Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus.

Adenovirus type 5 (Ad) based vectors transduce vascular endothelial cells (EC) and have been widely used for vascular gene transfer. However, many cell types express the Ad receptor (cox-sackievirus adenovirus receptor; CAR), preventing selective EC infection and precluding clinical use. We previously isolated the human EC-binding peptides SIGYPLP and LSNFHSS by phage display and demonstrated by means of a bispecific antibody that SIGYPLP directs efficient, high-level, EC-selective Ad-mediated gene transfer. We now generate genetically modified Ad fiber proteins with selective EC tropism by engineering these peptides into the HI loop of the Ad fiber. SIGYPLP, but not LSNFHSS, enhanced EC selectivity, demonstrating maintenance of peptide-cell binding fidelity upon incorporation into virions. Combining fiber mutations that block CAR binding (detargeting) with SIGYPLP insertion (retargeting) generated a novel Ad vector, AdKO1SIG, in a single component system. AdKO1SIG demonstrated efficient and selective tropism for EC compared with control Ad vectors. This is the first demonstration of genetic incorporation of a novel, mammalian, cell-selective ligand that retains its targeting fidelity in the Ad fiber HI loop, in combination with point mutations that abolish fiber-CAR interaction. This study demonstrates the potential for improving the cell-selectivity and safety of adenoviral vectors.

Structural analysis of a fiber-pseudotyped adenovirus with ocular tropism suggests differential modes of cell receptor interactions.

Adenovirus (Ad) entry into cells is initiated by the binding of the fiber knob to a cell surface receptor. The coxsackie- and adenovirus receptor (CAR) functions as the attachment receptor for many, but not all, Ad serotypes. Ad type 37 (Ad37), a subgroup D virus that causes keratoconjunctivitis in humans, does not infect cells via CAR despite demonstrated binding of the Ad37 knob to CAR. We have pseudotyped a fiber deletion Ad5 vector with the Ad37 fiber (Ad37f), and this vector retains the ocular tropism of Ad37. Here we present a cryo-electron microscopy reconstruction of Ad37f that shows the entire Ad37 fiber, including the shaft and knob domains. We have previously proposed that Ad37 may not utilize CAR for cell entry because of the geometric constraints imposed by a rigid fiber (E. Wu, J. Fernandez, S. K. Fleck, D. Von Seggern, S. Huang, and G. R. Nemerow, Virology 279:78-89, 2001). Consistent with this hypothesis, our structural results show that the Ad37 fiber is straight and rigid. Modeling of the interaction between Ad37f and host cell receptors indicates that fiber flexibility or rigidity, as well as length, can affect receptor usage and cellular tropism.

Adenovirus type 5 viral particles pseudotyped with mutagenized fiber proteins show diminished infectivity of coxsackie B-adenovirus receptor-bearing cells.

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.beta gal.Delta F, an E1-, E3-, and fiber-deleted adenoviral vector encoding beta-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector.

Signaling antibodies complexed with adenovirus circumvent CAR and integrin interactions and improve gene delivery.

Current adenoviral (Ad) vectors cannot be targeted to specific cell types due to the widespread distribution of the Ad receptor (CAR). Moreover, CAR and/or internalization receptors (alphav integrins) are absent or present at low levels on some cell types, rendering them resistant to Ad-mediated gene delivery. To address these problems, we have developed a novel vector targeting approach that takes advantage of the common cell signaling pathways initiated by ligation of alphav integrins and growth factor receptors. Recombinant growth factor/cytokines (TNF-alpha, IGF-1, EGF) which trigger phosphatidylinositol-3-OH kinase (PI3K) activation, a signaling molecule involved in adenovirus internalization, were fused to a monoclonal antibody specific for the viral penton base. Ad vectors complexed with these bifunctional mAbs increased gene delivery 10 to 50-fold to human melanoma cells lacking alphav integrins. The bifunctional mAbs also enhanced gene delivery by fiberless adenovirus particles which cannot bind to CAR. Improved gene delivery correlated with increased virus internalization and attachment as well as PI3K activity. The use of bifunctional mAbs to trigger specific cell signaling pathways offers a widely applicable method for bypassing the normal Ad receptors in gene delivery and potentially increasing the selectivity of gene transfer.

Adenovirus vector pseudotyping in fiber-expressing cell lines: improved transduction of Epstein-Barr virus-transformed B cells.

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.betagal.DeltaF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.betagal.DeltaF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.

A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles.

