Gene transfer via reversible plasmid condensation with cysteine-flanked, internally spaced arginine-rich peptides
ABSTRACT Nonviral gene transfer offers biosafety, stability, and expense advantages over viruses; however, it has suffered from poor efficiency. Because arginine-rich peptides facilitate uptake of macromolecules such as proteins, liposomes, and iron nanoparticles, we explored their potential in enhancing plasmid DNA delivery. In their unmodified form, known protein transduction sequences, including hepta-arginine and Tat(47-57), failed to support effective gene delivery. However, by flanking a core of consecutive arginines with amino- and carboxy-terminal cysteines in vitro gene transfer was observed. Furthermore, interspersing arginines with glycine and histidine residues achieved reversible plasmid condensation and dramatically increased transfection levels in a variety of cell types. Unlike most available cationic homopolymers that function only in vitro, these new peptides also increased gene expression in both murine and human tissue in vivo. Thus, cysteine-flanked, internally spaced arginine-rich (CFIS-R) peptides represent a new approach to efficient nonviral plasmid delivery using rationally designed protein transduction domains.
- SourceAvailable from: Christopher H Contag
[Show abstract] [Hide abstract]
- "The enzyme substrate luciferin was administered 48 hr later, followed by bioluminescent imaging (5 min after substrate administration). Pseudocolor images, representing bioluminescent signal intensity, were superimposed on gray-scale reference images of mice to better localize the signal  "
ABSTRACT: The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vivo applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vivo to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vivo delivery of imaging agents.Molecular Imaging 11/2003; 2(4):313-23. DOI:10.1162/153535003322750655 · 2.19 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Although most research on gene therapy has focused on the use of recombinant viruses to deliver genes to cells in vivo, progress also has been made toward developing nonviral, pharmaceutical formulations of genes for in vivo human therapy. Various methods for nonviral gene therapy have been proposed. Some approaches are aimed at developing "artificial viruses" that attempt to mimic the process of viral infection using synthetic materials. Others apply the theory and methods of advanced, particulate drug delivery to deliver DNA to select somatic targets. These approaches employ DNA complexes containing lipid, protein, peptide, or polymeric carriers as well as ligands capable of targeting the DNA complex to cell-surface receptors on the target cell and ligands for directing the intracellular trafficking of DNA to the nucleus. Nonviral systems have been used to deliver genes to the lung, liver, endothelium, epithelium, and tumor cells and have been shown to be generally safe. More than a dozen clinical trials are currently underway using nonviral systems for disease indications including cystic fibrosis and cancer. Future advances in nonviral systems will be based on an emerging appreciation of the biological constraints on the fate and function of DNA within the body and within the cell.Human Gene Therapy 10/1995; 6(9):1129-44. DOI:10.1089/hum.1995.6.9-1129 · 3.62 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The polycationic nanopeptide RKKRRQRRR is a fragment of HIV-Tat protein known to be a transfection enhancer. Such factors control the transfection efficiency of plasmid DNA complexed with tat peptide. However, their actions are poorly understood. Several cell lines and primary cells were used in transfection experiments. The optimal charge ratio of plasmid to the branched 8Tat peptide was 1:8 for all cell lines and primary cells tested except primary human skin fibroblasts where it was 1:4. The conditions of complex formation (volume, DNA-peptide-solution mixing order and the nature of solution (HEPES, 0.15 M NaCl or 5% dextrose)) influenced transfection efficiency. Chloroquine increased the transgene expression 50-fold in HeLa cells but had little impact on Cos7 cells or 3T3 fibroblasts. The integrity of plasmid DNA in the presence of DNase I was shown to be dependent on the DNA/Tat ratio and the nature of the complex solvent. Complexes with -/+ charge ratio of 1:8 formulated in NaCl and dextrose provided high degree of DNA resistance towards nuclease degradation. The conditions of DNA-peptide complex formation and DNA/Tat ratio have significant impact on the level of transgene expression and degree of DNA protection from nuclease attack. The level of elevation of gene delivery in the presence of chloroquine varied considerably between different cells, indicating possible different mechanisms of plasmid-peptide internalisation and intracellular trafficking. The efficiency of branched 8Tat peptide as a plasmid DNA carrier varied between different cell types and have to be optimised for each application.Journal of Drug Targeting 02/2004; 12(1):39-47. DOI:10.1080/106118604200041403 · 2.72 Impact Factor