Targeted conversion of the transthyretin gene in vitro and in vivo.

Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.

Injectable silicone implants as vaccine delivery vehicles.

Injectable silicone implants were assessed as vaccine delivery vehicles in sheep, using either the model antigen avidin or Clostridium tetani and Clostridium novyi toxoids. Two types of implant were compared, the matrix type, that has been shown to deliver antigen in vitro in a first-order profile over approximately 1 month, and the covered rod type, that delivers antigen for several months in a zero-order profile. The implants were prepared using lyophilized antigen and adjuvant (in this case, recombinant ovine interleukin-1beta; rovIL-1beta) and manufactured in the absence of extremes of temperature or pH or the use of organic solvents. Use of the matrix type implant was capable of inducing antibody responses equivalent to those induced by conventional vaccination with aluminium hydroxide adjuvant ("alum"). The use of the covered rod implants, that release very low levels of antigen over a long period, induced responses that were markedly enhanced over the alum control groups. The covered rod implant also favoured production of both IgG1 and IgG2 isotypes in contrast to responses of matrix-vaccinated sheep and conventionally vaccinated control sheep in which IgG1 predominated. Prolonged duration of the antibody response was also observed following vaccination with covered rod implants. Dose-response analysis using the matrix implant demonstrated a trend towards improved responses for lower antigen doses. Clostridial vaccination of sheep showed that protective antibody titres up to 4-fold higher than for alum-adjuvanted groups could be induced by administering the antigen in the covered rod implant. Responses elicited by all implant groups were dependent on the inclusion of adjuvant into the implant formulation.

Continuous antigen delivery from controlled release implants induces significant and anamnestic immune responses.

Two continuous delivery injectable silicone implants were tested to determine if they were capable of delivering vaccines in a single shot. The Type A implant delivers antigen in vitro over a 1-month-period and the Type B over several months. Vaccination studies in sheep were designed to compare the responses induced by the Type A and B implants, Alzet mini-osmotic pumps and conventional antigen delivery. A model antigen, avidin, was used along with IL-1beta or alum as adjuvants. Sheep were immunised with various formulations and the titre and isotype of the antigen specific antibodies monitored. The Type B implant induced antibody (Ab) titres of greater magnitude and duration than soluble vaccines or the Type A implant with adjuvant, but only if IL-1beta was included in the formulation. Both implants induced antibodies of IgG1 and IgG2 isotype. A memory response to soluble antigen challenge was induced by the Type B+IL-1beta implant, which was predominantly of an IgG1 isotype.

Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions.

We have previously demonstrated that Atelocollagen, used clinically for wound healing, is a reliable safe carrier for gene delivery. To obtain phenotypic changes by gene expression of cDNA, we developed an efficient technique for high-throughput gene transfer and expression screening in mammalian cells in microarrays by precoating a microplate with an Atelocollagen complexed with cDNA to which cells are then seeded. The complexes with a nanoparticle form were efficiently transduced into cells without use of any additional transfection reagent, and they allowed for long-term gene expression without apparent chromosomal integration. The complex spotted onto the well of a microplate was stable for a long period and allowed the cells to transduce and express reporter genes in a dose-dependent manner. We also showed that the present method using Atelocollagen-based gene transfer is applicable to gene medicines such as antisense ODNs and adenovirus vectors. These results suggest that Atelocollagen may be appropriate for general use in high-throughput screening of large sets of gene medicines for functional analyses in mammalian cells.

The application of biodegradable collagen minipellets as vaccine delivery vehicles in mice and sheep.

Collagen minipellets are injectable delivery vehicles that release antigen and adjuvant over several days in a first-order release profile. In vaccination experiments in mice, secondary antibody responses induced by minipellets formulated with avidin and IL-1beta as adjuvant were equivalent to those induced by a conventional immunization with avidin in alum. When no adjuvant was used, anti-avidin responses induced by minipellets were 10-20-fold higher than those induced by injection of avidin in saline. In sheep, conventional vaccination with avidin in alum induced antibody responses initially exceeding that induced by minipellets formulated with avidin and IL-1beta, while following a secondary vaccination, the minipellet antibody response was equal to or greater than the alum-adjuvanted control groups. Increasing levels of IL-1beta adjuvant resulted in enhanced persistence of the antibody response. When clostridial vaccine antigens were incorporated into the minipellets, total antibody responses induced in sheep were equivalent to those induced by vaccination with the clostridial antigens in alum. Neutralizing antibody titres exceeded those induced by conventional vaccination. No adverse site reactions were observed at the implant site, with immunohistological study showing that the cellular infiltrate was dominated by a transient influx of neutrophils. This is a typical response to delivery of bioactive IL-1beta. The minipellets were completely degraded within 35 days of implantation.

