Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.

Development and antitumor activity of a BCL-2 targeted single-stranded DNA oligonucleotide.

PNT100 is a 24-base, chemically unmodified DNA oligonucleotide sequence that is complementary to a region upstream of the BCL-2 gene. Exposure of tumor cells to PNT100 results in suppression of proliferation and cell death by a process called DNA interference. PNT2258 is PNT100 that is encapsulated in protective amphoteric liposomes developed to efficiently encapsulate the PNT100 oligonucleotide, provide enhanced serum stability, optimized pharmacokinetic properties and antitumor activity of the nanoparticle both in vivo and in vitro. PNT2258 demonstrates broad antitumor activity against BCL-2-driven WSU-DLCL2 lymphoma, highly resistant A375 melanoma, PC-3 prostate, and Daudi-Burkitt's lymphoma xenografts. The sequence specificity of PNT100 was demonstrated against three control sequences (scrambled, mismatched, and reverse complement) all encapsulated in a lipid formulation with identical particle characteristics, and control sequences did not demonstrate antiproliferative activity in vivo or in vitro. PNT2258 is currently undergoing clinical testing to evaluate safety and antitumor activity in patients with recurrent or refractory non-Hodgkin's lymphoma and additional studies are planned.

Antibody-linked spherical nucleic acids for cellular targeting.

Spherical nucleic acid (SNA) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through noncovalent functionalization with a complementary antibody-DNA conjugate. As a proof-of-concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.

Targeted nanoparticles enhanced flow electroporation of antisense oligonucleotides in leukemia cells.

Liposome nanoparticles (LNs) with a targeting ligand were used in a semi-continuous flow electroporation (SFE) device to enhance in vitro delivery of exogenous oligonucleotides (ODN). Nanoparticles comprising transferrin-targeted lipoplex encapsulating ODN G3139 were mixed with K562 cells (a chronic myeloid leukemia cell line) and incubated for half an hour to accomplish nanoparticle binding. The mixture was then flowed through a SFE channel where electric pulses were given. Better ODN delivery efficiency was achieved with an increase of ∼24% to the case in combination of non-targeted LNs and SFE, and ∼60% to the case using targeted LNs alone, respectively. The MTS assay results confirmed cell viability greater than 75%.

Amphoteric liposomes enable systemic antigen-presenting cell-directed delivery of CD40 antisense and are therapeutically effective in experimental arthritis.

OBJECTIVE: Mediation of RNA interference by oligonucleotides constitutes a powerful approach for the silencing of genes involved in the pathogenesis of inflammatory disease, but in vivo application of this technique requires effective delivery to immune cells and/or sites of inflammation. The aim of the present study was to develop a new carrier system to mediate systemic administration of oligonucleotides to rheumatoid arthritis (RA) joints, and to develop an antisense oligonucleotide (ASO)-based approach to interfere with CD40-CD154 interactions in an experimental model of RA. METHODS: A novel liposomal carrier with amphoteric properties, termed Nov038, was developed and assessed for its ability to systemically deliver an ASO directed against CD40 (CD40-ASO). Male DBA/1 mice with collagen-induced arthritis were treated with Nov038-encapsulated CD40-ASO, and the effects of treatment on various parameters of disease activity, including clinical score, paw swelling, lymph node responses, and inflammatory cytokine production in the joints, were assessed. RESULTS: Nov038 was well tolerated, devoid of immune-stimulatory effects, and efficacious in mediating systemic oligonucleotide delivery to sites of inflammation. In mice with collagen-induced arthritis, Nov038 enabled the therapeutic administration of CD40-ASO and improved established disease, while unassisted CD40-ASO was ineffective, and anti-tumor necrosis factor alpha (anti-TNFalpha) treatment was less effective in this model. Nov038/CD40-ASO efficacy was attributed to its tropism for monocyte/macrophages and myeloid dendritic cells (DCs), resulting in rapid down-regulation of CD40, inhibition of DC antigen presentation, and reduction in collagen-specific T cell responses, as well as decreased levels of TNFalpha, interleukin-6 (IL-6), and IL-17 in arthritic joints. CONCLUSION: Amphoteric liposomes represent a novel carrier concept for systemic and antigen-presenting cell-targeted oligonucleotide delivery with clinical applicability and numerous potential applications, including target validation in vivo and inflammatory disease therapeutics. Moreover, Nov038/CD40-ASO constitutes a potent alternative to monoclonal antibody-based approaches for interfering with CD40-CD40L interactions.

