Augmented adenovirus-mediated gene transfer to atherosclerotic vessels

Vascular endothelium is an important target for gene transfer in atherosclerosis. In this study, we examined gene transfer to normal and atherosclerotic blood vessels from two species, using an organ culture method. Using normal aorta, we determined optimal dose, duration of exposure to adenovirus, and duration of incubation of vessels in tissue culture. Aortas from normal and atherosclerotic monkeys were cut into rings and incubated for 2 hours with a recombinant adenovirus, carrying the reporter gene for beta-galactosidase driven by a cytomegalovirus (CMV) promoter. After 20 hours of incubation, transgene expression was assessed with a morphometric method after histochemical staining and a chemiluminescent assay of enzyme activity. Expression of beta-galactosidase after histochemical staining, expressed as percentage of total cells, was similar in adventitial cells of normal monkeys (21 +/- 4%, mean +/- SE%) and atherosclerotic monkeys (25 +/- 12%). Transgene expression in endothelium was higher in atherosclerotic than in normal vessel (53 +/- 3% versus 27 +/- 7%, P < .05). Chemiluminescent assay indicated greater beta-galactosidase activity (2.5 +/- 0.6 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.6 +/- 0.2 mU/mg of protein, P < .05). Aortas from normal (n = 6) and atherosclerotic (n = 5) rabbits also were examined. Transgene expression (after histochemical staining) in endothelium was much greater in atherosclerotic than normal rabbits (39 +/- 3% versus 9 +/- 2%, P < .05) and expression in adventitial cells was similar (normal 23 +/- 2%, atherosclerotic 24 +/- 4%). Chemiluminescent assay indicated greater beta-galactosidase activity (1.2 +/- 0.4 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.2 +/- 0.1 mU/mg protein, P < .05). These findings suggest that an adenoviral vector with a CMV promoter provides similar transgene expression in adventitia of both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus it may be possible to achieve greater transgene expression in atherosclerotic than in normal arteries.     

Enhanced in vivo adenovirus-mediated gene transfer to rat hepatocarcinomas by selective administration into the hepatic artery

Adenovirus-mediated gene therapy of experimental hepatocarcinoma is hindered by low transduction efficacy in vivo. We evaluated the extent of gene expression following various routes of administration of recombinant adenovirus AdCMVlacZ in diethylnitrosamine-induced rat hepatocarcinoma. We first characterized the vascularization of diethylnitrosamine-induced hepatocarcinomas using a computerized tomography scanner approach. The efficacy of gene transfer was then evaluated by three routes of administration: intraportal, selective injection through the hepatic artery and direct injection into the tumor. Diethylnitrosamine-induced hepatocarcinomas had predominantly an arterial blood supply, 67% of the total liver blood supply. Compared with intraportal administration, arterial injection improved gene transfer into tumors whereas that to the non-tumor areas was diminished. In addition, this route of injection allowed the efficient transduction of dysplastic nodules. Diethylnitrosamine-induced hepatocarcinoma in rats is a relevant model for the study of human hepatocarcinoma due to its vascularization. Arterial infusion improved the ratio of transduced tumorous to nontumorous cells and allowed targeting of gene transfer to dysplastic nodules. This will be useful in the design of gene therapy for hepatocarcinoma.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

p53 gene transfer to the injured rat carotid artery decreases neointimal formation

