Polyethylenimine but not cationic lipid improves antisense activity of 3'-capped phosphodiester oligonucleotides

Lipofectin, which is a mixture of neutral lipid with a cationic lipid, has been widely used to enhance cellular delivery of phosphorothioate, 2'-sugar-modified, and chimeric antisense oligonucleotides. Phosphodiester oligonucleotides delivered with Lipofectin usually do not elicit antisense activity probably because cationic lipid formulations do not sufficiently protect unmodified oligonucleotides from nuclease degradation. We show that a cationic polymer, polyethylenimine (PEI), improves the uptake and antisense activity of 3'-capped 20-mer and 12-mer antisense phosphodiester oligonucleotides (PO-ODN) targeted to different regions of Ha-ras mRNA and to the 3'-untranslated region (3'-UTR) of C-raf kinase. In contrast, PEI, which forms a very stable complex with the 20-mer phosphorothioate oligonucleotide (PS-ODN), does not enhance its antisense activity. Using fluorescently labeled carriers and ODN, we show that PEI-PS-ODN particles are very efficiently taken up by cells but PS-ODN is not dissociated from the carrier. Our results indicate that carrier-ODN particle size and stability and ODN release kinetics vary with the chemical nature of the ODN and the carrier being transfected into the cells. The very low cost of PEI compared with cytofectins and the increased affinity for target mRNA and decreased affinity for proteins of PO-ODN compared with PS-ODN make the use of PEI-PO-ODN very attractive.     

Propynylated phosphodiester oligonucleotides inhibit ICAM-1 expression in A549 cells on electroporation

Oligodeoxynucleotides (ODN) are used largely as either primers, antisense, or triplex-forming units. Phosphodiester ODN (PO-ODN), which are very rapidly degraded by exonucleases, must be protected at their ends. Even so, their life span inside cells is quite short. Phosphorothioate ODN (PS-ODN) are less sensitive to nucleases and are extensively used as antisense. Unfortunately, unlike PO-ODN, they interact with a number of molecules, including proteins, in addition to their specific nucleic acid targets. Their affinity for their target is lower than that of PO-ODN. PS-ODN containing propyne groups on C5 of pyrimidine have been shown to have a higher affinity toward their nucleic acid target. Here, we show that propynylated PO-ODN are more stable and much more efficient than their propyne-free counterparts. They are not efficient when they are used as lipoplexes, but they act as specific antisense on electroporation.     

Immunostimulation and anti-DNA antibody production by backbone modified CpG-DNA

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Phosphorothioate oligodeoxynucleotides inhibit basic fibroblast growth factor-induced angiogenesis in vitro and in vivo.

Angiogenesis is regulated by heparin-binding growth factors, such as basic fibroblast growth factor (bFGF). We investigated the effects of phosphorothioate-mediated oligodeoxynucleotides (PS-ODN) on bFGF-induced angiogenesis. Because PS-ODN are polyanions, they can also bind many heparin-binding proteins. On a basement matrix using a Matrigel matrix, we observed <50% tube formation by human umbilical endothelial cells with 10 microM bFGF, vascular endothelial growth factor, or nuclear factor-kappaB (NF-kappaB) antisense and sense PS-ODN, while phosphodiester oligodeoxynucleotides (PO-ODNs) were not affected. The PS-ODN, but not the PO-ODN, inhibited the bFGF-induced rabbit corneal neovascularization. In albino rats, the NF-kappaB antisense PS-ODN showed a low rescue score for bFGF-dependent photoreceptor rescue because of their degradation by constant light exposure. However, antisense PS-ODN active against bFGF inhibited angiogenesis more strongly than did the antisense NF-kappaB PS-ODN. Because of the important role bFGF plays in angiogenesis, some PS-ODN may serve as potent antiangiogenic compounds that act through a combination of polyanionic phosphorothioate effects and a sequence-specific antisense mechanism.     