Cell culture and xenograft-bearing animal studies of radiolabeled antisense DNA carrier nanoparticles with streptavidin as a linker.

UNLABELLED: Transmembrane transfectors (carriers) are increasingly being viewed as helpful or even necessary to improve cellular delivery in connection with antisense tumor targeting and other applications requiring cell membrane transport of DNAs, RNAs, and other oligomers. We are investigating streptavidin as a convenient linker for biotinylated carriers and oligomers because it requires only simple mixing for preparation. The goal of this study was to evaluate antisense DNA-streptavidin-carrier nanoparticles for accumulation in cell culture and in xenograft-bearing mice. METHODS: The 3 carriers were cholesterol, a 10-mer Tat peptide, and a 10-mer polyarginine peptide. A 20-mer DNA targeting the mdr1 messenger RNA coding for Pgp expression was used as the phosphodiester (PO) DNA as well as the phosphorothioate (PS) DNA. In all cases, the (99m)Tc radiolabel was on the DNA. The 8 nanoparticles were first tested in mdr1(++) KB-G2 and TCO-1 cells and in mdr1(+/-) KB-31 cells in culture for evidence of improved accumulation and antisense targeting. Thereafter, the PS DNA-streptavidin-Tat, PO DNA-streptavidin-Tat, and PS DNA-streptavidin-cholesterol nanoparticles were administered intravenously to KB-G2 xenograft-bearing mice, and tissue distributions were measured. RESULTS: In culture, the PO nanoparticles showed increased accumulation compared with the corresponding nanoparticles without the carrier in all 3 cell types; in contrast, with the PS nanoparticles, any similar carrier-mediated increase may have been obscured by the much higher protein-binding affinity of PS DNA. As evidence of antisense targeting, the Tat and cholesterol PS nanoparticles showed statistically significant accumulation at 23 h in cells in the descending order TCO-1, KB-G2, and KB-31, although there were no significant differences among the PO nanoparticles. In xenograft-bearing mice, the tissue accumulation of both forms of the PS nanoparticles greatly exceeded that of the PO nanoparticles and, including in the tumor, were similar to that obtained previously for naked PS DNA. CONCLUSION: The presence of the streptavidin linker had no obvious detrimental effect on the functions of the carriers and antisense DNAs. The higher protein-binding affinity of the PS nanoparticles than the PO nanoparticles was still apparent both in vitro and in vivo, the pharmacokinetics of the PS nanoparticles were similar to that of naked PS DNA, and the carriers improved cellular accumulation, at least for the PO nanoparticles. These observations, taken together with the higher accumulation of both forms of the antisense PS nanoparticles in mdr1(++) KB-G2 and TCO-1 cells than in mdr1(+/-) KB-31 cells, suggest that further effort is justified to confirm that the antisense properties of the DNAs were not compromised by the presence of streptavidin.

Improved delivery in cell culture of radiolabeled antisense DNAs by duplex formation.

PURPOSE: Delivery remains an unresolved problem in applications requiring intravenous administration of DNAs. Recently improved antisense translation interruption in cells was reported for an antisense (AS) oligomer as a duplex compared to singlet AS oligomer presumably because of improved delivery. The unstable phosphodiester backbone of the sense (S) oligomer and its shorter chain length apparently encouraged intracellular dissociation and release of the AS oligomer. We have investigated the mechanism involved to evaluate whether the approach may be useful for antisense radionuclide imaging. PROCEDURES: Duplexes were formed between an AS phosphorothioate DNA against the mdr1 mRNA and the uniform phoshorothioate or uniform phosphodiester sense (S) DNAs with either four or six mismatches. RESULTS: Accumulations in KB-G2 (Pgp++) cells of radiolabeled AS DNA as duplex accumulated threefold higher compared to singlet. Accumulation was still antisense as shown by reduced accumulations with the radiolabel on the S DNA. However, the DNA backbone had no clear influence on accumulations. CONCLUSIONS: Targeting of mRNAs with radiolabeled AS DNAs may be improved in cell culture if duplexed with an S DNA engineered for low hybridization affinity to encourage dissociation in the presence of the target mRNA.

Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics.

The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.

Initial observations of 99mTc labelled locked nucleic acids for antisense targeting.

BACKGROUND AND OBJECTIVE: The most recent DNA analogues to become commercially available are known as locked nucleic acids (LNAs). The aim of this study was to evaluate the properties of LNAs for antisense targeting. METHODS: A 15 mer LNA antisense to RIalpha mRNA was studied in cell culture. The antisense LNA (5'-amine linker-TGCCTCCTCACTGGC) was purchased along with its sense control LNA. Surface plasmon resonance was used to compare affinity constants with uniform 18 mer phosphorothioate (PS) DNA and uniform 18 mer phosphodiamidate morpholinos (MORFs, another DNA analogue). After radiolabelling with 99mTc via MAG3, the antisense and sense LNAs were added at 5 nM to wells containing ACHN cells in culture and accumulations measured over 24 h. Subcellular partition was determined after 16 h of incubation by separating membrane bound, cytoplasmic and nuclear fractions. The cell studies were conducted both with naked LNAs and with liposomes (oligofectamine) as carrier. RESULTS: Radiochemical purity was about 95% after purification on a P4 column and each LNA was radiolabelled at about 20 GBq.micromol(-1) (100.microCi.microg(-1)). The surface plasmon resonance results showed a more favourable dissociation constant for the duplex with DNA of the 15 mer LNA (0.55 x 10(-10).M(-1)) compared to the duplex with 18 mer DNA and 18 mer MORFS (2.05 and 1.06 x 10(-10).M(-1), respectively). Because of lower dissociation constants, the hybridization affinities of LNAs are therefore higher than those of uniform and identical PS DNAs or MORFs. The cellular accumulations suggested an antisense effect in that the antisense LNA accumulation was higher than sense both when added naked (1.8% vs. 0.4% at 24 h) and with liposome carrier (3.8% vs. 1.0% at 24 h). Thus while absolute cellular uptake was lower than that observed by this laboratory with other oligomers, the antisense/sense differential was higher. The number of antisense LNAs accumulating per cell specifically (i.e., antisense minus sense) was about 45,000 naked and about 100,000 with carrier. Subcellular partition showed that both LNAs were partitioned to each fraction with antisense accumulations greater than sense and carrier accumulations greater than naked as before. That as much as 2.9% of the antisense LNA (with carrier) was in the cytoplasmic or nuclear factions demonstrates that the LNA was internalized. CONCLUSIONS: LNAs appear to be attractive oligomers for antisense targeting and other radiopharmaceutical applications.

Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells.

Cancer therapy is currently limited by the difficulty of achieving efficient delivery into target cells. To investigate whether therapeutics can be delivered specifically to cancer cells, we have explored the possibility of selecting small peptides that bind specifically, or preferentially, to breast cancer cell lines. By using random peptide phage libraries and an experimental approach that allows the selection of internalized peptides, cell-specific binding peptides have been identified. The peptides define a major core motif (LTVXPWY) that was not found in negative phages. Phage displaying LTVSPWY peptide sequence exhibited a specific binding to breast cancer cells. None of the selected peptides bound to human primary cells from different tissue origin (e.g., epithelial, endothelial, hematopoetic). The potential of the selected peptides to mediate cellular internalization in the context of phages and recombinant GFP-peptide fusions was demonstrated. By linking the LTVSPWY peptide to an antisense phosphorothioate oligonucleotide against the ErbB2 receptor, specific delivery to cancer cells was achieved. In contrast to free antisense, the peptide-antisense conjugates inhibited ErbB2 gene expression. Thus, efficient delivery of antisense oligonucleotides can be achieved by coupling them to cancer cell-specific peptides, identified by a method that did not require any knowledge about their corresponding receptors.

Polymeric nanogels produced via inverse microemulsion polymerization as potential gene and antisense delivery agents.

