Gene delivery has numerous potential applications both clinically and for basic science research. Non-viral vectors represent the long-term future of gene therapy and biomaterials are a critical component for the development of efficient delivery systems. Biomaterial development combined with fundamental studies of virus function and cellular processes will enable the molecular level design of modular vectors. Vectors are being developed based on cationic polymers or lipids that contain functional groups to mediate appropriate interactions with the extracellular environment or to interface with specific cellular processes. This review describes recent progress on the development of biomaterials for non-viral vectors and highlights opportunities for future development. Ultimately, efficient vectors will expand the traditional applications of gene therapy within the clinic and may enable numerous other opportunities within diagnostics, biotechnology, and basic science research.
Background-Gene delivery by non-specific adsorption of non-viral vectors to protein-coated surfaces can reduce the amount of DNA required, and also increase transgene expression and the number of cells expressing the transgene. The protein on the surface mediates cell adhesion and vector immobilization, and functions to colocalize the two to enhance gene delivery. This report investigates the mechanism and specificity by which the protein coating enhances gene transfer, and determines if the protein coating targets the vector for internalization by a specific pathway.
Many organisms rely on antimicrobial peptides (AMPs) as a first line of defense against pathogens. In general, most AMPs are thought to kill bacteria by binding to and disrupting cell membranes. However, certain AMPs instead appear to inhibit biomacromolecule synthesis, while causing less membrane damage. Despite an unclear understanding of mechanism(s), there is considerable interest in mimicking AMPs with stable, synthetic molecules. Antimicrobial N-substituted glycine (peptoid) oligomers (“ampetoids”) are structural, functional and mechanistic analogs of helical, cationic AMPs, which offer broad-spectrum antibacterial activity and better therapeutic potential than peptides. Here, we show through quantitative studies of membrane permeabilization, electron microscopy, and soft X-ray tomography that both AMPs and ampetoids trigger extensive and rapid non-specific aggregation of intracellular biomacromolecules that correlates with microbial death. We present data demonstrating that ampetoids are “fast killers”, which rapidly aggregate bacterial ribosomes in vitro and in vivo. We suggest intracellular biomass flocculation is a key mechanism of killing for cationic, amphipathic AMPs, which may explain why most AMPs require micromolar concentrations for activity, show significant selectivity for killing bacteria over mammalian cells, and finally, why development of resistance to AMPs is less prevalent than developed resistance to conventional antibiotics.
At high bacterial cell density the gene expression program of Pseudomonas aeruginosa is regulated by quorum sensing. Among the gene products highly up-regulated by this system is an exoprotease, leucine aminopeptidase (PA-LAP), which is coexpressed with several known virulence factors and secreted as a proenzyme. We undertook a study of its activation by expressing the full-length proform of PA-LAP recombinantly in Escherichia coli (here termed, rLAP55) and characterizing individual steps in its conversion to an active enzyme. Activation is initiated with the proteolytic removal of a C-terminal prosequence. Removal of ϳ20 amino acids is accomplished by Pseudomonas elastase, which is also positively regulated by quorum sensing. Activation is also mediated by other proteases that cleave rLAP55 near its C terminus. The importance of the C terminus was confirmed by showing that C-terminal deletions of 1-24 amino acids produce a fully active enzyme. The removal of C-terminal prosequences either by proteolysis or deletion leads to an unusual autoprocessing event at the N terminus. Autoprocessing is apparently an intramolecular event, requires the active site of LAP, and results in the removal of 12 N-terminal amino acids. Furthermore, a detailed analysis of the C-terminal prosequence suggests that the proenzyme state is dependent on the presence of a basic side chain contributed by the last amino acid, lysine 536. Our data support a model whereby full-length PA-LAP is activated in a two-step process; proteolytic cleavage at the C terminus is followed by an intramolecular autocatalytic removal of a 12-amino acid propeptide at the N terminus.
Non-viral gene delivery by immobilization of complexes to cell-adhesive biomaterials, a process termed substrate-mediated delivery, has many in vitro research applications such as transfected cell arrays or models of tissue growth. In this report, we quantitatively investigate the efficiency of gene delivery by surface immobilization, and compare this efficiency to the more typical bolus delivery. The ability to immobilize vectors while allowing cellular internalization is impacted by the biomaterial and vector properties. Thus, to compare this efficiency between vector types and delivery methods, transfection conditions were initially identified that maximized transgene expres- sion. For surface delivery from tissue culture polystyrene, DNA complexes were immobilized to pre-adsorbed serum proteins prior to cell seeding, while for bolus delivery, complexes were added to the media above adherent cells. Mathematical modeling of vector binding, release, and cell association using a two-site model indicated that the kinetics of polyplex binding to cells was faster than for lipoplexes, yet both vectors have a half-life on the surface of approximately 17 min. For bolus and surface delivery, the majority of the DNA in the system remained in solution or on the surface, respectively. For polyplexes, the efficiency of trafficking of cell-associated polyplexes to the nucleus for surface delivery is similar or less than bolus delivery, suggesting that surface immobilization may decrease the activity of the complex. The efficiency of nuclear association for cell-associated lipoplexes is similar or greater for surface delivery relative to bolus. These studies suggest that strategies to enhance surface delivery for polyplexes should target the vector design to enhance its potency, whereas enhancing lipoplex delivery should target the material design to increase internalization.
The efficiency of biomaterial-based gene delivery is determined by the interaction of the material and the vector. For lipoplexes, surface immobilization has been used to transfect cells for applications such as cell arrays and model tissue formation through patterned transfection. Further increases in the delivery efficiency are limited by cellular internalization, which may be overcome by altering the material/vector interactions. In this report, we investigated the modification of the lipoplex physical properties through self-assembly with cationic peptides, and subsequently quantified cellular association, internalization and nuclear accumulation of DNA and transfection. Relative to lipid alone, peptide-lipoplexes enhanced transfection by up to 4.6-fold. The presence of the peptide in the lipoplex increased internalization efficiency by up to 4.5-fold, decreased the percentage of lysosomal DNA by 2.1-fold and increased the efficiency of nuclear accumulation by 3.0-fold. In addition, an analysis of internalization pathways for peptide-lipoplexes indicated a greater role of clathrin and caveolae-mediated endocytosis relative to macropinocytois, which was not observed for peptide-free lipoplexes. These results demonstrate peptide-induced enhancement of gene transfer by surface immobilization due to increased cellular internalization and nuclear accumulation, which has numerous applications ranging from cell-based assays to regenerative medicine.
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