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Adenovirus (Ad) types 2 and 5 encode at least five proteins within the E3 transcription unit that help the virus evade the immune system. Two such proteins, RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K), form the RID (receptor internalization and degradation) complex (formerly E3-10.4K/14.5K). RID mediates clearance from the cell surface and lysosomal degradation of a number of important members in the tumor necrosis factor receptor (TNFR) superfamily and the receptor tyrosine kinase receptor family. Affected receptors include Fas, TRAIL (TNF-related apoptosis-inducing ligand) receptor 1 (TR1), TR2, and epidermal growth factor receptor (EGFR). Degradation of Fas and TRAIL receptors protects Ad-infected cells from apoptosis. To investigate the mechanism of action of RIDalpha, 14 mutant RIDalpha proteins, each containing a three- to five-amino-acid deletion, were constructed and then expressed from the E3 region of a replication-competent recombinant Ad in the same context as wild-type RIDalpha. Each mutant protein was characterized with regard to five physical properties associated with wild-type RIDalpha, namely, protein stability, proteolytic cleavage, insertion into the membrane, complex formation with RIDbeta, and transport to the cell surface. Additionally, the mutant proteins were tested for their ability to mediate internalization and degradation of EGFR and Fas and to protect cells from Fas-mediated apoptosis. The majority of mutant RIDalpha proteins (8 out of 14) were physically similar to wild-type RIDalpha. With regard to functional characteristics, the cytoplasmic domain of RIDalpha is largely unimportant for receptor internalization and degradation and the extracellular domain of RIDalpha is important for down-regulation of EGFR but not Fas.

Background: Stent-induced coronary restenosis is a major clinical and public health problem. Proliferating cell nuclear antigen (PCNA) is an important regulator of cell division, and blocking of its expression after angioplasty may limit intimal proliferation., Methods and Results: We cloned the porcine PCNA gene and constructed a chimeric hammerhead ribozyme to a segment of the gene with human homology. In vitro studies with both cultured porcine and human vascular smooth muscle cells demonstrated uptake of ribozyme within the nucleus and significant inhibition of cellular proliferation. The ribozyme was then delivered locally into pig coronaries in a stent model. At 30 days, histomorphometric analysis showed neointimal thickness of 0.51+/-0.20 mm in the ribozyme group versus 0.71+/-0.27 and 0.66+/-0.25 mm in stent controls and scrambled ribozyme control, respectively (P=0.002, P=0.03). Quantitative angiographic analysis showed late loss of 1.4+/-0.5 mm for ribozyme versus 1.9+/-0.4 and 2.0+/-0.4 mm for the controls (P=0.05 and P=0. 02)., Conclusions: Chimeric hammerhead ribozyme to PCNA inhibits smooth muscle cell proliferation in vitro and reduces both histomorphometric and angiographic restenosis in the porcine coronary stent model when delivered locally.

Background: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants., Objectives: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector., Study Design: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model., Results: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes., Conclusions: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.

Two effective ribozymes (CR2 and CR4) that target HCV RNA 5' UTR and capsid gene regions were generated. Ribozyme cleavage was demonstrated in vitro, which can be enhanced by facilitator RNA molecules. In tissue culture cells, these two ribozymes can inhibit the expression of a cotransfected reporter gene containing HCV RNA target sequences. Furthermore, transduction of human hepatoma cells, HepG2, with retroviral vectors carrying CR2 or CR4 ribozymes enabled the cells to resist the infection by retroviral particles containing HCV target sequences. These results represent the first positive step towards the application of hairpin ribozymes in gene therapy for the treatment of HCV infection.