The adenovirus (Ad) fiber protein largely determines viral tropism through interaction with specific cell surface receptors. This molecule may also be involved in virion assembly or maturation, as some previously characterized fiber mutants were defective for processing of viral structural proteins. We previously described packaging cell lines that express Ad type 5 (Ad5) fiber and can complement the temperature-sensitive Ad fiber mutant H5ts142. We have now used these packaging cells to construct a new adenoviral vector (Ad5.betagal.DeltaF) with E1, E3, and L5 (fiber) deleted and analyzed the fiber null phenotype. Ad5.betagal.DeltaF growth was completely helper independent, and fiberless particles were produced by a single final round of growth in 293 cells. Cryoelectron microscopic studies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the structure and composition of these particles was nearly identical to those of first-generation Ad vectors. As expected, fiberless particles had reduced infectivity on epithelial cells, but they retained the ability to infect monocytic cells via an integrin-dependent pathway. These studies provide a novel approach to developing retargeted Ad gene therapy vectors.

Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein.

Adenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.

Inhibition of radiation cataractogenesis by WR-77913.

The radioprotective drug S-3-amino-2-hydroxypropylphosphorothioic acid (WR-77913) has been tested as an inhibitor of radiation cataractogenesis. Animals treated with 15 Gy whole-head 137Cs gamma radiation developed mature cataracts 10-12 weeks after irradiation. Intraperitoneal pretreatment with 815 mg/kg WR-77913 30 min before irradiation delayed the development of cataracts; mature cataracts required 42 weeks for development. Doses as low as 350 mg/kg, substantially below the toxic range, resulted in graded but incomplete protection and a significant delay in the development of cataracts. Drug treatment combined with radiation doses of 12.5 or 10 Gy showed less pronounced protection. The optimum time of drug delivery was found to be between 30 min and 2 h before irradiation; protective action diminished if longer times were used or if the drug was given after irradiation. These results are discussed in relation to those obtained with other chemical radioprotective agents and in terms of possible mechanisms of the action of the drug.

The Broad-Complex directly controls a tissue-specific response to the steroid hormone ecdysone at the onset of Drosophila metamorphosis.

In Drosophila, all of the major metamorphic transitions are regulated by changes in the titer of the steroid hormone ecdysone. Here we examine how a key regulator of metamorphosis and primary ecdysone response gene, the Broad-Complex, transmits the hormonal signal to one of its targets, the Sgs-4 glue gene. We show that Broad-Complex RNAs accumulate in mid third instar larval salivary glands prior to Sgs-4 induction, as expected for the products of a gene that regulates the timing of Sgs-4 activation. The Broad-Complex codes for a family of zinc finger transcriptional regulators. We have identified a number of binding sites for these proteins in sequences known to regulate the timing of Sgs-4 induction, and have used these sites to derive a binding consensus for each protein. Some of these binding sites are required in vivo for Sgs-4 activity. In addition, rbp+, a genetically defined Broad-Complex function that is required for Sgs-4 induction, acts through these Broad-Complex binding sites. Thus, the Broad-Complex directly mediates a temporal and tissue-specific response to ecdysone as larvae become committed to metamorphosis.

Ectopic expression of wild-type or a dominant-negative mutant of transcription factor NTF-1 disrupts normal Drosophila development.

The Drosophila melanogaster tissue-specific transcription factor NTF-1 was originally identified in vitro as a protein that could bind to and activate transcription from the Dopa decarboxylase (Ddc) gene. A structure-function analysis of NTF-1 led to the identification of a discrete amino-terminal activation domain. Here, we report that an NTF-1 mutant lacking the activation domain acts as a trans-dominant inhibitor of NTF-1 activation in tissue culture cells by forming inactive heterodimers with the full-length protein. Ectopically expressing this dominant-negative protein or the full-length protein in developing Drosophila embryos leads to dire developmental consequences. Overexpressing the trans-dominant NTF-1 leads to lethality, while overexpressing full-length NTF-1 results in both lethality and morphogenetic defects. Our results suggest that both the activity and the regulation of NTF-1 are critical for viability and proper development of the fly.

A transcriptional switch between the Pig-1 and Sgs-4 genes of Drosophila melanogaster.

Pig-1 and Sgs-4 are a pair of closely linked and divergently transcribed Drosophila melanogaster genes, which are both expressed in larval salivary glands but at different times during development. While Sgs-4 is expressed at high levels only at the end of the third instar, Pig-1 exhibits a major peak of expression during late second and early third instar. Thus, Pig-1 expression declines as Sgs-4 expression is induced. In this paper, we show that three adjacent elements located within the short region between these genes can account for the switch from Pig-1 to Sgs-4 expression. A 170-bp segment acts as an enhancer to direct Sgs-4 expression in late-third-instar salivary glands. A 64-bp sequence located just upstream from the enhancer can modify its temporal specificity so that it works throughout the third instar. Expression induced at mid-third instar by a combination of these two elements can be repressed by a negative regulatory sequence located still further upstream. We present evidence suggesting that the changing interactions between these regulatory elements and the Sgs-4 and Pig-1 promoters lead to the correct pattern of expression of the two genes.