PURPOSE: We studied the effect of adenovirus-mediated p53 gene transfer on the injured rat carotid artery to determine its ability to decrease the formation of neointima. METHODS: In vivo gene transfer was used in isolated segments of balloon-injured rat carotid arteries. Genetically modified adenovirus containing the gene encoding for wild-type p53 (AdWTp53) was applied in three concentrations: 8 x 10(10), 1.6 x 10(10), and 8 x 10(9) pfu/mL. Control rats received either adenovirus null (AdNull), 8 x 10(10) pfu/mL, or Medium-199 solution (vehicle). Expression of p53 was determined 4 days after gene transfer by Western blotting. Neointimal formation was assessed after 14 days by harvesting carotid arteries and determining the intima/media (I/M) ratio based on cross-sectional area measurement. Simultaneously, immunohistochemistry was done to detect the presence of p53 on smooth muscle cell nuclei. RESULTS: P53 expression was confirmed by Western blotting. There was a significant reduction in neointimal formation on all treated animals compared with controls. The highest dose of AdWTp53 (8 x 10(10) pfu/mL) resulted in a near-total arrest of neointimal formation (I/M = 0.09 +/- 0.03, mean +/- SEM) with P <. 0001 versus vehicle (I/M = 2.23 +/- 0.15) or AdNull (I/M = 2.12 +/-. 12). The intermediate dose of AdWTp53 (1.6 x 10(10) pfu/mL) resulted in an I/M value of 1.04 +/- 0.18, with P <.001 versus vehicle and P =.001 versus AdNull. The lowest dose (8 x 10(9) pfu/mL) resulted in an I/M value of 1.12 +/- 0.18, with P <.001 versus vehicle and P <. 002 versus AdNull. The immunohistochemistry was positive for the presence of p53 in rats infected with AdWTp53. CONCLUSIONS: Adenovirus-mediated gene transfer of p53 protein significantly decreases the formation of neointima in the rat carotid injury model. This may represent a potential therapy for restenosis in humans.     

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.     

Transgenesis by adenovirus-mediated gene transfer into mouse zona-free eggs

Zona-free mouse eggs at the pronucleus stage were infected with a replication-defective adenovirus vector containing a nuclear-targeted lacZ gene. Exogenous beta-galactosidase activity was detected in almost all eggs at the two-cell stage. Of 27 mice that developed from infected eggs, three carried the integrated exogenous gene mediated by the adenovirus. Two of the three expressed the lacZ gene, and all three mice transmitted the adenovirus-mediated transgene to F1 progeny Southern blot analysis was consistent with single copy integration. This finding should accelerate the development of new strategies for transgenesis and assist studies on the function of cloned genes in vivo.     

Apoptosis by retrovirus- and adenovirus-mediated gene transfer of Fas ligand to glioma cells: implications for gene therapy

Astrocytic tumors frequently express Fas/APO-1 (Fas), in sharp contrast to surrounding normal brain cells, providing a potential window through which selective killing of tumor cells could be pursued. To assess this possibility, we transduced Fas into U251, a glioma cell line resistant to anti-Fas antibody-mediated apoptosis, and obtained transfectants with high levels of Fas expression. Anti-Fas antibody showed significantly enhanced cytotoxicity for the transfectants, suggesting that U251 cells maintained an intercellular cascade of Fas-mediated apoptosis. When U251 transfectants with high-level Fas expression were transduced with Fas ligand-encoding gene via retrovirus, they were unaffected by exposure to anti-Fas antibody or Fas ligand adenovirus (Adeno-FL). Thus, retroviral induction of Fas ligand into the glioma cells with high levels of Fas led to the selection of cells that were resistant to Fas-dependent apoptosis. These resistant U251 transfectants were susceptible to FADD adenovirus (Adeno-FADD)-induced apoptosis, indicating that a cascade of death signals was blocked at the steps between Fas ligand and FADD. As for adenoviral transduction of Fas ligand into gliomas, gliomas with a relatively high level of expression of Fas were remarkably sensitive to Adeno-FL-induced apoptosis. Besides, Adeno-FADD induced pronounced apoptosis in all glioma cells. Our data suggest the possibility of using adenovirus-mediated transduction of Fas ligand and FADD genes as a therapeutic approach to target gliomas.     