The effects of surfactant on aerosolization of adenovirus and the distribution of aerosolized and instilled adenovirus in rabbit lungs were measured. Using 99Tc sulfur colloid as a marker for the aerosol, 33P-adenovirus with and without 3H-surfactant, we found that the aerosol had the same particle size distribution independent of surfactant and the 33P-adenovirus distributed proportionately to the other radiolabels in the aerosol. Rabbits received aerosolized or instilled suspensions of adenovirus and 99Tc-sulfur colloid with or without 10 mg/ml surfactant. Rabbit lungs were separated into large airways (trachea, carina, and primary bronchi) and more distal lung parenchyma either immediately after treatment for measurement of 99Tc distribution or 3 days after treatment for luciferase transgene expression. More instillate (94 +/- 1%) than aerosol (68 +/- 2%) was recovered in the parenchyma immediately after treatment (p < 0.01). Transgene expression in the parenchyma as a percent of total lung expression was lower for both instillation (72 +/- 8%) and aerosolization (30 +/- 8%) than for the initial distributions (p < 0.01). Surfactant did not change the distributions of 99Tc sulfur colloid or transgene expression. The adenovirus was aerosolized successfully but the distribution of the aerosol did not favor transgene expression in the lung parenchyma.

To define the toxicity of cystic fibrosis transmembrane conductance regulator gene (CFTR) gene therapy with a replication-deficient recombinant adenovirus (Av1Cf2) in a nonhuman primate model, 10(10) plaque forming units (pfu) were instilled directly through a bronchoscope into the right lung of 5 macaques, and a lower dose of 4 x 10(6) pfu was administered to the right lung of 1 macaque. One sham-treated control received phosphate-buffered saline (PBS). The macaques were evaluated sequentially by clinical examination, vital signs, weight, hematology, blood chemistry, chest radiography, pulse oximetry, and bronchoalveolar lavage (BAL) at baseline and 3-28 days post-treatment. After the period of observation, macaques were sacrificed for autopsy and histological examination. The macaques tolerated the experimental therapy clinically with no changes in body temperature, oxygen saturation, heart rate, body weight, or blood pressure. However, 1 macaque with visible evidence of aspiration at the time of initial bronchoscopy developed tachypnea with right lower lobe (RLL) pneumonia on chest radiograph and by histology. There were no changes in Hgb, Wbc, BUN, plasma electrolytes, bilirubin, or hepatic transaminases. In the macaques that received 10(10) pfu, there was a progressive increase in the number of CD8+ lymphocytes in BAL that was maximal at 28 days. Histological examination of the treated lungs of the high-dose macaques at 3 days showed marked peribronchial and perivascular cuffing by inflammatory cells and alveolar accumulation of neutrophils and macrophages. The alveolitis appeared to be resolving at 28 days, although the perivascular and peribronchial aggregates of mononuclear cells were still present. In the high-dose macaques, BAL interleukin-8 (IL-8) was increased at all time points (256-388 pg/ml versus 1-84 pg/ml at baseline and in control), whereas IL-1 beta was increased only at days 21 and 28 (341-852 pg/ml versus 30-92 pg/ml at baseline and in control). There were no increases in BAL cell counts, IL-1 beta or IL-8, and histological changes were mild in the macaque that received 4 x 10(6) pfu. Evaluation for Av1Cf2-derived human CFTR expression using RS-PCR demonstrated expression at 3, 10, and 21, but not 28 days in macaques treated with 10(10) pfu of Av1Cf2. In situ hybridization analysis demonstrated human CFTR mRNA in the alveolar regions of the lobes that received the vector at 10 and 21 days. There was no evidence of expression after treatment with 4 x 10(6) pfu. This study showed that high-dose adenoviral vector administration to the lung achieved CFTR gene transfer and expression but was associated with increased concentrations of cytokines in BAL and alveolar inflammation. A low dose, equivalent to the maximum clinical dose currently proposed for phase I trials in human subjects, was not associated with cellular or cytokine evidence of inflammation, and histological abnormalities were mild.