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid.

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.

Biomaterials for gene delivery: atelocollagen-mediated controlled release of molecular medicines.

Over the last decade, increasing attention has been paid to the development of systems to deliver drugs for long periods at controlled rates. Some of these systems can deliver drugs continuously for over one year. However, little effort has been given to developing systems for the controlled release of nucleic acids. Recently, a novel gene transfer method which allows prolonged release and expression of plasmid DNA in vivo in normal adult animals was established. In this system, a biocompatible natural polymer such as collagen or its derivatives acts as the carrier for the delivery of DNA vectors. The biomaterial carrying the plasmid DNA was administered into animals and, once introduced, gradually released plasmid DNA in vivo. A single injection of plasmid DNA/biomaterial produced physiologically significant levels of gene-encoding proteins in the local/systemic circulation of animals and resulted in prolonged biological effects. These results suggest that the biomaterials carrying plasmid DNA may enhance the clinical potency of plasmid-based gene transfer, facilitating a more effective and long-term use of naked plasmid vectors for gene therapy. Furthermore, the biomaterials can be removed surgically, minimizing the effect of gene products if some unexpected side effects should be observed after application. The application of these systems to expand the bioavailability of molecular medicine, including antisense oligonucleotides and adenovirus vectors, and to aid in stem cell transplantation in the context of DNA-based tissue engineering will be discussed.

Cell-substrate and cell-cell interactions differently regulate cytoskeletal and extracellular matrix protein gene expression.

In this study, cellular events during tissue formation were investigated at the mRNA level using the Northern blot technique. The levels of expression of mRNAs encoding specific proteins (beta-actin, fibronectin, and laminin) during tissue formation on tissue culture dishes were quantitatively assessed using a Northern blot technique with autoradiography. The level of beta-actin mRNA increased with incubation time and reached a maximal level near the confluent state, followed by reduced beta-actin mRNA expression at a later stage of tissue formation. The time course of beta-actin mRNA expression corresponded well to the time course of morphologic changes and cytoskeletal organization in adherent cells. Expression of the mRNAs encoding the extracellular matrix proteins fibronectin and laminin was initiated at the proliferation stage. After maximum expression levels of these two mRNAs were reached at the confluent stage, a gradual decrease in their expression levels was seen during long-term culture. Expression patterns of mRNAs encoding cytoskeletal and extracellular matrix proteins strongly depended on the type of artificial substrates used; a mRNA expression pattern similar to that observed during tissue formation on tissue culture dishes was observed on a cell-adhesive substrate during tissue formation, whereas reduced expression was seen during tissue formation on a less adhesive substrate. Thus, the dynamic changes occurring during tissue formation were quantified to investigate the roles of artificial substrates in tissue formation at the mRNA level.

In situ detection of DNA fragmentation and expression of bcl-2 in human neuroblastoma: relation to apoptosis and spontaneous regression.

UNLABELLED: Spontaneous regression occurs in some cases of neuroblastoma, especially stage IVS. The incidence of neuroblastoma has been reported to be increasing since the mass screening program was introduced in Japan. This would indicate that the screening is detecting regressing tumors. However, the mechanism of regression is still unknown. To evaluate the hypothesis that the regression might be related to apoptosis, the authors examined apoptosis by in situ end-labeling of fragmented DNA and expression of the apoptosis-suppressing protein bcl-2. MATERIALS AND METHODS: One hundred eighteen neuroblastoma cases were available for examination. Eighty (67.8%) were detected by the mass screening program. Serial sections were cut from paraffin-embedded tumors. A modified TdT-mediated dUTP nick end-labeling (TUNEL) method was performed to detect apoptosis. Immunohistochemical analysis was performed to detect bcl-2 expression. RESULTS: In cases under 1 year of age or with a favorable clinical stage, the incidence of apoptosis was significantly high. Expression of bcl-2 was associated with N-myc amplification and unfavorable histology (Shimada classification). Tumors in patients under 1 year of age often had areas where cellularity was markedly decreased, and apoptosis was often observed while bcl-2 expression was reduced. In such cases, there was a negative correlation between occurrence of apoptosis and bcl-2 expression. This suggests that apoptosis may be related to spontaneous regression in neuroblastoma.

mRNA expression induced by cell-substrate interaction. A two-dimensional tissue formation process.

