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Biomedical subjects

D V Schaffer

Publications and source records attributed to D V Schaffer.

6 recordsLinked to original sources
PubMed2006

Antiviral RNAi therapy: emerging approaches for hitting a moving target.

The field of directed RNA interference (RNAi) has rapidly developed into a highly promising approach for specifically down regulating genes to alleviate disease pathology. This technology is especially well-suited to treating viral infections, and numerous examples now illustrate that a wide range of viruses can be inhibited with RNAi, both in vitro and in vivo. One principle that has arisen from this work is that antiviral RNAi therapies must be tailored to the unique life cycle of each pathogen, including the choice of delivery vehicle, route of administration, gene(s) targeted and regulation and duration of RNAi induction. Although effective strategies will be customized to each virus, all such therapies must overcome similar challenges. Importantly, treatment strategies must compensate for the inevitable fact that viral genome sequences evolve extremely rapidly, and computational and bioinformatics approaches may aid in the development of therapies that resist viral escape. Furthermore, all RNAi strategies involve the delivery of nucleic acids to target cells, and all will therefore benefit from the development of enhanced gene design and delivery technologies. Here, we review the substantial progress that has been made towards identifying effective antiviral RNAi targets and discuss strategies for translating these findings into effective clinical therapies.

Genetic Engineering↗
PubMed2000

Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery.

Ligand-conjugated polymer (polyplex) gene delivery vectors have strong potential as targeted, in vivo gene transfer vehicles; however, they are currently limited by low delivery efficiency. A number of barriers to polyplex-mediated delivery have been previously identified, including receptor binding, internalization, endosomal escape, and nuclear localization. However, based on understanding of viral gene delivery systems, yet another potential barrier may exist; a limited ability to unpackage the plasmid DNA cargo following localization to the nucleus. We have developed a model system that employs a cationic polymer linked to epidermal growth factor (EGF) as a ligand to target delivery of plasmid DNA encoding the green fluorescent protein to mouse fibroblasts bearing the EGF receptor. Using fluorescence microscopy to simultaneously trace both the plasmid and polymer during gene delivery in combination with an in vitro transcription assay, we provide evidence that plasmid unpackaging can indeed be a limiting step for gene expression for sufficiently large polymer constructs. Short-term expression is significantly enhanced by using short polycations that dissociate from DNA more rapidly both in vitro and in vivo. Finally, we describe a thermodynamic model that supports these data by showing that shorter polycations can have a higher probability of dissociating from DNA. This work demonstrates that vector unpackaging should be added to the list of barriers to receptor-mediated polyplex gene delivery, thus providing an additional design principle for targeted synthetic delivery vehicles.

Animals↗
PubMed2000

Targeted synthetic gene delivery vectors.

Synthetic gene delivery vehicles have made significant progress in the past decade in demonstrating strong potential for targeted delivery to specific cells, low toxicity and immunogenicity and large carrying capacity. However, significant advances must still be made to increase the efficiency of both polymer and lipid vehicles. Furthermore, techniques to generate more effective targeting moieties for a variety of cell types, as well as means to consistently assemble vectors containing these targeting ligands, are areas for further improvement. This review focuses on significant recent advances in generating a number of novel targeted vectors, and discusses progress in the development of new genetic and chemical systems to enhance the targeting, assembly and biocompatibility of synthetic vectors.

Animals↗
PubMed1998

Optimization of cell surface binding enhances efficiency and specificity of molecular conjugate gene delivery.

Molecular conjugates, or polyplexes, are promising synthetic vectors for targeted, in vivo gene delivery, if their efficiency can be improved. Gaining mechanistic information on conjugate gene delivery can potentially yield significant improvements in transfer efficiency by revealing barriers to conjugate transfer from the cell surface to the nucleus. We have developed an experimental system that employs epidermal growth factor as the ligand to direct delivery of DNA encoding the green fluorescent protein to mouse fibroblasts. We report here that the initial step of delivery, binding of the conjugate to the cell surface, is a barrier to gene transfer. We examined the effects of conjugate charge, ligand cross-linker spacer length, and ligand valency on polyplex cell surface binding, internalization, and gene transfer. We find that delivery is both efficient and specific only within a relatively narrow window of conjugate charge, results that correlate with binding and internalization of radiolabeled conjugate. In addition, increasing the cross-linker length can improve binding affinity and delivery. Finally, there is a significant optimum in gene delivery as a function of ligand valency, due to saturation of receptor binding and internalization. Optimizing parameters that affect surface binding therefore improves the efficiency and specificity of molecular conjugate gene delivery.

3T3 Cells↗
PubMed1994

Purification and characterization of alpha-L-fucosidase from Chinese hamster ovary cell culture supernatant.

In this study, alpha-L-fucosidase from Chinese hamster ovary (CHO) cell culture supernatant was purified 11 200-fold to apparent homogeneity to assess the rate of fucose hydrolysis from oligosaccharide and glycoprotein substrates. The fucosidase migrated as a single band of 51 kDa on SDS-PAGE and is a glycoprotein, as determined by retention on concanavalin A-Sepharose, and by lectin blotting with concanavalin A. Hydrolysis of the artificial substrate 4-methyl-umbelliferyl-alpha-L-fucoside (4MU-Fuc) followed simple Michaelis-Menten kinetics, and was competitively inhibited by free fucose and by two known fucosidase inhibitors, fucosylamine and deoxyfuconojirimycin. Hydrolysis of fucose from oligosaccharides including 2'-fucosyllactose, 3-fucosyllactose, Fuc alpha(1,6)GlcNAc and pooled gp120 oligosaccharides with the Fuc alpha(1,6)GlcNAc linkage also followed simple Michaelis-Menten kinetics. However, activity toward 4MU-Fuc was optimal near pH 7, while activities toward the oligosaccharide substrates were optimal near pH 5. No fucose was released from the recombinant CHO cell-produced glycoproteins gp120 or soluble CD4 with the Fuc alpha(1,6)GlcNAc linkage, or from human serum alpha 1-acid glycoprotein with the Fuc alpha(1,3)GlcNAc linkage. Enzymatic removal of sialic acid and galactose from gp120 oligosaccharides did not alter the susceptibility of gp120 to fucosidase attack. These data suggest that released CHO cell fucosidase does not contribute to the heterogeneity of fucosylation that has been observed in CHO cell culture-produced glycoproteins.

Animals↗