Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro

We have developed a system for targeting foreign DNA to hepatocytes in vitro using a soluble DNA carrier that takes advantage of receptor-mediated endocytosis to achieve internalization. The idea is based on the fact that hepatocytes possess a unique receptor that binds and internalizes galactose-terminal (asialo)glycoproteins. To create a targetable carrier system that could bind DNA in a nondeforming manner, we used poly(L-lysine) to bind DNA in a strong but noncovalent interaction. An asialoglycoprotein, asialoorosomucoid (AsOR), was chemically coupled to poly(L-lysine) to form an asialoorosomucoid-poly(L-lysine) conjugate. Various proportions of conjugate to DNA were tested to determine conditions that maximized DNA content in a soluble complex and that limited solubility of complexes. To test the targetable gene delivery system, AsOR-poly(L-lysine) conjugate was complexed to the plasmid pSV2 CAT containing the gene for chloramphenicol acetyltransferase (CAT) driven by an SV-40 promoter. We tested this complex using a model system consisting of human hepatoma cell line Hep G2 [asialoglycoprotein receptor (+)], hepatoma SK-Hep 1, IMR-90 fibroblasts, and uterine smooth muscle [receptor (-)] cells. Each cell line was incubated with 0.2 micron filtered AsOR-poly(L-lysine)-DNA complex or controls consisting of DNA plus AsOR, DNA plus poly(L-lysine), or DNA alone. Cells were assayed for the presence of CAT activity as a measure of gene transformation. SK-Hep 1, IMR-90, and smooth muscle [receptor (-)] cells produced no detectable acetylated chloramphenicol derivatives under any of these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity

Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy.     

Enhancement of immune responses by DNA vaccination through targeted gene delivery using mannosylated cationic liposome formulations following intravenous administration in mice

The present study investigated the potency of the mannosylated cationic liposomes (Man liposomes) that we have developed in novel DNA vaccine carrier. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. The potency of the Man liposome/pCMV-OVA complex was compared with naked pCMV-OVA and that complexed with DC-Chol liposomes. In cultured mouse peritoneal macrophages, MHC class I-restricted antigen presentation of the Man liposome/pCMV-OVA complex was significantly higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. After intravenous administration, OVA mRNA expression and MHC class I-restricted antigen presentation on CD11c+ cells and inflammatory cytokines, such as TNF-alpha, IL-12, and IFN-gamma, that can enhance the Th1 response of the Man liposome/pCMV-OVA complex were higher than that of naked pCMV-OVA and that complexed with DC-Chol liposomes. Also, the spleen cells from mice immunized by intravenous administration of the Man liposome/pCMV-OVA complex showed the highest proliferation response and IFN-gamma secretion. These findings suggest that the targeted delivery of DNA vaccine by Man liposomes is a potent vaccination method for DNA vaccine therapy.     

Targeted adenovirus-mediated gene delivery to T cells via CD3.

T cells are primary targets in numerous gene therapy protocols. However, the use of subgroup C adenovirus serotype 2 or 5 (Ad2 or Ad5) as a vector to transduce T cells is limited by its poor transduction efficiency for these cells. In this report we show that poor T-cell transduction results from these cells lacking both the primary Ad2-Ad5 receptor, used in attachment, and the secondary Ad receptor, which mediates entry of most adenovirus serotypes. These deficiencies were overcome by using a bispecific antibody (bsAb) with specificities for human CD3 and for a FLAG epitope genetically introduced into Ad5 (Ad.FLAG) to redirect the virus to human T cells. The anti-FLAG x anti-CD3 bsAb increased Ad.FLAG binding 30-fold, induced the efficient uptake of Ad.FLAG into the cells, and led to a 100- to 500-fold increase in the transduction of resting T cells. Moreover, fluorescence-activated cell sorter analysis showed that 25 to 90% of the T cells were transduced by the bsAb-complexed Ad.FLAG at multiplicities of infection between 20 and 100 active particles per cell. These results demonstrate that bsAbs can target Ad to non-Ad receptors on cells that are normally resistant to Ad, resulting in their efficient and specific transduction.     

Liposomal delivery of CTL epitopes to dendritic cells

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.     

