP-7 expression also suppressed myofibroblast generation, appearance of monocytes/macrophages and expression of MCP-1, TGF-βs, and collagen I α2 chain in the affected stroma. Ectopic BMP-7 did not suppress stromal neovascularization throughout the interval studied and also did not reduce VEGF mRNA expression at Day 10. Ectopic BMP-7 in burned corneal tissue resulted in activation of Smad1/5/8 signaling and partial suppression of the phospho-Smad2 signal. These data suggest that overexpression of BMP-7 is an effective strategy for treatment of ocular alkali burns.

Delayed angiogenesis in aging rats and therapeutic effect of adenoviral gene transfer of VEGF

We have studied an age-related impairment in angiogenesis and evaluated the effect of overexpressing VEGF in this situation. Polyvinyl alcohol sponges were implanted subcutaneously into aged (24-month), adult (12-month), and young (2-month) rats. Blood vessel ingrowth and proliferative activity in the sponges were assessed by histology with immunostaining for von Willebrand's factor and proliferating cell nuclear antigen (PCNA), respectively. The percentage of total sponge area filled with ingrowing fibrovascular tissue was minimal in aged rats, intermediate in adult rats and highest in young rats. A similar pattern was observed for the total blood vessel numbers in the sponges from old to young animals. The percentage of total sponge endothelial cells (ECs) showing proliferative activity (PCNA positive) was lowest in the aged animals, intermediate in the adult rats and highest in the young rats. To further explore the mechanism of impaired angiogenesis in aged animals, we investigated and found a reduced level of endogenous VEGF protein expression in 12-month-old rats compared to that in 2-month-old rats. VEGF121 gene transfer significantly enhanced blood vessel and fibrovascular tissue ingrowth in adult/aged rats. Adenoviral-VEGF gene transfer also significantly stimulated EC proliferation in aged and adult rats. However, identical treatment failed to further stimulate the already more robust angiogenesis in young animals. The different angiogenic response in adult vs. young rats was not due to differences in gene transfer efficiency, since similar levels of human VEGF121 protein was detected in adult and young rats. Our results indicate that the decreased angiogenic response with aging is associated with reduced EC proliferation and reduced endogenous VEGF production. Adenoviral-VEGF121 gene transfer is effective in augmenting angiogenesis, particularly in older animals.

Overexpression of Basic Fibroblast Growth Factor and Bcl-xL with Adenoviral Vectors Protects Primarily Cultured Neurons against Glutamate Insult

Excitatory amino acid (EAA) toxicity seems to be an important mechanism of neuronal cell death after cerebral infarction. We examined the inhibitory effects of neuronal cell death caused by EAA in vitro by means of adenoviral gene transfer of neurotrophic basic fibroblast growth factor (bFGF) and antiapoptotic Bcl-xL. Recombinant adenoviral vectors expressing human bFGF gene with secretory signals of interleukin-2 and human Bcl-xL gene were constructed. Primarily cultured rat neuronal cells were treated with glutamate to cause EAA, and the neuroprotective effects of gene transfer by these adenoviral vectors were investigated at several time points of infection. Each adenoviral infection to primarily cultured neuronal cells exhibited neuroprotective effects against EAA caused by glutamate. Both gene transfer of bFGF with secretory signal and Bcl-xL transfer to neuronal cells exhibited the synergistic neuroprotective effects against EAA. These effects were most prominent with gene transfer 4 hours before glutamate insult; gene transfer performed simultaneously with and up to 4 hours after the insult exhibited definite neuroprotective effects. These experiments revealed marked neuroprotective effects of adenoviral gene transfer of bFGF and Bcl-xL into neuronal cells in vitro. The findings may lead to new approaches for treating occlusive cerebrovascular disease.

Chances and shortcomins of adenovirus-mediated ATP7B gene transfer in Wilson disease: proof of principle demonstrated in a pilot study with LEC rats.

Wilson disease (WD) is an autosomal recessively inherited copper storage disease due to mutations in the ATP7B gene. It results in impaired biliary copper excretion followed by liver injury leading to cirrhosis. In parallel, copper accumulates in other tissues e. g. basal ganglia of the brain inducing motoric disorders. Phenotypical cure of Wilson disease by liver transplantation raised the question whether gene therapy may represent a successful alternative treatment procedure. To examine the principle feasibility of this approach we investigated the effects of gene transfer using an adenoviral vector construct expressing the human ATP7B cDNA in an established rodent model for WD, the Long-Evans Cinnemon rat (LEC). Transduction efficiency was assessed by RT-PCR, Western blot and immunofluorescence analysis. The therapeutic effect was estimated by analyzing holoceruloplasmin and its ferroxidase activity in serum, and the copper content of excrements. Changes in copper homeostasis were determined by positron emission tomography (PET). Successful, but temporary gene transfer was clearly detectable on RNA and protein levels. In parallel the temporary therapeutic effect was documented by restoration of serum holoceruloplasmin and of its ferroxidase activity. Additionally the Ad-ATP7B treated LEC rat revealed a higher (64)Cu content in stool. PET was able to visualize differences in (64)Cu distribution between wild type and LEC rats, indicating its principle usefulness as analytical tool. The data demonstrate proof of principle of successful gene therapy in an experimental model of WD. As a consequence of successful but only transient therapeutic effect of adenoviral gene transfer we can now focus more efficient and permanent gene transfer strategies.