To evaluate the role of cell-mediated immunity during gene transfer to the respiratory epithelium, the time course of luciferase activity was assessed after intratracheal administration of Av1Luc1, an E1a-E3-deleted adenoviral (Ad5) vector expressing firefly luciferase, to FVB/N, BALB/c and BALB/c-nu/nu adult mice. Adenovirus-mediated luciferase activity was rapidly lost from the respiratory tract between 2 and 14 days after treatment of both FVB/N and BALB/c wild-type mice. In the wild-type mice, loss of luciferase activity was associated with an early inflammatory response consisting of infiltration with macrophages and polymorphonuclear leukocytes and a more prolonged response characterized by lymphocytic infiltration. In the immune-deficient nu/nu mice, luciferase activity was maintained at higher levels than in immune-competent mice after exposure to virus and was associated with a distinct pattern of inflammation, consisting primarily of macrophages and polymorphonuclear cells but lacking the lymphocytic infiltrates typical of the inflammation in wild-type mice. Adenoviral DNA was rapidly cleared from the lungs of both nu/nu and wild-type mice. Markedly increased expression of proliferating cell nuclear antigen (PCNA) was observed in bronchiolar and alveolar epithelial cells and in inflammatory cells after exposure to Av1LUc1. The proliferative response of the respiratory epithelium was more extensive and persistent in wild-type than in nu/nu mice. To assess further the impact of the immune system on adenovirus-mediated gene expression, cotton rats treated with cyclosporin A or dexamethasone were exposed to Av1Luc1. Both agents decreased lung inflammation and significantly increased lung luciferase activity. The loss of lung luciferase activity is dependent, in part, on the immune-mediated clearance of respiratory epithelial cells, which may limit the extent and duration of gene expression with recombinant adenoviral vectors.

Cystic fibrosis (CF) is a common, fatal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene manifested by abnormalities in the regulation of chloride ion (Cl-) secretion across the apical membrane of epithelial cells throughout the body. Adenovirus-mediated delivery of the normal CFTR cDNA and correction of the CF epithelial cell Cl- secretory phenotype suggests the feasibility of gene therapy for CF lung disease. Few studies, however, have focused on the evaluation of the safety of the adenovirus-mediated gene transfer approach. This study presents in vitro data on the efficacy and safety of adenovirus-mediated transfer of the human CFTR cDNA using Av1Cf2. Av1Cf2-mediated transfer of the human CFTR cDNA complemented the abnormal cAMP-regulated Cl- permeability of cells with the CF epithelial phenotype. Av1 vectors did not replicate infectious virus in HeLa cells infected in vitro, although trace vector DNA synthesis was observed at very high multiplicity of infection. Expression of the adenoviral late gene for the hexon capsid protein was observed at trace levels in Av1 vector-infected HeLa cells, but not in freshly isolated human bronchial epithelial cells, consistent with the pattern of DNA synthesis observed in these different target cells. Although, these observations support the efficacy and safety of use of Av1Cf2 for treatment of the fatal pulmonary component of CF.

Cystic fibrosis (CF) is a common, fatal hereditary disease resulting from mutations of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene in which epithelial cells throughout the body manifest altered regulation of apical membrane chloride secretion. Although the disease affects multiple organs throughout the body, over 90% of patients die of complications of the lung involvement. The feasibility of adenovirus-derived vectors for in vivo delivery of the human CFTR cDNA to treat the pulmonary component of CF is currently being evaluated using in vitro and in vivo approaches. Defining the therapeutic window between biological efficacy and toxicity is an important part of this work. Here we present data regarding the preclinical evaluation of the safety of in vivo delivery of the human CFTR cDNA to the cotton rat airway epithelium using the replication-deficient adenoviral vector Av1Cf2 or a similar vector, Av1LacZ4, expressing the Escherichia coli LacZ gene as a histologic marker. Gene transfer to the respiratory epithelium was efficient, as demonstrated by in situ hybridization and histochemical staining. Administration of these vectors resulted in a mild, transient, dose-dependent cellular inflammatory response similar in character to that seen with adenovirus 5 (Ad5), but far less in intensity, which was not associated with structural lung damage or mortality. Av1Cf2 DNA sequences were easily detected in the lung after pulmonary administration, but could not be demonstrated in organs other than the lung. These preclinical observations suggest that adenovirus-mediated gene transfer to the airway epithelium can be achieved efficiently, but is accompanied by a dose- and time-dependent inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviral vectors have an important role as in vivo gene delivery vehicles in developing human gene therapy for the fatal pulmonary component of cystic fibrosis. In this study we evaluated the immune responses to wild-type adenovirus and replication-deficient, first generation adenoviral (Av1) vectors in the cotton rat (Sigmodon hispidus) and then quantitatively evaluated the efficiency of gene delivery and expression of single and repeated in vivo administration of Av1 vectors to the respiratory tract. Av1 vector reporter gene expression was quantitatively evaluated by employing a luciferase expression vector (Av1Luc1) and measuring luciferase activity in whole lung tissue homogenates by routine luminometry. Gene transfer and expression in naive animals (e.g. first Av1 vector dose) was efficient. A repeat dose also resulted in successful gene transfer and expression, although at a significantly reduced level (p < 0.01) compared with naive animals. This reduction inversely correlated with serum human adenovirus neutralizing antibody (HANA) titers. Importantly, increasing doses of Av1Cf2, an Av1 vector expressing the human CFTR cDNA, resulted in a graded HANA response consistent with a lack of in vivo replication. These observations have significant implications for repeated administration of adenoviral vectors to the lungs of individuals with cystic fibrosis.