Pegylated polyethylenimine-Fab' antibody fragment conjugates for targeted gene delivery to human ovarian carcinoma cells

Specific targeting of ovarian carcinoma cells using pegylated polyethylenimine (PEG-PEI) conjugated to the antigen binding fragment (Fab') of the OV-TL16 antibody, which is directed to the OA3 surface antigen, was the objective of this study. OA3 is expressed by a majority of human ovarian carcinoma cell lines. To demonstrate the ability of the PEG-PEI-Fab' to efficiently complex DNA, an ethidium bromide exclusion assay was performed. Comparison with PEG-PEI or PEI 25 kDa showed only minor differences in the ability to condense DNA. Since conjugation of Fab' to PEG-PEI might influence complex stability, this issue was addressed by incubating the complexes with increasing amounts of heparin. This assay revealed stability similar to that of unmodified PEG-PEI/DNA or PEI 25 kDa/DNA complexes. Complexes displayed a size of approximately 150 nm with a zeta potential close to neutral. The latter property is of particular interest for potential in vivo use, since a neutral surface charge reduces nonspecific interactions. Binding studies using flow cytometry and fluorescently labeled DNA revealed a more than 6-fold higher degree of binding of PEG-PEI-Fab'/DNA complexes to epitope-expressing cell lines compared to unmodified PEG-PEI/DNA complexes. In OA3-expressing OVCAR-3 cells, luciferase reporter gene expression was elevated up to 80-fold compared to PEG-PEI and was even higher than that of PEI 25 kDa. The advantage of this system is its specificity, which was demonstrated by competition experiments with free Fab' in the cell culture media during transfection experiments and by using OA3-negative cells. In the latter case, only a low level of reporter gene expression could be achieved with PEG-PEI-Fab'.     

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.     

Salmonella enterica serovar Typhimurium interaction with dendritic cells: impact of the sifA gene

Salmonella enterica serovar Typhimurium (S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA, invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-gamma production compared to S. Typhimurium aroC derivatives.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

Structural advantage of dendritic poly(L-lysine) for gene delivery into cells

This study aimed to investigate the relationships between structures of gene carrier molecules and their activities for gene delivery into cells. We compared 2 types of poly(L-lysine) as carriers, that is, dendritic poly(L-lysine) (KG6) and linear poly(L-lysine) (PLL). KG6 formed a neutral DNA complex, and its DNA compaction level was weaker than that of PLL. The amount of DNA binding and uptake into cells mediated by PLL was 4-fold higher than that with KG6. However, KG6-mediated gene expression was 100-fold higher than that by PLL. Since pK(a) values of terminal amines of KG6 were lowered even though small amounts of DNA were internalized into cells, sufficient DNA amounts for effective gene expression escaped to the cytosol due to the proton sponge effect in the endosome. In addition, weakly compacted DNA with KG6 was advantageous in accessing RNA polymerase in the cell nucleus. On the other hand, PLL did not show the proton sponge effect in the endosome and resulted in strong compaction of DNA. Even though large DNA amounts were internalized into cells, most of the DNA would not take part in gene expression systems in the nucleus. Amount of induced cytokine production after intravenous injection of DNA complexes with KG6 and PLL was low, and was similar to the case when DNA was injected alone. Therefore, no significant difference in effects on cytokine production was observed between KG6 and PLL.     

Magnetic dipole interaction of endogenous magnetic nanoparticles with magnetoliposomes for targeted drug delivery

Dynamics of magnetoliposomes binding to the tumor cells and the efficiency of their recognition for targeted drug delivery is largely determined by physical interaction. In this paper we assess the strength of magnetic dipole interaction that occurs between endogenous magnetic nanoparticles in tumor cells and exogenous magnetic nanoparticles as a component of magnetoliposomes, and compare it with the forces of specific binding of the antigen-antibody complex. To assess the strength of magnetic dipole interaction the model of chains of identical particles was used, and an order of magnitude, 10^?9 N, was obtained. Thus, the indicated force has an order of magnitude close to the forces of specific binding, and even more. The force of magnetic dipole interaction between a magnetically marked dosage form and tumor cells is virtually the additional specific binding force — “passive targeting” for targeted drug delivery in consequence of the fact that tumor cells tend to contain the number of biogenic nanoparticles of magnetite (Fe₃O₄) by an order of magnitude greater than normal.     

