(LYS)(16)-based reducible polycations provide stable polyplexes with anionic fusogenic peptides and efficient gene delivery to post mitotic cells

Extracellular stability, endocytic escape, intracellular DNA release and nuclear translocation of DNA are all critical properties of non-viral vector/DNA particles. We have evaluated a (Lys)(16)-based linear, reducible polycation (RPC) in combination with an acid-dependent, anionic fusogenic peptide for gene delivery to dividing and post-mitotic cells. The RPC was formed from Cys(Lys)(16)Cys monomers. Molecular weight was 24,000 Da, corresponding to an average of 10.5 peptide monomers per RPC. Non-reducible polylysine (PLL) (27,000 Da) and monomeric (Lys)(16) peptide were evaluated for comparison. (Lys)(16)/DNA particles were disrupted at fusogenic peptide concentrations well below those used for gene delivery. By contrast, RPC/DNA an PLL/DNA particles were stable in the presence of high concentrations of the anionic peptide. Addition of 10% serum virtually abolished the transfection ability of (Lys)(16)/DNA/fusogenic peptide particles, but had little effect on RPC/DNA/fusogenic peptide particles. RPC/DNA/fusogenic peptide particles were highly effective for gene delivery to both cell lines and post-mitotic corneal endothelium. PLL/DNA/fusogenic peptide particles were moderately effective on cell lines, but gave no gene delivery with corneal endothelial cells. We conclude that (Lys)(16)-based RPC/DNA/fusogenic peptide particles provide a gene delivery system which is potentially stable in the extracellular environment and, on reductive depolymerisation, can release DNA plasmids for nuclear translocation.     

A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.     

Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery

The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)(16)-containing peptide/DNA particles. Eight (Lys)(16)-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to - charge ratio of 2.8:1 with 10 microg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate approximately 60 peptide/DNA nanoparticles per target cell, each containing approximately 70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.     

Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution

BACKGROUND: The major barrier to the clinical application of hydrodynamic gene delivery to the liver is the large volume of fluid required using standard protocols. Regional hydrodynamic gene delivery via branches of the portal vein has not previously been reported, and we have evaluated this approach in a rat model. METHODS: The pGL3 plasmid with the luciferase reporter gene was used at 50 micro g/ml in isotonic solutions, and was administered with a syringe pump for precise control of the hydrodynamic conditions evaluated. Gene expression was individually measured in six anatomically distinct liver lobes. The effect of systemic chloroquine to promote endocytic escape and a (Lys)(16)-containing peptide to condense the DNA into approximately 100-nm nanoparticles was also evaluated. RESULTS: Hydrodynamic conditions for excellent gene delivery were obtained by using 3-ml volumes ( approximately 12 ml/kg) of isotonic DNA solution delivered at 24 ml/min to the right lateral lobe ( approximately 20% of the liver mass). Under these conditions, >95% of gene delivery usually occurred in the targeted right lateral lobe. Outflow obstruction was essential for gene delivery, both at optimal and at very low levels of hydrodynamic gene delivery. The use of systemic chloroquine to promote endocytic escape did not augment hydrodynamic gene delivery, while condensation of DNA in non-ionic isotonic solutions (5% dextrose) to nanoparticles of approximately 100 nm completely abolished gene delivery. CONCLUSIONS: Regional hydrodynamic gene delivery via a branch of the portal vein offers a physiological model of liver gene therapy, for experimental and clinical application.     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), 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 (DH) 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 alpha-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 C2C12 mouse myotubes, using gene expression systems containing the beta-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 C2C12 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 (approximately 18 ng/ml).     

A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA

BACKGROUND: Animal viruses such as enveloped virus carry multi-functional proteins in the virion that can mediate more than two distinct steps of a gene delivery process during the transfer of viral genome into host cells. We tested if the aspects of the viral gene delivery mechanism could be mimicked by forming composite formulae from multi-functional synthetic gene carriers having complementary action modes. METHODS: Polyethylenimine (PEI) was chosen as the component responsible for endosome escape and DNA condensation and KALA for cellular entry and DNA condensation. Compact DNA-carrier particles consisting of the core part where DNA chains were tightly condensed by PEI and the outer layer lined with KALA were formulated, characterized and compared with monolithic cationic formulae in terms of gene delivery efficiency and mechanism. RESULTS: High-level gene expression was observed when C2C12 cells were transfected with DNA that was first partially condensed with PEI and, then, fully with KALA. In these formulae KALA mediated enhanced cellular entry of DNA by facilitating endocytic vesicle formation, while PEI provided an effective endosomolytic capacity. An optimal PEI/KALA formula showed transfection efficiencies better than or comparable to the commercial cationic liposome in various cell types in culture and in vivo. CONCLUSIONS: Gene delivery by combining the membrane-active property of KALA with the endosomolytic activity of PEI can be more efficient than that by either of the properties alone. It appears that, in these formulae, the predominant role of KALA is to facilitate cellular entry of DNA by providing a fusogenic capability, rather than an endosomolytic activity.     

