AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

A hypoxic inducible factor-1 alpha hybrid enhances collateral development and reduces vascular leakage in diabetic rats.

BACKGROUND: Diabetes mellitus is a common comorbidity of atherosclerosis. Hypoxia-inducible factor-1 (HIF-1) is the master regulator of the angiogenic response to hypoxia. METHODS: We studied the effects of adenoviral vectors expressing a constitutively active HIF-1 alpha hybrid (Ad2/HIF-1 alpha/VP16) or vascular endothelial growth factor (Ad2/VEGF) on collateral development and vascular leakiness in a diabetic rat model of hindlimb ischemia. RESULTS: After the removal of the right femoral artery, the mRNA levels of VEGF, angiopoietin-1 and angiopietin-4 in the calf muscles, as measured by Taqman reverse transcriptase-polymerase chain reaction, were transiently elevated in Zucker lean (ZL) but not Zucker diabetic fatty (ZDF) rats. The angiographic score, as determined by post-mortem angiography, was significantly lower in ZDF animals 35 days after surgery compared to their ZL counterparts. In separate animals, intramuscular injection of Ad2/HIF-1a/VP16 and Ad/2VEGF into the thigh muscles significantly increased the angiographic score and capillary density 21 and 35 days after the injection compared to Ad2/CMVEV (a vector expressing no transgene) or vehicle. After the injection of Ad2/CMVEV or vehicle, the Evans-blue dye content in the thigh muscles was significantly higher in ZDF rats than their ZL counterparts. Ad2/HIF-1 alpha/VP16 but not Ad2/VEGF reduced tissue Evans blue dye content. CONCLUSIONS: The endogenous angiogenic response to ischemia was impaired in ZDF rats, possibly due to down-regulation of angiogenic factors. Ad2/HIF-1 alpha/VP16 enhanced collateral development and reduced vascular leakage in the ischemic hindlimb of ZDF rats indicating that hybrid HIF-1 alpha angiogenic therapy may be efficacious for peripheral vascular disease with a diabetic comorbidity.

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor alpha/retinoid X receptor.

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid beta-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid beta-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1alpha/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for beta-oxidation. Furthermore, adenovirus-mediated expression of HIF-1alpha/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor alpha (PPARalpha)/retinoid X receptor (RXR), in the presence or absence of a PPARalpha ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPARalpha/RXR.

A constitutively active hypoxia-inducible factor-1alpha/VP16 hybrid factor activates expression of the human B-type natriuretic peptide gene.

Hypoxia-inducible factor-1 (HIF-1) is a primary regulator of the physiological response to hypoxia. A recombinant adenovirus expressing a constitutively active hybrid form of the HIF-1alpha subunit (Ad2/HIF-1alpha/VP16) is being evaluated as a gene therapy for the treatment of peripheral vascular disease. Ad2/HIF-1alpha/VP16 up-regulates known HIF-1-responsive genes, including those involved in angiogenesis. Expression profile analysis revealed that the brain natriuretic peptide (BNP) gene was significantly up-regulated in response to HIF-1alpha/VP16 in human fetal cardiac cells. Real-time reverse transcription-polymerase chain reaction analyses confirmed transcriptional activation of the BNP gene by HIF-1alpha/VP16 in human but not rat cardiac cells. Because hypoxia itself did not increase human BNP gene expression in these analyses, the mechanism of the HIF-1alpha/VP16 effect was determined. Analyses of promoter deletion mutants suggested that the cis-acting sequence in the human BNP promoter mediating activation by HIF-1alpha/VP16 was a putative HIF-1 responsive element (HRE) located at -466. An SV40 basal promoter-luciferase plasmid containing a minimal BNP HRE was up-regulated by HIF-1alpha/VP16, whereas a similar construct carrying a mutation within the HIF-1 binding site was not. Mutation of an E-box motif within the BNP HRE reduced HIF-1alpha/VP16-mediated transcriptional activation by 50%. Gel-shift analyses showed that both the native HIF-1alpha and HIF-1alpha/VP16 are able to bind to a probe containing the HIF-1 binding site. These experiments demonstrate the existence of a functional HRE in the BNP promoter and further define the scope and mechanism of action of Ad2/HIF-1alpha/VP16.

Adenovirus-mediated expression of beta-adrenergic receptor kinase C-terminus reduces intimal hyperplasia and luminal stenosis of arteriovenous polytetrafluoroethylene grafts in pigs.