This study aimed to develop a quantitative assay method of determining cellular events at the mRNA level during tissue formation. Quantitative assessment of mRNAs coding specific proteins (beta-actin, fibronectin, laminin) during tissue formation on tissue culture dishes was attained using a Northern blot technique with autoradiography. The amount of beta-actin mRNA, the expression of which is initiated shortly after adhesion, greatly increased with incubation time and reached maximal levels near the confluent state, followed by a reduced expression at a later stage of tissue formation. The time course of beta-actin mRNA expression, which reflects cytoskeletal organization, corresponded well to morphologic changes in adherent cells. Expression of mRNAs coding the extracellular matrix proteins, fibronectin and laminin, was initiated at the proliferation stage. After maximum expression levels were observed at the confluent stage, a gradual decrease in the expression of both mRNAs was seen after longer culture periods. Expression patterns of mRNAs coding cytoskeletal and extracellular matrix proteins greatly depended upon the type of artificial substrates. Thus, the dynamic changes in tissue formation were quantified to elucidate the significance of artificial substrates in tissue formation at the mRNA level.

A total delivery system of genetically engineered drugs or cells for diseased vessels. Concept, materials, and fabricated prototype device.

The development of a percutaneous procedure using a catheterized system for diseased vessels has been increasingly in demand in conjunction with gene therapy using genetically engineered drugs (antisense) and cells. The authors' strategic concept realizes revascularization at narrowed, diseased sites and delivery of drugs or cells into the diseased tissues or targeted cells. An inflatable, drug-releasing double balloon is installed at the tip of a catheter. The outer balloon, fabricated with micropores (diameters of 20 and 30 mm) by an excimer laser ablation technique, releases a viscous solution containing a photoreactive polymer and drug or cells on inflation of the inner balloon. A photoresponsive water-soluble polymer, molecularly designed for its ability to achieve prolonged local residency of antisense DNA at the tissue level and enhanced transmembrane transport at the cellular level, is premixed with antisense oligonucleotide drug. On light irradiation, the nonionic polymer is reversibly converted to a positively charged polymer that can be complexed with highly negatively charged antisense DNA (c-myb), which may enhance the transmembrane delivery of antisense. On cessation of irradiation, the complex slowly dissociates to function intracellularly as an antisense drug, resulting in inhibition of cell proliferation. Thus, our integrated, dual-function balloon system may contribute to mechanical dilatation gene therapies at diseased vessels.

Detection of specific base sequences in solution using DNA probes labeled with d- and/or 15N-substituted spin-labels.

In order to enhance the sensitivity and the accuracy of the detection by the DNA probe method in which spin-labeled oligonucleotides are used as probes, 4-amino-2,2,6,6-tetramethylpiperidine-15N-oxyl-d16 (4-amino-TEMPO-15N-d16) and 4-amino-2,2,6,6-tetramethylpiperidine-14N-oxyl-d16 (4-amino-TEMPO-14N-d16) were incorporated into the phosphate linkage of oligonucleotides by the hydrogen phosphonate method and these DNA probes were used for the detection of target DNAs in solution. The intensities of the electron paramagnetic resonance (EPR) signals of the oligonucleotides labeled with 4-amino-TEMPO-14N-d16 and 4-amino-TEMPO-15N-d16 were 3-fold and 4-fold larger than that of the oligonucleotide labeled with 4-amino-TEMPO-14N, respectively. Also, the EPR lines of these labeled oligonucleotides do not overlap each other, allowing the detection of two different regions in the same target DNA by the single EPR measurement.

Oligonucleotides site-specifically spin-labeled at 5'-terminal or internucleotide linkage and their use in gene analyses.