Polymeric nanogel vectors were developed for cellular gene and antisense delivery. Inverse microemulsion polymerization was utilized to synthesize biocompatible nanogels with controlled size, morphology, and composition. The chemical composition, size, polydispersity, stability, and swelling behavior of the nanogels were investigated by NMR, light scattering, transmission electron microscopy, and atomic force microscopy. The cell viability, uptake, and physical stability of nanogel-DNA complexes were evaluated under physiological conditions. Monodisperse nonionic and cationic nanogels were produced with controllable sizes ranging from 40 to 200 nm in diameter. The nanogels demonstrated extended stability in aqueous media and exhibited low toxicity in cell culture. Cationic nanogels formed monodisperse complexes with oligonucleotides and showed enhanced oligonucleotide uptake in cell culture. The nanogels synthesized in this study demonstrate potential utility as carriers of oligonucleotides and DNA for antisense and gene delivery.

Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer.

The present study was designed to determine the maximum tolerated dose (MTD), toxicity profile, pharmacokinetics (PKs), and antitumor activity of the protein kinase C-alpha antisense oligonucleotide ISIS 3521 (ISIS Pharmaceuticals, Inc., Carlsbad, CA) when administered in combination with 5-fluorouracil (5-FU) and leucovorin (LV). Patients with refractory solid tumors received ISIS 3521 as a 21-day continuous infusion administered simultaneously with 5-FU and LV given daily for 5 days repeated every 4-5 weeks (one cycle). 5-FU and ISIS 3521 PK analysis were performed on samples taken during the first cycle in all patients. Fifteen patients received ISIS 3521 at one of three dose levels: (a) 1.0 (n = 3 patients); (b) 1.5 (n = 3 patients); and (c) 2.0 (n = 9 patients) mg/kg/day. All patients simultaneously received 5-FU (425 mg/m(2)/day) and LV (20 mg/m(2)/day) for 5 consecutive days. Grade 1-2 toxicities included alopecia, fatigue, mucositis, diarrhea, anorexia, nausea/vomiting, and tumor pain. One patient had grade 3 chest pain considered to be related to 5-FU therapy, another patient had dose-limiting grade 3 mucositis resolving in <7 days, and one patient with a history of gastritis had an acute upper gastrointestinal bleed thought to be 5-FU-induced toxicity. Five patients developed cycle 1 grade 4 neutropenia, which resolved without colony-stimulating factors before the next treatment cycle. There were no effects on prothrombin time and activated partial thromboplastin time. A clinically defined MTD was not reached. The character and severity of these toxicities do not seem to be dose related, and, as such, there was no classical dose-limiting toxicity defining the MTD. ISIS 3521 PKs in the presence of 5-FU was consistent with those reported previously. 5-FU PK parameters were also similar in the presence or absence of ISIS 3521. Six of 14 patients ( approximately 43%) across all dose cohorts had an improvement in measurable tumor response ranging from minor reduction in tumor size (4 patients) to objective partial response (>50% reduction in tumor size, 2 patients). ISIS 3521 is tolerable at its recommended single-agent dose when given with 5-FU and LV. There is no apparent PK interaction between ISIS 3521 and 5-FU and LV. Antitumor activity was observed with the combination; however, it is uncertain whether clinical activity is a result of enhanced drug interaction. Our study warrants further exploration of efficacy in a Phase II and/or Phase III clinical trial setting.

Tumor targeting and imaging of intraperitoneal tumors by use of antisense oligo-DNA complexed with dendrimers and/or avidin in mice.

To establish an effective nonviral gene delivery and a corresponding imaging method for i.p.-disseminated tumors, various oligonucleotide-carrier complexes were synthesized, and their in vitro and in vivo properties were examined. The 20-mer multiamino-linked oligonucleotide (oligo), synthesized as antisense against the c-erbB-2 sequence, and the 3'-biotinylated form of the same oligonucleotide (oligo-Bt) were (111)In labeled through a diethylenetriaminepentaacetic acid chelate. (111)In-oligo was mixed with generation 4 polyamidoamine dendrimer (G4) or with biotinylated G4 (G4-Bt), which are positively charged to form electrostatic complexes. (111)In-oligo/G4-Bt and (111)In-oligo-Bt were conjugated to avidin ((111)In-oligo/G4-Av and (111)In-oligo-Av, respectively). (111)In-oligo/G4, (111)In-oligo/G4-Av, (111)In-oligo-Av, and carrier-free (111)In-oligo (2.96 kBq/22.4-45.9 ng of oligo) were examined for internalization in vitro in human ovarian cancer cells (SHIN3). Biodistribution of (111)In-oligo-carrier complexes or (111)In-oligo was examined in normal (n = 4-7) or i.p. SHIN3 tumor-bearing (n = 6-10) mice 2-24 h after i.p. injection (74 kBq/125-300 ng). Scintigraphy of i.p. tumor-bearing and normal mice was performed at various times postinjection of (111)In-oligo-carrier complex or (111)In-oligo (1.85 MBq/2.2 ng). (111)In-oligo-carrier complexes bound to the tumor cells were internalized at a rate of 34-56% at 24 h. In vivo, G4, G4-Av, and Av significantly enhanced tumor delivery of (111)In-oligo [9.1, 14.5, and 24.4% of injected dose per g of tissue (ID/g) at 24 h; P < 0.05, < 0.01, and < 0.0001, respectively] compared with delivery without carrier (0.8% ID/g). Scintigrams of (111)In-oligo delivered to the i.p.-disseminated tumors by the carriers were successfully obtained. In conclusion, G4, G4-Av, and Av can effectively deliver (111)In-oligo to i.p.-disseminated tumors. (111)In-oligo-carrier complexes also have potential as tracers for imaging and monitoring of gene delivery.