Susceptibility to chronic thromboembolic pulmonary hypertension may be conferred by miR-759 via its targeted interaction with polymorphic fibrinogen alpha gene

A deletion/insertion (Del/Ins) polymorphism of 28 base pairs (bp) in the 3′ untranslated region (UTR) of fibrinogen alpha gene (FGA) was associated with thromboembolic diseases, but the underlying mechanisms remain unknown. Computational predication reveals that the 28 bp polymorphic fragment is complementary to the sequence of a microRNA, miR-759. In this study, we aim to investigate the association and implicated mechanisms between FGA polymorphisms and the susceptibility to chronic thromboembolic pulmonary hypertension (CTEPH). The Del/Ins polymorphism was analyzed in 190 patients with CTEPH and 628 controls. The FGA 3′UTR and miR-759 interaction was investigated using luciferase assay and quantitative RT-PCR method. Expression of miR-759 and FGA in human tissues was investigated by RT-PCR. The results reveal that the allele frequency of Ins was significantly higher in the patients than in the controls (55.8 vs. 47.1%, P = 0.003, odds ratio = 1.42, 95% confidence interval: 1.13–1.79). Both miR-759 and FGA were expressed in human liver. Co-transfection of miR-759 decreased the expression and mRNA stability of reporter gene containing the FGA 3′UTR. The effect of miR-759 was stronger on the Ins allele than on the Del allele. These observations suggest that the expression of FGA was regulated by miR-759 through its interaction at the polymorphic 3′UTR sequence, which was associated with the susceptibility to CTEPH.     

A surface-modified baculovirus vector with improved gene delivery to B-lymphocytic cells

A short peptide motif from gp350/220 of Epstein-Barr virus, EDPGFFNVEI, which was known to bind to CD21, a surface protein on B-lymphocyte, was inserted into the baculovirus surface protein gp64. The recombinant virus carrying the hybrid gp64/gp350 gene, vAc-gp350EGFP, was obtained, and the expression of gp64/gp350 protein was confirmed with immunoblot using anti-gp350 antibody. When compared with a control virus with wild type gp64, vAc-gp350EGFP showed increased transduction efficiency in B cell lines Raji, HR1, B95-8, BJAB, and DG75, regardless of their being EBV-positive or EBV-negative. No such increase was seen in non-B cell lines HEK293 and HeLa. When Raji cells were transduced with increased amount of vAc-gp350EGFP, transduction became saturated when the multiplicity of infection was higher than 20pfu/cell. The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody. These results showed that vAc-gp350EGFP entered B cells by interacting with CD21.     

Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose–siRNA (CMM–siRNA) conjugates to macrophages and dendritic cells (DCs). CMM–siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.     

Protein phosphatase-1 is targeted to DNA polymerase delta via an interaction with the p68 subunit

Protein phosphatase-1 (PP1) is a Ser/Thr protein phosphatase that participates in the phosphorylation/dephosphorylation regulation of a diverse range of cellular processes. The PP1 catalytic subunit (PP1) achieves this by its ability to interact with many targeting subunits such that PP1 activity is thereby specified against phosphoprotein substrates in the microvicinity of its targeting subunit. DNA polymerase delta (Pol delta) is a key enzyme in mammalian chromosomal replication. It consists of four subunits, p125, p50, p68, and p12. We identify p68 as a novel PP1 targeting subunit. PP1 was shown to associate with human DNA polymerase delta by affinity chromatography and coimmunoprecipitation assays from mammalian cell lysates and in vitro by pull-down assays. The binding domain for PP1 was identified as the sequence KRVAL, a variant of the canonical RVxF PP1 binding motif. These studies provide the first evidence for the targeting of PP1 to DNA polymerase delta. We also show that CK2 phosphorylates the Pol delta p125, p68, and p12 subunits and that these phosphorylated subunits are substrates for PP1. These findings identify a new role for p68 as a PP1 targeting subunit that implicates PP1 in the dephosphorylation of Pol delta. Our findings also show that CK2 is a strong candidate for the protein kinase involved in the in vivo phosphorylation of p68.     

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.