Spin-labeled oligonucleotides (S-probes) were synthesized and examined as DNA probes to monitor hybrid formation. TEMPO was introduced either at the internucleotide linkage of 5'-terminus (Type 1) or at the 5'-terminal hydroxyl group (Type 2) and both types of S-probes were used in this study. The presence of target DNA was detected in solution by EPR spectroscopy for both types of S-probes. Hybridization of the S-probes resulted in notable broadening of EPR line width, accompanied by a decrease in the EPR signal height ratio for I(-1)/I(0).I(-1)/I(0) of S-probes having no spacer between oligonucleotide and TEMPO decreased more markedly than that of S-probes with a spacer, indicating that TEMPO should be introduced to an oligonucleotide directly to monitor hybrid formation. When M13mp8 single-stranded DNA with or without an EcoRI recognition site was selected as a target DNA, hybrid formation was detected only for DNA containing EcoRI site in solution using spin-labeled oligonucleotides.

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.

Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.

Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides.

Characterization of antisense DNA derivatives having stereoisomeric linkages.

The isomer separation of oligonucleoside phosphoramidates (OPA) by RPLC was studied. All stereoisomers of OPA-tetramer (dATCG) modified with isopropylamine could be separated and they showed different CD spectra each other. OPA-decamer (dGGGCATCGTC) modified with 3-amino-1-propanol could be separated into five fractions. Each fraction was found to have different ability to form hybrids with its complementary oligonucleotide, indicating that it is possible to exclude stereoisomers which can hardly bind to target nucleic acids.

Structural analysis of nucleic acids by labeled oligonucleotides.

In this report, the characterization of labeled oligonucleotides was discussed from the view points of base sequence analysis and structural analysis of nucleic acids in solution. Oligonucleotides site specifically spin labeled with TEMPO and fluorescent labeled with fluorescein were prepared and used for those analyses. The changes of ESR lines and rotational correlation time (tau) of the spin labeled oligonucleotide (S-probe) were dependent on the base sequence of S-probe, diastereoisomers, and the manner of hybridization. These results suggest that the conformation of the hybrid largely affected the local mobility of TEMPO and that tau value of S-probe reflected the local structure of the hybrid. When S-probe which was complementary to a single strand region of 5S RNA, was mixed with 5S RNA, tau value largely changed, indicating that the S-probe could form hybrid with 5S RNA in solution. Similar results were also obtained in the fluorescence depolarization analysis using fluorescent labeled oligonucleotide (F-probe). These results suggest that S-probe and F-probe are capable for the recognition of the secondary structure of 5S RNA in solution and useful for the analysis of the secondary structure of other nucleic acids in solution.

ESR behaviour of spin-labelled oligoDNAs.

Oligodeoxyribonucleotides (oligoDNAs) were spin-labelled at the 5'-end internucleotide linkage, directly or through the spacers of different lengths, with TEMPO and the labelled oligoDNAs were explored for the ESR spectroscopical behaviour in solution. Oxidation of H-phosphonate intermediates in CCl4 solutions of TEMPO and diamines was taken for the introduction of TEMPO and spacers, respectively. ESR lines of TEMPO attached to oligoDNAs, directly or through spacers, were already broadened compared to those of free TEMPO, and the broadening decreased with increasing chain length of the spacer and increased with increase in the chain length of the oligoDNAs. The ESR lines of the oligoDNA labelled directly were further broadened in the presence of a DNA complementary to the labelled oligoDNA. In addition, the broadened lines were further broadened in the presence of a larger DNA. This observation implies that spin labelling at the internucleotide linkage is useful in the DNA probe method to avoid B/F separation.

A study on spin-labelled oligonucleotide synthesis and its electron spin resonance behavior in solution.

An oligonucleotide spin-labelled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) at the internucleotide bond (d-Tp(L)TpTpTpT) prepared by oxidation of the pentanucleotide containing the H-phosphonate diester (d-Tp(H)TpTpTpT) in the presence of 4-amino-TEMPO, was separated and identified by high-performance, reverse-phase liquid chromatography combined with detection by electron spin resonance spectroscopy. This spin-labelled oligonucleotide produced a triplet with the slightly broadened M1 = -1 ESR component, while a triplet with almost equal intensities was obtained from the spin-label. The M1 = -1 component from the labelled oligonucleotide was further broadened in the presence of poly(A) which forms a complementary double strand with this molecule.