Angiotensin II AT(1A) receptor antisense lowers blood pressure in acute 2-kidney, 1-clip hypertension.

To test the effectiveness of antisense oligonucleotides targeted to the angiotensin type 1A (AT(1A)) receptor mRNA on blood pressure reduction, the 2-kidney, 1-clip (2K1C) Goldblatt model of hypertension was studied in the acute phase of hypertension, when the peripheral renin-angiotensin system is overactive. A single injection of AT(1A) receptor antisense oligodeoxynucleotides significantly reduced systolic blood pressure for a period of 8 days in 2K1C rats after clipping, from 157.5+/-5 mm Hg on day 7 to 141.3+/-3.0 mm Hg on day 15 after clipping (P<0.01). The AT(1A) receptor antisense oligonucleotide labeled with fluorescein shows that the antisense oligonucleotide at 24 hours was taken up into aorta, mesenteric artery, liver, kidney glomeruli, and medulla, remaining up to 6 days. The AT(1A) receptor number in fmol/g tissue was significantly decreased after AT(1A) receptor antisense oligonucleotide treatment in the dorsal aorta, mesenteric artery, renal cortex, and renal medulla (P<0.05) compared with that of the AT(1A) receptor-scrambled antisense oligonucleotide control-treated group. The data clearly demonstrate a prolonged antihypertensive effect of AT(1A) receptor antisense oligonucleotide in the 2K1C renovascular model of hypertension when it is administered intravenously in a single low dose (0.33 mg/kg(-1)). It also shows that the AT(1A) receptor antisense oligonucleotide is actively taken up by AT(1A) target tissues and that there is a significant decrease in receptor density. We conclude that in the acute phase of 2K1C hypertension, antisense to AT(1A) receptor decreases AT(1A) receptor density, which attenuates the vascular vasoconstrictive effects of high plasma angiotensin II levels and in the kidney elicits natriuresis. The decrease in renal AT(1A) receptor density may also lead to sodium loss and reduction of extracellular volume.

Antisense knockdown of inducible nitric oxide synthase inhibits the relaxant effect of VIP in isolated smooth muscle cells of the mouse gastric fundus.

1. Our previous results showed that the non-selective nitric oxide synthase (NOS) inhibitor L-N(G)-nitroarginine (L-NOARG) and the selective inducible NOS (iNOS) inhibitor N-(3-(acetaminomethyl)-benzyl)acetamidine (1400W) inhibited the relaxant effect of vasoactive intestinal polypeptide (VIP) in isolated smooth muscle cells of the mouse gastric fundus, suggesting the involvement of iNOS. The identity of the NOS isoform involved in the VIP-induced relaxation in isolated smooth muscle cells of the mouse gastric fundus was now further investigated by use of antisense oligodeoxynucleotides (aODNs) to iNOS. 2. Incubation of isolated smooth muscle cells with fluorescein isothiocyanate (FITC)-labelled aODNs showed that nuclear accumulation occurs quickly and reaches saturation after 60 min. The in vivo intravenous administration of aODNs to iNOS, 24 and 12 h before murine tumour necrosis factor alpha (mTNFalpha) challenge, significantly reduced the nitrite levels induced by the mTNFalpha challenge. 3. Intravenous administration of aODNs to iNOS in mice, 24 and 12 h before isolation of the gastric smooth muscle cells, decreased the inhibitory effect of the NOS inhibitors L-NOARG and 1400W on the relaxant effect of VIP, whereas neither saline nor sODNs had any influence. 4. Preincubation of the isolated smooth muscle cells with aODNs almost abolished the inhibitory effect of L-NOARG and 1400W on the VIP-induced relaxation, whereas sODNs failed. 5. These results illustrate that the inhibitory effect of NOS inhibitors in isolated smooth muscle cells of the mouse gastric fundus is due to inactivation of iNOS. iNOS, probably induced by the isolation procedure of the smooth muscle cells, seems involved in the relaxant effect of VIP in isolated gastric smooth muscle cells.

Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA.

We have shown recently that cell accumulation in culture of antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one antisense phosphorothioate DNA by three different methods. The 18-mer antisense DNA against the RIalpha subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37 degrees C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIalpha mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine) >MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the results suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling.

Antisense properties of peptide nucleic acids.

PNA is a nucleic acid analog with an achiral polyamide backbone consisting of N-(2-aminoethyl)glycine units (figure 1). The purine or pyrimidine bases are linked to the each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid (4). PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules (5-7). The uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes. The non-natural character of the PNA makes PNA oligomers highly resistant to protease and nuclease attacks (8). These properties of PNA oligomers suggest that they could potentially serve as efficient antisense or antigene reagents. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional (9) and translational level (10,11), and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells (12). However, contrary to the "normal" nucleic acid analogs, PNA oligomers are not efficiently delivered into the cytoplasm of the cell, and until recently this has hindered the application of PNA oligomers as antisense reagents. In this work we summarize some recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.

Use of capillary electrophoresis methods to characterize the pharmacokinetics of antisense drugs.

As antisense drugs become mature for clinical trials, analytical techniques to analyze antisense DNA in biological media for characterization of their pharmacokinetics will be in demand. Due to the superior resolving power of capillary gel electrophoresis (CGE), CGE will likely be a preferred method in quantifying intact oligonucleotides as well as the putative metabolic products. Nonetheless, biological mediums can influence the stability of the gel column, making a CGE assay time-consuming. In one approach, high-performance liquid chromatography (HPLC) was used to quantify the total amount of antisense compounds to increase the sample throughput and CGE was used to determine the relative percentage of the intact and metabolic species on specific samples. Alternatively, extensive sample pretreatment procedures were performed and the samples were quantified and characterized directly by CGE alone with the use of an internal standard. Both methods have been used to characterize the pharmacokinetics of antisense compounds. This review focuses on the instrumental and technical aspects of analyzing antisense DNA in biological mediums using CGE either as a single or a combined method towards better understanding of the pharmacokinetics of antisense DNA. Moreover, the newer analytical technologies of capillary electrophoresis (CE), which hold great potential to be used for pharmacokinetic applications, such as the replenishable sieving matrix combined with an innovative coupling approach and microchip CE, will also be explored.

[Control of in vivo characteristics of gene and antisense DNA disposition].

In order to construct a strategy for control of in vivo disposition characteristics of gene and antisense DNA, in vivo stability and basic pharmacokinetic properties of DNA were investigated. A model gene, plasmid DNA (pCAT), and model oligonucleotide (thymidine 10-mer; T10) derivatives with phosphoroamidate substitution at the 3' and/or 5'-terminal internucleotide linkage, were rapidly degraded in vivo after intravenous injection into mice. The degradation rates were much faster than those observed in in vitro experiments using plasma and whole blood, suggesting that they underwent enzymatic degradation in other compartments than the blood pool. More than 70% of injected pCAT was taken up by the liver within 5 minutes, and the uptake clearance was almost identical to the plasma flow rate in the liver. On the other hand, T10 derivatives were rapidly excreted in the urine and taken up by the kidney and liver. The urinary excretion clearance was close to the glomerular filtration rate. In an attempt to control T10 disposition characteristics, the oligonucleotide was conjugated to a macromolecular carrier, carboxymethyl dextran (CMD). The T10-CMD conjugate exhibited increased in vivo stability and prolonged plasma circulation time. Thus, the present study has shown that macromolecular conjugation is a useful approach to improve in vivo disposition of antisense oligonucleotides.