[Envelope-type nano device for gene delivery].

We introduced a new concept "Programmed Packaging" for the rational development of non viral gene delivery system. There are many non-viral gene delivery system, however, their utility is limited in the in vitro experiments. The major reason is considered that it is difficult to package many functional devices into a nanoparticle.

Intranuclear disposition of exogenous DNA in vivo: silencing, methylation and fragmentation.

The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed.

Improved cell viability of linear polyethylenimine through gamma-cyclodextrin inclusion for effective gene delivery.

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation.

Quantitative comparison of intracellular trafficking and nuclear transcription between adenoviral and lipoplex systems.

To develop nonviral gene vectors that are sufficient for clinical application, it is necessary to understand why and to what extent nonviral vectors are inferior to viral vectors, which in general show a more efficient transfection activity. This study describes a systematic and quantitative comparison of the cellular uptake and subsequent intracellular distribution (e.g.

Non-linear pharmacodynamics in a non-viral gene delivery system: positive non-linear relationship between dose and transfection efficiency.

A remarkable non-linearity was found between dose and transfection activities of non-viral gene delivery systems, such as a Lipofectamine/DNA complex and an octaarginine-modified multifunctional envelope-type nano device (R8-MEND). We measured the nuclear delivery of pDNA to distinguish the non-linearity in intracellular pharmacokinetics or pharmacodynamics after transfection with R8-MEND at different doses. A remarkable positive non-linearity was found in the pharmacodynamics when the dose was increased.

High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression.

The mechanism of the arginine-rich peptide-mediated cellular uptake is currently a controversial issue. Several factors, including the type of peptide, the nature of the cargo, and the linker between them, appear to affect uptake. One of the less studied factors, which may affect the uptake mechanism, is the effect of peptide density on the surface of the cargo.

Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with micro (mu) and NLS-micro by cytoplasmic and nuclear microinjection: a comparative study with poly-L-lysine.

Background: The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLS(SV40)).

Effect of methylated adenine in plasmid DNA on transgene expression in mice.

Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N6-methyladenine (N6-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N6-MeA, when complexed with cationic lipids.

Correction of frameshift mutations with single-stranded and double-stranded DNA fragments prepared from phagemid/plasmid DNAs.

We recently found that a heat-denatured, double-stranded DNA fragment, prepared from plasmid DNA (dsHES), and a sense single-stranded DNA fragment, prepared from single-stranded phagemid DNA (fSense), corrected an inactivated hygromycin-resistance and enhanced green fluorescence protein fusion (Hyg-EGFP) gene containing a base substitution (G:C to C:G) mutation 2-fold and more than 10-fold, respectively, more efficiently than the conventional PCR fragment (pcrHES), in the small fragment homologous replacement method. In this study, we tested the abilities of these new DNA fragments to correct Hyg-EGFP genes inactivated by one base insertion (+G) and deletion (-C) mutations. In contrast to its activity with the substitution mutation, the fSense fragment showed similar efficiencies to those of the dsHES fragment in the correction of frameshift mutations.

Development of efficient packaging method of oligodeoxynucleotides by a condensed nano particle in lipid envelope structure.

An efficient delivery system is required if antisense oligodeoxynucleotides (ODN) are to be utilized for gene therapy. We report herein on the development of a novel ODN delivery system, ODN-encapsulated nano particles (ODN-ENP) using an efficient and simple packaging method. The ODN-ENP consists of a condensed ODN particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of ODN with a small diameter (150 nm).

Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment.

A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H.

Pharmacokinetics and brain uptake of lactoferrin in rats.

Lactoferrin (Lf) is an iron-binding glycoprotein belonging to the transferrin (Tf) family. Lf was reported to cross the blood brain barrier (BBB) via receptor-mediated transcytosis in an in vitro model of the BBB. In the present study, we compared the in vivo brain uptake of Lf with that of OX26, an anti-Tf receptor antibody, and Tf.

Pharmacokinetic analysis of the tissue distribution of octaarginine modified liposomes in mice.

We recently found that octaarginine modified liposomes (R8-Lip) can be efficiently internalized by cultured cells. The purpose of the present study was to quantitatively determine the effect of R8-density on the tissue distribution of R8-Lip in mice, using their clearance as an index. R8 was introduced in the form of stearylated R8 (STR-R8).

Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy.

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells.

Mutagenesis induced by oxidized DNA precursors: roles of Y family DNA polymerases in Escherichia coli.

To reveal the roles of Y family DNA polymerases in the mutagenesis induced by oxidatively damaged DNA precursors, 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP (8-OH-dGTP) were introduced into Escherichia coli strains deficient in the Y family polymerases, DNA polymerase IV (pol IV, encoded by the dinB gene) and DNA polymerase V (pol V, encoded by the umuDC locus). The mutation induced by 2-OH-dATP, but not that induced by 8-OH-dGTP, occurred less frequently in the dinB- strain than in the wild-type (wt) strain, suggesting the involvement of pol IV in the mutagenesis by 2-OH-dATP. Expression of pol IV from plasmid enhanced the mutagenesis by 2-OH-dATP in the dinB- strain.

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes.

Background: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application.

A multifunctional envelope-type nano device for novel gene delivery of siRNA plasmids.

A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-L-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study.

Kinetic analysis of protein production after DNA transfection.

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively.

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells.

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides.

New NTP analogs: the synthesis of 4'-thioUTP and 4'-thioCTP and their utility for SELEX.

The synthesis of the triphosphates of 4'-thiouridine and 4'-thiocytidine, 4'-thioUTP (7; thioUTP) and 4'-thioCTP (8; thioCTP), and their utility for SELEX (systematic evolution of ligands by exponential enrichment) is described. The new nucleoside triphosphate (NTP) analogs 7 and 8 were prepared from appropriately protected 4'-thiouridine and -cytidine derivatives using the one-pot method reported by J. Ludwig and F.

Construction of a multifunctional envelope-type nano device by a SUV*-fusion method.

A novel assembly method "SUV*-fusion method" was developed for the construction of a small and homogenous multifunctional envelope-type nano device (MEND) by utilizing a detergent-rich small unilamellar vesicle (SUV*). The method consists of three steps: (1) DNA condensation with a polycation, (2) electrostatic interaction of the SUV* with the DNA/polycation complex (DPC) and (3) lipid coating of DPC by SUV* fusion via removal of the detergent. We confirmed the construction of the MEND by sucrose density gradient centrifugation, and isolated the MEND only from the boundary between 25% and 40% sucrose.

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding.

Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: the use of a nuclear transfer score.

In the present study, the nuclear delivery of a green fluorescence protein (GFP)-encoding pDNA condensed by protamine was investigated in terms of trans-gene expression after cytoplasmic (E(cyt)) and nuclear (E(nuc)) microinjection. To compare the nuclear transfer process, a novel parameter; the nuclear transfer (NT) score was introduced. The E(cyt) value for protamine/pDNA particles increased in a charge ratio-dependent manner.

In vivo mutagenicities of damaged nucleotides produced by nitric oxide and ionizing radiation.

To evaluate the in vivo mutagenicities of damaged DNA precursors (deoxyribonucleoside 5'-triphosphates) produced by exposure to nitric oxide (NO) and ionizing radiation, five damaged deoxyribonucleotides (deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, dUTP, and 8-hydroxy-dATP) were introduced into competent Escherichia coli cells. Their mutagenic potentials were assayed using the chromosomal rpoB gene as a mutagenesis target. In contrast to 8-hydroxy-dGTP and 2-hydroxy-dATP, which were examined in an earlier study, none of these damaged deoxyribonucleotides significantly increased the rpoB mutant frequency.