High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles.

One limit to successful receptor-mediated gene delivery is the exit of the endocytosed material from the endosome. We demonstrate here the delivery of marker genes to tissue culture cells using a modification of the receptor-mediated gene delivery technique that exploits the endosomolytic activity of defective adenovirus particles. In particular, greater than 90% of the transfected-cell population is found to express a beta-galactosidase gene, and, most importantly, this high level of expression can be obtained with psoralen-inactivated virus particles. Furthermore, because the delivered gene is not carried within the genome of the adenovirus particle, the size constraints are relieved, and we can, therefore, show the delivery of a 48-kilobase cosmid DNA molecule

Binding-incompetent adenovirus facilitates molecular conjugate-mediated gene transfer by the receptor-mediated endocytosis pathway

Molecular conjugate vectors may be constructed that accomplish high efficiency gene transfer by the receptor-mediated endocytosis pathway. In order to mediate escape from lysosomal degradation, we have incorporated adenoviruses into the functional design of the conjugate. In doing so, however, we have introduced an additional ligand, which can bind to receptors on the cell surface, undermining the potential for cell specific targeting. To overcome this, we have treated the adenovirus with a monoclonal anti-fiber antibody, which renders the virus incapable of binding to its receptor. The result is a multi-functional molecular conjugate vector, which has preserved its binding specificity while at the same time being capable of preventing lysosomal degradation of endosome-internalized conjugate-DNA complexes. This finding indicates that adenoviral binding is not a prerequisite for adenoviral-mediated endosome disruption

Coupling of adenovirus to transferrin-polylysine/DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes.

We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells

Species D human adenovirus type 9 exhibits better virus-spread ability for antitumor efficacy among alternative serotypes

Species C human adenovirus serotype 5 (HAdV-C5) is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR), HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR)-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5

Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells

No abstract available

The Circumstellar Structure and Excitation Effects around the Massive Protostar Cepheus A HW 2

We report SMA 335 GHz continuum observations with angular resolution of ~0.''3, together with VLA ammonia observations with ~1'' resolution toward Cep A HW 2. We find that the flattened disk structure of the dust emission observed by Patel et al. is preserved at the 0.''3 scale, showing an elongated structure of ~$0.''6 size (450 AU) peaking on HW 2. In addition, two ammonia cores are observed, one associated with a hot-core previously reported, and an elongated core with a double peak separated by ~1.''3 and with signs of heating at the inner edges of the gas facing HW 2. The double-peaked ammonia structure, as well as the double-peaked CH3CN structure reported previously (and proposed to be two independent hot-cores), surround both the dust emission as well as the double-peaked SO2 disk structure found by Jimenez-Serra et al. All these results argue against the interpretation of the elongated dust-gas structure as due to a chance-superposition of different cores; instead, they imply that it is physically related to the central massive object within a disk-protostar-jet system.Comment: 12 pages, 3 figures; accepted for publication in the Astrophysical Journa