BACKGROUND: Hemodialysis vascular access dysfunction is the single most important cause of morbidity in kidney hemodialysis patients. Failure of an arteriovenous polytetrafluoroethylene (PTFE) graft, the most common form of hemodialysis access, is primarily due to intimal hyperplasia and thrombosis at the venous anastomosis. METHODS AND RESULTS: This study was aimed at evaluating the efficacy and safety of an adenoviral vector (Ad2/betaARKct) encoding the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct) in a pig model of arteriovenous PTFE graft failure. Transduction of the external jugular vein with Ad2/betaARKct (5E9, 5E10, or 5E11 particles per vein) did not result in systemic toxicity, as measured by clinical and pathological assessments. Ad2/betaARKct significantly reduced neointimal hyperplasia in the graft/vein anastomosis. It also improved the graft patency rate and angiographic score, as measured histologically and angiographically, compared with vehicle or empty viral vector controls. CONCLUSIONS: Our results suggest that local administration of adenoviral vectors encoding betaARKct into the jugular vein represents a viable strategy to treat AV graft hemodialysis vascular access failure.

Innate immunity and angiogenesis.

Activation of an innate immune response is among the first lines of defense after tissue injury. Restoring blood flow to the site of injured tissue is often a necessary prerequisite for mounting an initial immune response to pathogens and for subsequent initiation of a successful repair of wounded tissue. The multiple links among pathogen recognition and suppression, increased angiogenesis, and tissue repair are the topics of this review, which examines of the roles of antimicrobial peptides, mammalian toll-like receptors (TLRs), inflammatory cytokines, and putative "danger" signals, among other signaling pathways, in triggering, sustaining, and then terminating an angiogenic response.

Expression of constitutively stable hybrid hypoxia-inducible factor-1alpha protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury.

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxia-inducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-1alpha on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/HIF-1alpha/herpes simplex virus protein VP16 and Ad2/HIF-1alpha/nuclear factor-kappaB (NF-kappaB)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1alpha/VP16 and Ad2/HIF-1alpha/NF-kappaB mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1alpha protein levels. These results suggest that adenovirus-mediated expression of HIF-1alpha/VP16 or HIF-1alpha/NF-kappaB, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries.

BACKGROUND: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis. METHODS: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.

Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors.

Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1alpha mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1alpha/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1alpha, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1alpha/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1alpha/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia- or Ad2/HIF-1alpha/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability.

Differential effects of membrane and soluble Fas ligand on cardiomyocytes: role in ischemia/reperfusion injury.

Cardiomyocyte apoptosis by Fas ligand (FasL)/Fas signaling is associated with various pathophysiological conditions, such as ischemia/reperfusion injury and congestive heart failure. In this study, we tested the hypothesis that shedding of membrane FasL is a mechanism for downregulating FasL/Fas signaling and both membrane and soluble FasL are involved in cardiomyocyte hypoxia/reoxygenation (H/R) injury. We also examined the relative importance of mitochondrial damage and direct cleavage of the executioner caspases by activated initiator caspase 8 in the propagation of FasL/Fas signaling activated by either recombinant membrane FasL or H/R. We demonstrated that in neonatal rat cardiomyocytes maintained under normal culture conditions, recombinant human soluble FasL increased caspase 3 activation by twofold but did not reduce cell viability. In contrast, infection with a recombinant adenoviral vector expressing the non-cleavable human FasL (Ad2/nchFasL) resulted in cardiomyocyte death that was attenuated by soluble FasL. H/R increased the mRNA levels of both FasL and Fas and activated caspases 8, 9 and 3, indicating the activation of FasL/Fas signaling. Z-IETD.fmk and Z-LEHD.fmk, selective inhibitors for caspases 8 and 9, respectively, abolished caspase 3 activation induced by Ad2/nchFasL or H/R. Z-IETD.fmk also significantly reduced Ad2/nchFasL- or H/R-induced cardiomyocyte death. H/R potentiated membrane FasL-induced cell death. These results suggest that shedding of membrane FasL downregulates FasL/Fas signaling in cardiomyocytes and both membrane and soluble FasL contribute to H/R injury. Activation of FasL/Fas signaling by either recombinant membrane FasL under normal culture conditions or H/R causes cardiomyocyte death mainly through the mitochondrial damage/caspase 9 activation pathway.

Hypoxia up-regulates expression of peroxisome proliferator-activated receptor gamma angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1alpha.

