The efficiency of first-generation adenoviral vectors as gene delivery tools is often limited by the short duration transgene expression, which can be related to immune responses and toxic effects viral proteins. In addition, readministration usually ineffective unless animals are immunocompromised or a different adenovirus serotype used. Recently, devoid all coding sequences (helper-dependent gutless vectors) have been developed avoid expression mice, liver-directed transfer with AdSTK109,...

Replication-defective recombinant adenovirus (Ad) vectors are under development for a wide variety of gene therapy indications. A potential limiting factor associated with virus requiring repeated treatment is the humoral immune response to vector by host. In animal models, there dose-dependent rise in neutralizing antibodies after primary administration, which can preclude effective repeat administration. The strategy we have developed circumvent neutralization mask their surface covalent...

Intact DNA molecules extracted from HSV-1 (herpes simplex virus 1, human herpes 1) strain MP virions have a molecular weight of approximately 97 X 10(6), but cleavage with the HinIII restriction enzyme yields fourteen fragments summed weights 160 10(6). Six "major" occur once in every molecule population and account for 60% genetic information. Four "minor" are present amounts equivalent to one copy two genomes (0.5 molar ratio) other four only (0.25 ratio). The minor can be arranged into...

Electron microscope studies on self-annealed intact single strands and partially denatured molecules show that herpes simplex virus 1 DNA consists of two unequal regions, each bounded by inverted redundant sequences. Thus the region L (70 percent contour length DNA) separates left terminal a1b from its repeat b'a'1, which comprises 6 DNA. The S (9.4 right cas (4.3 a'sc'. regions termini are repeated itnernally differ in topology. Thus, is guanine plus cytosine rich, whereas only region,...

The utility of adenovirus vectors for gene therapy is limited by the transience expression that has been observed in various vivo models. Immunological responses to viral targets can eliminate transduced cells and cause loss transgene expression. We previously described characterization an E4 modified adenovirus, Ad2E4ORF6, which replication defective cotton rats. reasoned transfer based on Ad2E4ORF6 would have a reduced potential might be beneficial achieving persistence E1 replacement...

AAV2-sFLT01 is a vector that expresses modified soluble Flt1 receptor designed to neutralize the proangiogenic activities of vascular endothelial growth factor (VEGF) for treatment age-related macular degeneration (AMD) via an intravitreal injection. Owing minimal data available route administration adeno-associated virus (AAV), we initiated 12-month safety study administered intravitreally at doses 2.4 × 10(9) genomes (vg) and 10(10) vg cynomolgus monkeys. Expression sFlt01 protein peaked...

Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production clinical-grade rAAV for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard homogeneity, purity, and manufacturing consistency. Here, we demonstrate the application ultracentrifugation (AUC) characterize homogeneity preparations vectors. We show single sedimentation velocity run detected quantified number different...

Recombinant adenoviruses are currently being used as vectors for gene delivery to a wide variety of cells and tissues. Although generally efficacious transfer in vitro, improvement the efficiency vector vivo may aid several therapy applications. One major obstacle is lack high-affinity viral receptors on surface certain that targets therapy. In principle, incorporation avid, cell-specific ligands into virion could markedly improve entry desired We have developed strategy addressing this...

We describe the construction and characterization of an adenovirus type 2 vector, Ad2E4ORF6, which has been modified in E4 region to contain only open reading frame 6. When assayed cultured cells, Ad2E4ORF6 virus replication is slightly delayed but viral DNA synthesis, host-cell protein synthesis shut-off, yield are indistinguishable from wild type. Late normal with exception fiber reduced approximately 10-fold. Despite particles appear a full complement protein. Virus cotton rats indicates...

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production such vectors, functions supplied in trans from a stable cell line. Virtually all Ad used clinical studies produced the 293 cell, human embryonic kidney line expressing an integrated segment left end type 5 (Ad5) genome. Replication-competent vector variants that have regained sequences been observed within populations grown on cells....

The successful application of gene therapy for the treatment genetic diseases such as Fabry is reliant on development vectors that are safe and facilitate sustained expression therapeutic levels transgene product. Here, we report intravenous administration a recombinant AAV2 vector encoding human alpha-galactosidase A under transcriptional control liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher in BALB/c mice than could be realized using ubiquitous CMV...

Bifunctional PEG (polyethylene glycol) molecules provide a novel approach to retargeting viral vectors without the need genetically modify vector. In previous report we showed that modification of capsid by addition peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery those cells entry pathway. Here demonstrate further versatility this method coupling protein, FGF2, surface an adenovirus (Ad). This results in elimination endogenous tropism virus...

One potential limitation of adenovirus (Ad)-based vectors for the gene therapy cystic fibrosis (CF) and other genetic diseases is transience expression observed in most vivo systems. In this study, influence various factors on persistence transgene lung was investigated. absence immune pressure, such as nude mouse, genomic structure vector found to be predominant determining expression; Ad constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or transmembrane...

Adeno-associated virus (AAV) is a human parvovirus currently being developed as vector for gene therapy applications. Because the transfer commonly retains only AAV terminal repeats, propagation of recombinant (rAAV) requires that viral replication (Rep) and capsid (Cap) proteins be supplied in trans. In an effort to optimize production these vectors, panel helper plasmids was constructed determine if expression rep and/or cap genes limiting factor rAAV packaging. Expression Rep Cap...

Native DNA from four strains of herpes simplex virus 1 (HSV-1) circularized after digestion with the lambda exonuclease, indicating that molecules were terminally repetitious. In two strains, terminal repetition was evident in nearly 50% molecules. Maximal circularization observed when only 0.25 to 0.5% depolymerized by suggesting minimal size repetitious regions is range 400 800 bases pairs. More extensive exonuclease treatment resulted a reduction frequency circularization. To determine...

Inhibition of vascular endothelial growth factor (VEGF) for the management pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required optimal treatment. We have previously shown that novel, secreted anti-VEGF molecule sFLT01 delivered by injection an AAV2 vector (AAV2-sFLT01) gives persistent expression and efficacious in murine model retinal...

Adeno-associated virus (AAV) producer cell lines represent an effective method for large-scale production of AAV vectors. We set out to evaluate and characterize the use abbreviated protocol generate “masterwells” (MWs; a nonclonal population) as platform research preclinical vector production. In this system, single plasmid containing three components, sequence, rep, cap genes, selectable marker gene is stably transfected into HeLaS3 cells. Producer generating AAV2 expressing secreted form...

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic trans-membrane conductance regulator (CFTR) gene, is most common lethal genetic illness Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads transient production of mRNA and, some studies, correction epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses adenoviral vector have been reported, particularly at high...

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) administered by bronchoscopic instillation or aerosolization to lungs of (CF) patients. In this paper, we evaluate efficiency Ad2/CFTR-mediated transduction bronchial airway cells. The ability an Ad2/CFTR vector transduce cells first evaluated patients whom instillation. Cells at administration site were collected 2 days after treatment brushing....