Polyelectrolyte complex micelles composed of c-raf antisense oligodeoxynucleotide-poly(ethylene glycol) conjugate and poly(ethylenimine): effect of systemic administration on tumor growth

An antisense oligodeoxynucleotide (ODN) delivery system based on polyelectrolyte complex (PEC) micelles composed of an ODN-poly(ethylene glycol) (PEG) conjugate and polyethylenimine (PEI) was demonstrated. The PEC micelles having a core/shell structure were spontaneously formed in an aqueous solution by ionic interactions between ODN part in the conjugate and PEI. The ODN/PEI polyelectrolyte complex formed an inner core while PEG chains surrounded it as a shell. The morphology of the micelles was visualized as a separate sphere by atomic force microscopy (AFM). When the micelles containing a c-raf antisense ODN were intravenously administered into tumor-bearing nude mice, significant antitumor activities against human lung cancer were observed. The intravenously injected micelles also showed significantly higher accumulation level in the solid tumor region compared to that of naked ODN.     

Analysis of post-lysosomal compartments

Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.     

特异寡聚核苷酸对猪瘟病毒在细胞中增殖抑制作用的研究

本实验探讨了寡聚核苷酸对CSFV复制的影响以及作为抗CSFV新型药物的可行性。实验结果表明针对CSFV5'um端非编码区NS3蛋白丝氨酸蛋白酶功能区的寡聚核酸对CSFV复制均有显著的抑制作用,而针对CSFV3' 端非编码区寡聚核苷酸仅有轻微抑制作用,5'端寡聚核苷酸具有最佳抑制作用,同时发现相应序列中正义聚核苷酸的作用要优于反义寡聚核苷酸;脂质体介导转染能显著提高寡聚核苷酸对CSF复制的抑制作用。这些初步结果也提示,CSFV5'端和3'端非编码区对CSFV复制的重要性和作用有所不同。     

Antisense oligodeoxynucleotide-conjugated hyaluronic acid/protamine nanocomplexes for intracellular gene inhibition

Green fluorescent protein (GFP) antisense oligodeoxynucleotide (ODN) was covalently conjugated to hyaluronic acid (HA) via a reducible disulfide linkage, and the HA-ODN conjugate was complexed with protamine to increase the extent of cellular uptake and enhance the gene inhibition efficiency of GFP expression. The HA-ODN conjugate formed more stable polyelectrolyte complexes with protamine as compared to naked ODN, probably because of its increased charge density. The higher cellular uptake of protamine/HA-ODN complexes than that of protamine/naked ODN complexes was attributed to the formation of more compact nanosized complexes (approximately 200 nm in diameter) in aqueous solution. Protamine/HA-ODN complexes also showed a comparable level of GFP gene inhibition to that of cytotoxic polyethylenimine (PEI)/ODN complexes. Since both HA and protamine are naturally occurring biocompatible materials, the current formulation based on a cleavable conjugation strategy of ODN to HA could be potentially applied as safe and effective nonviral carriers for ODN and siRNA nucleic acid therapeutics.     

Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts

We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD-SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6-NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.     

Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.     

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.     

Phosphorothioate backbone modification modulates macrophage activation by CpG DNA

Macrophages respond to unmethylated CpG motifs present in nonmammalian DNA. Stabilized phosphorothioate-modified oligodeoxynucleotides (PS-ODN) containing CpG motifs form the basis of immunotherapeutic agents. In this study, we show that PS-ODN do not perfectly mimic native DNA in activation of macrophages. CpG-containing PS-ODN were active at 10- to 100-fold lower concentrations than corresponding phosphodiester ODN in maintenance of cell viability in the absence of CSF-1, in induction of NO production, and in activation of the IL-12 promoter. These enhancing effects are attributable to both increased stability and rate of uptake of the PS-ODN. By contrast, PS-ODN were almost inactive in down-modulation of the CSF-1R from primary macrophages and activation of the HIV-1 LTR. Delayed or poor activation of signaling components may contribute to this, as PS-ODN were slower and less effective at inducing phosphorylation of the extracellular signal-related kinases 1 and 2. In addition, at high concentrations, non-CpG PS-ODN specifically inhibited responses to CpG DNA, whereas nonstimulatory phosphodiester ODN had no such effect. Although nonstimulatory PS-ODN caused some inhibition of ODN uptake, this did not adequately explain the levels of inhibition of activity. The results demonstrate that the phosphorothioate backbone has both enhancing and inhibitory effects on macrophage responses to CpG DNA.     

