Read-through Activation of Transcription in a Cellular Genomic Context

Read-through transcription from the adjacent E1a gene region is required for wild-type (wt) activity of the downstream adenovirus E1b promoter early after infection (read-through activation). However, whether a cellular chromosomal template can support read-through activation is not known. To address this issue, read-through activation was evaluated in the context of stably expressed templates in transfected cells. Inhibition of read-through transcription by insertion of a transcription termination sequence between the E1a and E1b promoters reduced downstream gene expression from stably integrated templates. The results indicate that the mechanism of read-through activation does not depend on the structure of early adenovirus nucleoprotein complexes, a structure that is likely to be different from that of cellular chromatin. Accordingly, this regulatory interaction could participate in the coordinated control of the expression of closely linked cellular genes

Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes)

Attenuation of Vaccinia Tian Tan Strain by Removal of Viral TC7L-TK2L and TA35R Genes

Vaccinia Tian Tan (VTT) was attenuated by deletion of the TC7L-TK2L and TA35R genes to generate MVTT3. The mutant was generated by replacing the open reading frames by a gene encoding enhanced green fluorescent protein (EGFP) flanked by loxP sites. Viruses expressing EGFP were then screened for and purified by serial plaque formation. In a second step the marker EGFP gene was removed by transfecting cells with a plasmid encoding cre recombinase and selecting for viruses that had lost the EGFP phenotype. The MVTT3 mutant was shown to be avirulent and immunogenic. These results support the conclusion that TC7L-TK2L and TA35R deletion mutants can be used as safe viral vectors or as platform for vaccines

A novel attenuated replication-competent adenovirus for melanoma therapy.

To generate a replication-competent adenovirus (Ad) with specificity for melanoma, we constructed a tissue-specific promoter restricting E1A expression to melanoma cells. The combination of four copies of a mouse tyrosinase enhancer element (TE) fused to the human tyrosinase promoter (TP) yielded up to 2000-fold higher luciferase reporter activity in tyrosinase-expressing melanoma cells than in nonmelanoma cells. Insertion of the composite TETP construct upstream of the E1A gene was combined with deleting as far as possible the intertwined endogenous Ad enhancer/promoter (EP). The resulting AdDeltaEP-TETP vector, also deleted for the E3 region, was found to replicate in tyrosinase-positive melanoma cells, such as SK-Mel23 as efficiently as wild-type Ad5, but at a more than 50-fold reduced level in nonmelanoma tumour cells and primary human cells. Injection of AdDeltaEP-TETP into xenotransplanted melanomas, but not into HeLa-derived tumours led to long-lasting tumour regression in nude mice. This AdDeltaEP-TETP virus might be useful for the treatment of accessible lesions in advanced melanoma patients