Effects of adenoviral gene transfer of wild-type, constitutively active, and kinase-defective protein kinase C-lambda on insulin-stimulated glucose transport in L6 myotubes.

We used adenoviral gene transfer methods to evaluate the role of atypical protein kinase Cs (PKCs) during insulin stimulation of glucose transport in L6 myotubes. Expression of wild-type PKC-lambda potentiated maximal and half-maximal effects of insulin on 2-deoxyglucose uptake, but did not alter basal uptake. Expression of constitutively active PKC-lambda enhanced basal 2-deoxyglucose uptake to virtually the same extent as that observed during insulin treatment. In contrast, expression of kinase-defective PKC-lambda completely blocked insulin-stimulated, but not basal, 2-deoxyglucose uptake. Similar to alterations in glucose transport, constitutively active PKC-lambda mimicked, and kinase-defective PKC-lambda completely inhibited, insulin effects on GLUT4 glucose transporter translocation to the plasma membrane. Expression of kinase-defective PKC-lambda, in addition to inhibition of atypical PKC enzyme activity, was attended by paradoxical increases in GLUT4 and GLUT1 glucose transporter levels and insulin-stimulated protein kinase B enzyme activity. Our findings suggest that in L6 myotubes, 1) atypical PKCs are required and sufficient for insulin-stimulated GLUT4 translocation and glucose transport; and 2) activation of protein kinase B in the absence of activation of atypical PKCs is insufficient for insulin-induced activation of glucose transport.

Effects of Adenoviral Gene Transfer of Wild-Type, Constitutively Active, and Kinase-Defective Protein Kinase C- on Insulin-Stimulated Glucose Transport in L6 Myotubes

Effects of Adenoviral Gene Transfer of Wild-Type, Constitutively Active, and Kinase-Defective Protein Kinase C- on Insulin-Stimulated Glucose Transport in L6 Myotubes

Functional consequences of adenovirus-mediated murine pancreatic gene transfer.

Pancreatic adenoviral gene transfer can be achieved with high efficiency; however, questions concerning tissue injury from this commonly used vector have not been addressed. In these experiments, the effects of adenoviral gene transfer on pancreatic exocrine function were evaluated. Direct pancreatic injection with an adenoviral vector containing the Escherichia coli beta-galactosidase (beta-Gal; lacZ) transgene (H5.010CBlacZ) resulted in a high level of transgene expression (64 +/- 6% of pancreatic cells expressed beta-Gal) at 3 days following infection. However, amylase levels in four of five different subcellular pancreatic fractions were significantly decreased at this time point. Direct pancreatic injection with either saline or psoralen/UV-inactivated adenovirus did not have this effect, whereas both transduction with an adenoviral vector containing a different transgene and transduction with a homologous transgene resulted in decreased pancreatic amylase. The decrease in subcellular amylase levels persisted at 7 days post-transduction, and then returned to baseline at 21 days post-transduction. There was associated histologic damage (increased edema, inflammation, cell destruction, and vacuolization) at 3 and 7 days post-transduction, which resolved by 21 days. In summary, adenoviral transduction of the pancreas results in increased viral transgene expression and a uniform decrease in host amylase production throughout the pancreas. The normalization of amylase levels and histology suggest that organ recovery occurs. Gene transfer technology as a novel strategy for pancreatic diseases such as diabetes, pancreatitis, and cystic fibrosis is feasible but will benefit from continued approaches to limit toxicity.

Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum calcium ATPase in isolated rat myocytes.

In myocardial cells, relaxation is governed primarily by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase transporting enzyme, which regulates Ca2+ sequestration into the SR. Human and experimental cardiomyopathies are associated with reduced SR Ca(2+)-ATPase activity. To modify intracellular calcium mobilization, we created a recombinant adenovirus designed to over-express the cardiac SR Ca(2+)-ATPase (SERCA2a) under the control of the Rous sarcoma virus (RSV). In neonatal rat myocytes, Ad.RSV.SERCA2a increased the expression of SERCA2a in a concentration-dependent and time-dependent fashion. Enhancement of SR Ca(2+)-ATPase activity was even greater than increases in SERCA2a protein content in cells infected with Ad.RSV.SERCA2a for 48 hours at a multiplicity of infection (MOI) from 0.1 to 10.0 pfu/cell. Intracellular calcium transients measured in the neonatal cells infected with Ad.RSV.SERCA2a were characterized by an abbreviation of the relaxation phase, an increase in peak [Ca2+]i release, and a decrease in resting [Ca2+]i levels. Ad.RSV.SERCA2a also enhanced the contraction of the myocardial cells as detected by shortening measurements. We found that adenovirus-mediated gene transfer of SR Ca(2+)-ATPase can modify intracellular calcium handling and shortening in myocardial cells. Such vectors should be useful in examining the role of reduced SERCA2a activity in the pathophysiology of heart failure and in developing strategies for gene therapy.