Cutting edge: species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides

Different DNA motifs are required for optimal stimulation of mouse and human immune cells by CpG oligodeoxynucleotides (ODN). These species differences presumably reflect sequence differences in TLR9, the CpG DNA receptor. In this study, we show that this sequence specificity is restricted to phosphorothioate (PS)-modified ODN and is not observed when a natural phosphodiester backbone is used. Thus, human and mouse cells have not evolved to recognize different CpG motifs in natural DNA. Nonoptimal PS-ODN (i.e., mouse CpG motif on human cells and vice versa) gave delayed and less sustained phosphorylation of p38 MAPK than optimal motifs. When the CpG dinucleotide was inverted to GC in each ODN, some residual activity of the PS-ODN was retained in a species-specific, TLR-9-dependent manner. Thus, TLR9 may be responsible for mediating many published CpG-independent responses to PS-ODN.     

The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.     

Binding, internalization, and lysosomal association of 125I-human growth hormone in cultured human lymphocytes: a quantitative morphological and biochemical study

125I-human growth hormone (125I-hGH) binds specifically to receptors on cultures human lymphocytes (IM-9). When this process is studied by use of quantitative EM radioautography, under conditions of incubation at 15 degrees C for 5 min, the ligand is localized to the plasma membrane of the cell. At 30 degrees and 37 degrees C, however, 125I-hGH is progressively internalized by the cell as a function of time. The internalized ligand is found predominantly in the Golgi region of the cells, with a five-fold preferential localization to membrane-bounded structures with the morphological and cytochemical characteristics of lysosomes. Up to 59% of these lysosome-like structures are positive for the acid phosphatase reaction under the conditions of incubation at 37 degrees C for 120 min. When the cell associated radioactivity after 15- 120 min of incubation at 37 degrees C is extracted in 1 M acetic acid and filtered on a Sephadex G-100 column, 58-73% of the material elutes as intact hGH. When cells are incubated with 125I-hGH at 37 degrees C for 15-120 min, separated from the incubation medium, and washed and diluted 100-fold, the percent 125I-hGH dissociable decreases as a function of increasing time of incubation. When cells are incubated with 125I-hGH for 15 min at 37 degrees C and the radioactivity that dissociates from the cells during 15-90 min is studied, the labeled material appearing in the incubation medium is progressively degraded as a function of time of incubation. When the dissociation process is studied radioautographically, grains are found both in plasma membrane and intracelluar compartments after 30 min of association, but after 30 and 120 min of dissociation a higher proportion of grains are in the intracellular compartment. After 120 min of association, there is less dissociation from either compartment and a preferential increase of grains in the intracellular compartment. These data suggest that receptor-linked internalization of a polypeptide hormone provides a mechanism that couples degradation of the ligand with loss of the cell surface receptor.     

Intracellular localization and degradation of diphtheria toxin

The internalization of surface-bound diphtheria toxin (DT) in BS-C-1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon preincubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomal fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the ligand appeared in the medium as TCA-soluble radioactivity. EGF degradation was partially leupeptin-sensitive and markedly NH4Cl-sensitive, indicating lysosomal degradation. In contrast, DT A-fragment degradation was resistant to these inhibitors, while B-fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.     

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes.

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of 125I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca2+ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca2+ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more than 30 min. When incubations were carried out for more than 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations

BACKGROUND: Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified. METHODS AND RESULTS: The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes. CONCLUSIONS: The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.