Translocating peptides and proteins and their use for gene delivery

A dramatic surge in the development of peptides for gene delivery in vitro and in vivo has been witnessed in the past decade. A better understanding of the structural and mechanistic properties of peptides has been an important step for the rational design of optimal peptide-based gene delivery systems. Research has focused on the design of short synthetic peptides that overcome both extracellular and intracellular limitations of other gene delivery systems by binding reversibly and condensing DNA, specifically targeting cells and/or tissues, rapidly releasing plasmids into the cytoplasm and mediating efficient nuclear translocation.     

Low molecular weight disulfide cross-linking peptides as nonviral gene delivery carriers

Cross-linking peptides have been developed by inserting multiple Cys residues into a 20 amino acid condensing peptide that polymerizes through disulfide bond formation when bound to DNA resulting in small, highly stable DNA condensates that mediate efficient in vitro gene transfer [McKenzie et al. (2000) J. Biol. Chem. 275, 9970-9977]. In the present study, a minimal peptide of four Lys and two terminal Cys residues was found to substitute for Cys-Trp-(Lys)(17)-Cys, resulting in DNA condensates with similar particle size and gene expression in HepG2 cells. Substitution of His for Lys residues resulted in an optimal peptide of Cys-His-(Lys)(6)-His-Cys that, in addition to the attributes described above, also provided buffering capacity to enhance in vitro gene expression in the absence of chloroquine. The reported structure-activity relationships systematically explore peptides with combinations of Lys, Cys, and His residues resulting in low molecular weight peptides with improved gene transfer properties.     

Enhancement of gene delivery to human airway epithelial cells in vitro using a peptide from the polyoma virus protein VP1

BACKGROUND: Current liposome-based gene delivery methods for therapeutic benefit are limited by their low efficiency. One possible way to improve gene expression is to include a peptide with a nuclear localization signal (NLS) to enhance the movement of the transfection complex from the cytoplasm to the nuclei of target cells. We have tested a synthetic peptide based on the amino terminal region of the polyoma virus VP1 protein. This region has non-overlapping motifs for DNA binding and nuclear localization. METHODS: Luciferase gene transfer efficiency was evaluated using this peptide and a control peptide with a mutated NLS in subconfluent, confluent and polarized human bronchial epithelial (16HBE) cells compared to lipoplex alone. RESULTS: Gene transfer efficiency with a lipopolyplex containing the VP1 peptide enhanced gene delivery compared to lipoplex. Transfection with a lipopolyplex containing the control peptide failed to enhance gene delivery. The VP1 peptide increased the amount of plasmid associated with the nucleus while the mutant VP1 peptide did not. The order of lipopolyplex formation was important, with greatest enhancement when peptide was added to the plasmid before addition of the liposome. A bipartite peptide with the VP1 sequence and an integrin-binding motif (RGD) resulted in a reduction in gene transfer efficiency compared to lipoplex. Cell adhesion studies showed that the integrin binding associated with the RGD motif was lost when it was attached to the VP1 sequence. The combination of the two peptide sequences in cis may have compromised the function of both. CONCLUSIONS: Our results indicate that the VP1 peptide represents a strategy to enhance liposome-mediated gene delivery to airway epithelia in vitro. Comparison of transfection efficiencies between the VP1 and the mutant VP1 peptides and the direct measurement of plasmid associated with the nucleus suggests that this enhancement is caused by the NLS signal sequence in the peptide.     

Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Human-derived NLS enhance the gene transfer efficiency of chitosan

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.     

Nanoparticles of compacted DNA transfect postmitotic cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.     

Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.     

Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.     

The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver

In response to phenobarbital (PB) and other PB-type inducers, the nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the translocation mechanism, fluorescent protein-tagged human CAR (hCAR) was expressed in the mouse livers using the in situ DNA injection and gene delivery systems. As in the wild-type hCAR, the truncated receptor lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to the nucleus, indicating that the PB-inducible translocation is AF2 independent. Deletion of the 30 C-terminal residues abolished the receptor translocation, and subsequent site-directed mutagenesis delineated the PB-inducible translocation activity of the receptor to the peptide L313GLL316AEL319. Ala mutations of Leu313, Leu316, or Leu319 abrogated the translocation of CAR in the livers, while those of Leu312 or Leu315 did not affect the nuclear translocation. The leucine-rich peptide dictates the nuclear translocation of hCAR in response to various PB-type inducers and appears to be conserved in the mouse and rat receptors.     