Gene therapy strategies designed to combat haemophilia B, caused by defects in clotting factor IX, have so far concentrated on ex vivo approaches. We have now evaluated adenoviral vector-mediated expression of human factor IX in vivo. Injection of the vector Av1H9B, which encodes human factor IX cDNA, into the tail veins of mice resulted in efficient liver transduction and plasma levels of human factor IX that would be therapeutic for haemophilia B patients. However, levels slowly declined to baseline by nine weeks and were not re-established by a second vector injection. These results address both the advantages and obstacles to the use of adenoviral vectors for treatment of haemophilia B.

The D variant of encephalomyocarditis virus (EMCV-D) produces a disease syndrome that mimics insulin-dependent diabetes mellitus (IDDM) in certain mouse strains. Benign EMCV-B interferes with the ability of EMCV-D to produce IDDM. Because EMCV-B induces the production of relatively large amounts of interferon (IFN), it has been hypothesized that the interference by EMCV-B with the pathogenesis of EMCV-D is due to IFN. However, we have previously reported that in outbred ICR Swiss and inbred BALB/cByJ mice, interference by EMCV-B with the development of IDDM in response to infection with EMCV-D does not appear to involve IFN. We have isolated a subvariant of EMCV-B (EMCV-B1) which, preliminary experiments indicate, does not induce the production of detectable levels of IFN in cell culture. Studies were initiated using this subvariant to determine more conclusively if IFN is involved in interference by EMCV-B with the pathogenesis of EMCV-D. The data in the present study show that EMCV-B1 does not induce the production of detectable levels of IFN either in cell culture or in mice, but retains other reported characteristics of the parent EMCV-B, including the ability to interfere with the production of IDDM by EMCV-D in ICR Swiss male mice. These observations strengthen the hypothesis that protection of pancreatic beta cells in ICR Swiss mice by EMCV-B occurs by a mechanism other than IFN.

We reviewed the clinical results and radiographs of four patients who had revision of the acetabular component of a failed total hip arthroplasty with a bipolar socket supplemented by autoallograft to reconstruct massive bony defect. These patients were followed up from twelve to seventeen months. The clinical score was assessed by a modified d'Aubigne's rating score that assigned a maximum of 18 points. The preoperative clinical score ranged from 1 to 5 points (mean: 3.25 points). The postoperative score improved to a range of 13-17 points (mean: 15.25 points). Of the hips examined, nearly all were pain-free. The largest possible bipolar cup was used with solid structural allografting augmented by morseled cancellous autograft. These have been uniformly trouble-free with acetabular reconstitution. Cup migration was minimal or none. The follow-up periods were short and some migration is probably to be anticipated in the future. Although this technique was originally conceived as part of a staged procedure, it seemed as a definitive procedure yielding acceptable results for dealing with a severely deficient acetabulum. Larger series and longer periods of follow-up are demanded to support this assertion.