Synthetic PEGylated Glycoproteins and Their Utility in Gene Delivery (original) (raw)
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Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes
Pharmaceutical Research, 2000
Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using 125I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (−/+/−)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(−/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (−/+/−))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (−/+/−)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.
Designer peptide delivery systems for gene therapy
European Journal of Nanomedicine, 2015
Gene therapy has long been hailed as a revolutionary approach for the treatment of genetic diseases. The enthusiasm that greeted the harnessing of viruses for therapeutic DNA delivery has been tempered by concerns over safety. These concerns led to the development of alternative strategies for nucleic acid delivery to cells. One such strategy is the utilization of cationic peptides for the condensation of therapeutic DNA for delivery to its target. However, success of DNA as a therapy relies on its delivery to the nucleus of target cells, a process that is complicated by the many hurdles encountered following systemic administration. Non-viral peptide gene delivery strategies have sought inspiration from viruses in order to retain DNA delivering potency, but limit virulence. This review summarizes the progression of peptide-based DNA delivery systems, from rudimentary beginnings to the recent development of sophisticated multi-functional vectors that comprise distinct motifs with de...
Peptides in DNA delivery: current insights and future directions
Drug Discovery Today, 2008
Peptides are emerging as attractive alternatives to cationic polymers and lipids for nonviral DNA delivery. Their remarkable properties such as efficient condensation of DNA, translocation across the cellular membrane, pH-sensitive membrane disruption, and efficient targeting of attached cargoes to the nucleus make them lucrative for researchers to explore their application in DNA delivery. In this review article, we focus on how the chemical nature, structural features and DNA complexation strategies of different peptides have been utilized for efficient DNA delivery. We also discuss their potential problems hindering in vivo application.
Biophysical Journal, 1998
Novel synthetic peptides, based on carrier peptide analogs (YKAK n WK) and an amphipathic peptide (GLFEA-LLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (D H ) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its ␣-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C 2 C 12 mouse myotubes, using gene expression systems containing the -galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C 2 C 12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (ϳ18 ng/ml).
Enhanced gene transfer activity of peptide-targeted gene-delivery vectors
Journal of Drug Targeting, 2005
We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.
An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector
The Journal of Gene Medicine, 2000
Background Due to their chemical de®nition and reduced size, the use of peptides as gene delivery systems is gaining interest as compared to the more common polymeric non-viral vectors. To achieve gene transfer ef®ciencies that would make peptides a realistic alternative to existing methods, we have evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design, synthesis and characterization of a 23-residue cationic peptide which we term RAWA.
Journal of Controlled Release, 2021
The design of a non-viral gene delivery system that can release a functional nucleic acid at the intracellular destination site is an exciting but also challenging proposition. The ideal gene delivery vector must be non-toxic, non-immunogenic, overcome extraand intra-cellular barriers, protect the nucleic acid cargo from degradation with stability over a range of temperatures. A new 15 amino acid linear peptide termed CHAT was designed in this study with the goal of delivering DNA with high efficiency into cells in vitro and tissues in vivo. Rational design involved incorporation of key amino acids including arginine for nucleic acid complexation and cellular uptake, tryptophan to enhance hydrophobic interaction with cell membranes, histidine to facilitate endosomal escape and cysteine for stability and controlled cargo release. Six linear peptides were synthesised with strategic sequences and amino acid substitutions. Data demonstrated that all six peptides complexed pDNA to produce cationic nanoparticles less than 200 nm in diameter, but not all peptides resulted in successful transfection; indicating the influence of peptide design for endosomal escape. Peptide 4, now termed CHAT, was non-cytotoxic, traversed the plasma membrane of breast and prostate cancer cell lines, and elicited reporter-gene expression following intra-tumoural and intravenous delivery in vivo. CHAT presents an exciting new peptide for the delivery of nucleic acid therapeutics.
Targeted delivery of DNA for gene therapy via receptors
Trends in Biotechnology, 1993
The general concept of targeted delivery of therapeutic agents was first recognized by Paul Ehrlich at the turn of the century I. Only recently has this strategy been applied to the delivery of DNA-protein complexes to cells 2. Most of the research on DNA delivery by this approach has been performed by targeting the liver-specific asialoglycoprotein receptor (ASGPr) (Refs 3-10) and the relatively ubiquitous transferrin receptor,1 19. DNA delivery to hepatocytes has also been effected using insulin-polylysine conjugates 2°, and to antigen-bearing cells using an antibody 21. The majority of these experiments use a covalently crosslinked receptor ligand-polycation conjugate to bind DNA in an electrostatic complex 5 . Receptormediated endocytosis of the ligand carries the bound DNA into a cell, allowing subsequent expression of the foreign DNA ). This technique, which has been demonstrated in animals using the ASGPr (Refs 5-8), is promising for the delivery of therapeutic DNAs as well as antisense oligonucleotides2L