Peroxisome proliferator-activated receptors (PPAR), especially the PPARalpha and PPARgamma, are associated with an extraordinary diverse spectrum of cardiovascular diseases including hypertension, angiogenesis, cardiac hypertrophy, and atherosclerosis. PGAR (for PPAR gamma angiopoietin-related gene) is a recently identified PPAR target gene which is associated with adipose differentiation, systemic lipid metabolism, energy homeostasis, and possibly angiogenesis. We report here that WY-14643, a selective PPARalpha ligand up-regulated PGAR expression in neonatal rat cardiomyocytes. In parallel to activating the expression of vascular endothelial growth factor and glucose transporter-4, hypoxia increased PGAR mRNA levels. PGAR expression was also increased by desferrioxamine and CoCl(2), but not by sodium cyanide, results consistent with the pharmacological features of hypoxia-responsive genes. These studies are the first to demonstrate that hypoxia increases the mRNA levels of a PPAR target gene in cardiomyocytes. Furthermore, infection with adenoviral vectors encoding the wild-type or a hybrid form of HIF-1alpha highly increased PGAR mRNA levels. In contrast, neither hypoxia nor overexpression of HIF-1alpha affected the mRNA levels of PPARalpha, PPAR gamma, and muscle carnitine palmitoyltransferase, a known PPARalpha target gene. These results suggest that hypoxic activation of PGAR expression is likely mediated by HIF-1 but not the PPARalpha/RXR pathway.

Gene expression profiles in human cardiac cells subjected to hypoxia or expressing a hybrid form of HIF-1 alpha.

The cellular response to hypoxia depends on rapid posttranslational modifications of proteins as well as regulation of gene expression. We performed serial analysis of gene expression (SAGE) on human cardiac cells under normoxia, subjected to hypoxia, or infected with Ad2/HIF-1alpha/VP16 (an adenoviral vector expressing a stable hybrid form of hypoxia-inducible factor 1alpha) or Ad2/CMVEV (an empty vector). Of the 97,646 SAGE tags that were sequenced, 27% matched GenBank entries, while an additional 32% matched expressed sequence tags (ESTs) in UniGene. We analyzed 161 characterized genes or ESTs with a putative identification. Expression of 35, 11, and 46 genes was increased by hypoxia, infection with Ad2/EVCMV, or infection with Ad2/HIF-1alpha/VP16, respectively, compared with normoxia; conversely, 20, 11, 38 genes, respectively, were expressed at lower levels. Genes regulated by hypoxia were associated with transcription, biosynthesis, extracellular matrix formation, glycolysis, energy production, cell survival, and cell stress. Changes following infection with Ad2/HIF-1alpha/VP16 mimicked the hypoxic response to a certain extent. Infection with Ad2/CMVEV affected expression of genes that were associated with extracellular matrix formation and membrane trafficking. Differential expression of select genes was confirmed using TaqMan in additional human cardiac cells and rat neonatal ventricular myocytes. These data provide insight into gene expression underlying the diverse and complex cellular response to hypoxia, expression of HIF-1alpha/VP16, or adenoviral infection.

Stabilization of vascular endothelial growth factor mRNA by hypoxia-inducible factor 1.

Hypoxia regulates expression of vascular endothelial growth factor (VEGF) by increasing its transcription and by stabilizing its mRNA. Despite the pivotal role of hypoxia-inducible factor 1 (HIF-1) in transcriptional activation of hypoxia-responsive genes, it is not known whether HIF-1 mediates hypoxia-induced stabilization of VEGF mRNA. We constructed adenoviral vectors expressing either the wild-type HIF-1 alpha (Ad2/HIF-1 alpha/FL), a constitutively stable hybrid form of HIF-1 alpha (Ad2/HIF-1 alpha/VP16), or no transgene (Ad2/CMVEV). In rat glioma (C6) cells and human cardiac, vascular smooth muscle, and endothelial cells, infection with Ad2/HIF-1 alpha/VP16 or Ad2/HIF-1 alpha/FL increased VEGF expression at both the mRNA and protein levels. Under normoxic conditions, the half-life of VEGF mRNA was 42 min in C6 cells. Hypoxia and Ad2/HIF-1 alpha/VP16 increased the half-life of VEGF mRNA to 3.3 and 2.7 h, respectively, while Ad2/CMVEV had no effect. These studies are the first to demonstrate that overexpression of HIF-1 alpha increases VEGF mRNA stability. Our results also suggest that stabilization of VEGF mRNA by hypoxia is mediated, at least in part, by HIF-1.

Harnessing the response to tissue hypoxia: HIF-1 alpha and therapeutic angiogenesis.

In most cell types a family of transcriptional regulatory proteins termed hypoxia-inducible factors, of which HIF-1 is the most prevalent, mediates the physiologic response to hypoxia. Although much has been learned over the past decade since the discovery of the first HIF family member, the proximal signaling events linking a decline in oxygen concentration to the activation of HIF-dependent signaling are only now being clarified. Activation of HIF-1 in eukaryotes induces expression of many genes that assist in adapting the organism to an environment in which oxygen is limiting, such as of genes involved in new blood vessel formation, including isoforms of vascular endothelial growth factor and angiopoietins, among others. Targeted expression of constitutively active HIF transgenes to ischemic tissues may be beneficial as a form of therapeutic angiogenesis.