Repositioning of ETO gene in cells treated with VP-16, an inhibitor of DNA-topoisomerase II

The translocation t(8;21)(q22;q22) affecting AML1 and ETO genes is known to be one of the frequent chromosome translocations in acute myeloid leukemia. But no data have been available up to date concerning mutual positioning of these particular genes in the nucleus of a living cell as well as the mechanism of their rapprochement and realignment. Here we show that there is no proximity between these two genes in the primary nuclei of normal human male fibroblasts and moreover that these genes are located in different nuclear layers. But we further show that treatment of cells with VP-16 (etoposide), an inhibitor of DNA topoisomerase II widely used in anticancer chemotherapy, causes the ETO gene repositioning which allows AML1 and ETO genes to be localized in the same nuclear layer. Inhibitor studies demonstrate that such an effect is likely to be connected with the formation of stalled cleavable complexes on DNA. Finally, inhibition of ETO gene repositioning by 2,3-butanedione monoxime (BDM) suggests that this process depends on nuclear myosin. Together, our data corroborate the so called "breakage first" model of the origins of recurrent reciprocal translocation.     

Improving gene replacement by intracellular formation of linear homologous DNA

BACKGROUND: Gene targeting is a potential tool for gene therapy but is limited by the low rate of homologous recombination. Using highly homologous linear DNA improves gene targeting frequency but requires microinjection into nuclear cells to be effective. Because transfection of circular DNA is more efficient than transfection of linear DNA and adaptable to viral vectors, we developed a system for the intracellular release of linear fragments from circular plasmids. METHODS: Only one cutting site inside the "donor" DNA was not convenient because it led to integration of exogenous sequences into the target. So we constructed several "donor" plasmids containing the homologous sequences flanked by two I-Sce I recognition sites. Expression of I-Sce I allowed intracellular delivery of "ends-out" (replacement) vectors. We compared the efficiency of different constructions to correct a mutated gfp target. RESULTS: Co-transfection of "donor" plasmids and an I-Sce I expression vector into CHO cells enhanced the correction of an extrachromosomal mutated gfp target by at least 10 times. Maximum correction was observed with the greatest homology size and maximum effect of I-Sce I was obtained when the long hemi-sites of the duplicated I-Sce I sites were contiguous to the homologous sequence. Unexpectedly, the reverse orientation of I-Sce I sites provided little or no effect, probably due to the asymmetrical activity of the I-Sce I meganuclease. CONCLUSIONS: Releasing homologous DNA fragments with I-Sce I enhances gene replacement. This work provides the basis for the future design of viral vectors for gene replacement.     

Development of a self-assembling nuclear targeting vector system based on the tetracycline repressor protein

The ultimate destination for most gene therapy vectors is the nucleus and nuclear import of potentially therapeutic DNA is one of the major barriers for nonviral vectors. We have developed a novel approach of attaching a nuclear localization sequence (NLS) peptide to DNA in a non-essential position, by generating a fusion between the tetracycline repressor protein TetR and the SV40-derived NLS peptide. The high affinity and specificity of TetR for the short DNA sequence tetO was used in these studies to bind the NLS to DNA as demonstrated by the reduced electrophoretic mobility of the TetR.tetO-DNA complexes. The protein TetR-NLS, but not control protein TetR, specifically enhances gene expression from lipofected tetO-containing DNA between 4- and 16-fold. The specific enhancement is observed in a variety of cell types, including primary and growth-arrested cells. Intracellular trafficking studies demonstrate an increased accumulation of fluorescence labeled DNA in the nucleus after TetR-NLS binding. In comparison, binding studies using the similar fusion of peptide nucleic acid (PNA) with NLS peptide, demonstrate specific binding of PNA to plasmid DNA. However, although we observed a 2-8.5-fold increase in plasmid-mediated luciferase activity with bis-PNA-NLS, control bis-PNA without an NLS sequence gave a similar increase, suggesting that the effect may not be because of a specific bis-PNA-NLS-mediated enhancement of nuclear transfer of the plasmid. Overall, we found TetRNLS-enhanced plasmid-mediated transgene expression at a similar level to that by bis-PNA-NLS or bis-PNA alone but specific to nuclear uptake and significantly more reliable and reproducible.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.