Extensive beta-glucuronidase activity in murine central nervous system after adenovirus-mediated gene transfer to brain

Mucopolysaccharidosis type VII (MPS VII), caused by beta-glucuronidase deficiency, is a classic lysosomal storage disease. In the central nervous system (CNS), there is widespread pathology with distention of vacuoles in neurons and glia. An approach to therapy for MPS VII would require extensive delivery of enzyme to the CNS and subsequent uptake by the affected cells. In this study we show that intrastriatal injection of recombinant adenovirus encoding beta-glucuronidase (Ad betagluc) to MPS VII or wild-type mice results in focal, intense beta-glucuronidase mRNA expression near the injection site. Further, histochemical staining for enzyme activity showed that beta-glucuronidase activity extended well beyond transduced cells. Activity was detected throughout the ipsilateral striatum as well as in the corpus callosum, ventricles, and bilateral neocortex. Similarly, after injection into the right lateral ventricle or cisterna magna, enzyme activity was present in the ependymal cells of the ventricles, in the subarachnoid spaces, and also in the underlying cortex (150-500 microm from ependyma). The distribution of enzyme was most extensive 21 days after gene transfer to normal mouse brain, with more than 50% of the hemisphere positive for beta-glucuronidase activity. Eighty-four days after adenovirus injection a substantial level of enzyme expression remained (>40% of hemisphere positive for beta-glucuronidase activity). Histological sections from striatum of beta-glucuronidase-deficient mice injected with Ad betagluc showed a marked reduction in the number of distended vacuoles in both neurons and glia, as compared with uninjected striatum. Importantly, correction was noted in both hemispheres. Our finding that a relatively small number of transduced cells produce enzyme that reaches a large proportion of the CNS has favorable implications in developing direct gene transfer therapies for lysosomal storage disorders.     

Increased contact time improves adenovirus-mediated CFTR gene transfer to nasal epithelium of CF mice

By combining retrograde and anterograde tracing, evidence for a bineuronal connection from the suprachiasmatic nucleus (SCN) to the intermediolateral cell column in the spinal cord (IML) was obtained. The retrograde tracer cholera toxin subunit B (ChB) was pressure-injected into the spinal cord and the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was iontophoretically injected into the SCN. The two tracers were visualized simultaneously by a double immunohistochemical procedure. In the hypothalamus, ChB injections gave rise to retrogradely labeled cell bodies in the paraventricular nucleus, retrochiasmatic area, perifornical region, lateral hypothalamic area, and the posterior hypothalamic area. The SCN were found to project to all of these areas. Furthermore, spinal-projecting neurons were found in the brain stem, but no efferents from the SCN were observed to innervate these areas. In the most sparsely innervated areas, the lateral hypothalamic area and the perifornical region, only occasionally a PHA-L fiber in close apposition to a ChB-ir cell body was observed. This was also the case in the retrochiasmatic area and posterior hypothalamic area, although these areas received a moderate number-immunoreactive (ir) PHA-L-ir fibers. The highest number of closely apposed PHA-L-ir fibers and ChB-ir cell bodies was observed in the dorsal parvicellular and in the ventral division of the medial parvicellular paraventricular nucleus, which were also the areas receiving the densest input from the SCN. By anterograde tracing from the paraventricular nucleus of the hypothalamus, the exact topography of the terminal field formed by descending paraventricular neurons was established. Thus, it was confirmed that the paraventricular nucleus of the hypothalamus predominantly innervates the IML. The present study suggests the existence of a bineuronal link between the SCN and the IML, possibly involved in transmission of circadian signals from the endogenous clock to the pineal gland and other organs receiving sympathetic afferents.     

Systemic action of human growth hormone following adenovirus-mediated gene transfer to rat submandibular glands

1. The purpose of this study was to compare the effect of NIK-247 on muscarinic receptor subtypes with that of tacrine (THA) in rats. 2. NIK-247 and tacrine dose dependently inhibited the binding of [3H]pirenzepine (M1), [3H]AF-DX 384 (M2), and [3H]4-DAMP (M3). The IC50 values for NIK-247 were 4.4 x 10(-6) M, 1.1 x 10(-5) M, and 1.5 x 10(-5) M, respectively, whereas those for tacrine were 5.8 x 10(-7) M, 2.0 x 10(-6) M, and 5.8 x 10(-6) M, respectively. 3. Gpp[NH]p, a GTP analogue, slightly shifted the curve of displacement of [3H]AF-DX. 384 binding for NIK-247 to the right. However, Gpp[NH]p did not shift the curve of displacement of [3H]pirenzepine and [3H]4-DAMP binding to the right. 4. NIK-247 moderately decreased the rate of beating in right atrial preparations, but did not decrease it below 50% of control level. 5. These findings indicate that NIK-247 is an M1 antagonist, M2 partial agonist, and M3 antagonist.     

Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts: method and efficacy of adenovirus-mediated transduction

OBJECTIVE: Allograft-targeted immunosuppressive gene therapy may inhibit recipient immune activation and provide an alternative to systemic immunosuppression. We studied the optimal technique and efficacy of intracoronary gene transfer of viral interleukin-10 and human transforming growth factor-beta 1 in a rabbit model of heterotopic heart transplantation. METHODS: Replication-defective adenoviral vectors were constructed, expressing viral interleukin-10 (AdSvIL10) or transforming growth factor-beta 1 (AdCMVTGF-beta 1). Intracoronary delivery of vectors was accomplished ex vivo by either bolus injection or slow infusion. The allografts were implanted heterotopically in recipient rabbits and collected 4 days after the operation. Vector dose was 4 x 10(9) to 6 x 10(10) pfu/gm of donor heart. Transfer was confirmed by DNA amplification for both genes. Gene product expression in tissue was quantified by immunoassay and visualized by immunohistochemical staining. RESULTS: Allograft viral uptake was only 9.9% +/- 2.4% with bolus injection, but increased to 80.5% +/- 6.8% at 1 ml/min infusion rate (p = 5 x 10(-14)). Uptake ratio was not affected by vector quantity or slower infusion rates. Transforming growth factor-beta 1 was consistently detected in allografts infected with AdCMVTGF-beta 1, but not with control adenovirus or AdSvIL10. Expression was proportional to infused vector quantity and reached 10 ng/gm of allograft at infused 10(10) pfu/gm. Transforming growth factor-beta 1 was also detected in recipient's serum at less than 1 ng/ml. Viral interleukin-10 was detected in minor amounts only (< 1 ng/gm) in allografts infected with AdvIL10 up to 5 x 10(10) pfu/gm. Nevertheless, it was detected in recipient serum at concentrations up to 0.4 ng/ml. CONCLUSIONS: Intracoronary gene transfer of immunosuppressive cytokines to cardiac allografts during cold preservation is feasible. Slow infusion is superior to bolus injection. In vivo effects on allograft rejection remain to be determined.     

Interferons impair early transgene expression by adenovirus-mediated gene transfer in muscle cells

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.     

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.     

Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract

Two genes for Ca2+-dependent protein kinases, PCaPK-alpha and PCaPK-beta, were isolated from a Paramecium genomic DNA library. The coding region of PCaPK-alpha encoded 481 amino acids and that of PCaPK-beta encoded 493 amino acids, predicting molecular masses of 55603 Da and 57131 Da for each putative protein. The sequences of the protein kinase catalytic domains of PCaPK-alpha and PCaPK-beta were closely related to those of the Ca2+-dependent protein kinases (CDPKs) from Plasmodium, Eimeria, and several plants, and the catalytic region of the Ca2+/calmodulin-dependent protein kinase family (35-48% identity). In the junction region between the catalytic and regulatory regions, only 9 of 31 amino acid residues are the same in the two Paramecium genes, and the sequences encoded in the Paramecium genes differ from those in the plant CDPK genes in about 20 of 31 residues in the junction region. The C-terminal region of the Paramecium kinases shared sequence similarity with Paramecium calmodulin (30-34% identity). Two Ca2+-dependent protein kinases previously characterized from Paramecium (52 kDa CaPK-1, and 50 kDa CaPK-2) are activated by Ca2+ in the micromolar concentration range and they directly bind Ca2+ in a 45Ca2+ overlay blot assay. The size predicted from the genes, the presence of four putative Ca2+-binding motifs encoded in PCaPK-alpha and PCaPK-beta, and the immunological cross-reaction of expressed cloned fragments of these genes with CaPK-2, suggest that they encode proteins of the same family.