Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

Induced dystrophin exon skipping in human muscle explants.

Antisense oligonucleotide (AO) manipulation of pre-mRNA splicing of the dystrophin gene is showing promise in overcoming Duchenne muscular dystrophy (DMD)-causing mutations. To date, this approach has been limited to studies using animal models or cultured human muscle cells, and evidence that AOs can induce exon skipping in human muscle has yet to be shown. In this study, we used different AO analogues to induce exon skipping in muscle explants derived from normal and DMD human tissue. We propose that inducing exon skipping in human muscle explants is closer to in vivo conditions than cells in monolayer cultures, and may minimize the numbers of participants in Phase I clinical studies to demonstrate proof of principle of exon skipping in human muscle.

Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies.

PURPOSE: The phosphoprotein p53 is involved in transcriptional regulation and is detected in hematologic malignancies. In vitro incubation of acute myelogenous leukemia with OL(1)p53, a 20-mer phosphorothioate oligonucleotide complementary to p53 mRNA, results in leukemic cell death. A phase I dose-escalating trial was conducted to determine the toxicity of OL(1)p53 following systemic administration to patients with hematologic malignancies. PATIENTS AND METHODS: Sixteen patients with either refractory acute myelogenous leukemia (n = 6) or advanced myelodysplastic syndrome (n = 10) participated in the trial. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for 10 days by continuous intravenous infusion. RESULTS: No specific toxicity was directly related to the administration of OL(1)p53. One patient developed transient nonoliguric renal failure. One patient died of anthracycline-induced cardiac failure. Approximately 36% of the administered dose of OL(1)p53 was recovered intact in the urine. Plasma concentrations and area under the plasma concentration curves were linearly correlated with dose. Leukemic cell growth in vitro was inhibited as compared with pretreatment samples. There were no clinical complete responses. CONCLUSION: A phosphorothioate oligonucleotide, OL(1)p53, can be administered systemically without complications. This type of modified oligonucleotide can be administered without complete degradation, as it was recovered from the urine intact. This oligonucleotide may be useful in combination with currently available chemotherapy agents for the treatment of malignancies.

Transdermal delivery of antisense compounds.

Antisense technology holds tremendous promise for therapeutic applications and the study of gene function. A broadly applicable route of administration that would provide for non-invasive, simple, and convenient delivery is highly desirable. Application of oligonucleotides to the skin may represent a solution to the delivery question for both local treatment of skin disease and for systemic delivery. The iontophoretic mode of delivery for phosphorothioate oligonucleotides across hairless mouse skin reveals the potential limitation in the delivery of sufficient oligonucleotide to provide for efficacy. A potential solution to this problem is the use of significantly more potent C-5 propyne base modifications in a phosphorothioate oligonucleotide. The combination of the iontophoretic delivery mode with potent oligonucleotides resulted in selective inhibition of the CYP3A2 gene expression in the rat liver. Alternatively, oligomers with neutral charge combined with passive modes of transdermal delivery may also be feasible and represent an even more broadly applicable technology. Future studies will focus on specific applications of local and systemic therapy of antisense oligonucleotide in animal models for the design of treatment regimens.

An experimental model for interpreting percutaneous penetration of oligonucleotides that incorporates the role of keratinocytes.

Oligonucleotides have been extensively studied for their potential as therapeutic agents. Phosphorothioate oligonucleotides have been demonstrated to be particularly useful due to their stability against nucleases, their ability to be internalized by many cell types, and the ease with which they hybridize with target mRNA. These compounds have previously been delivered across the skin with the aid of iontophoresis. During transdermal delivery, the first viable cells exposed to the oligonucleotides are the keratinocytes. The purpose of this study was to determine the relationship between internalization of these compounds by keratinocytes and their transport across the skin. The in vitro uptake of 15 different fluorescently labeled phosphorothioate oligonucleotides into human keratinocytes was quantitatively measured with a fluorometer. Photomicrographs of keratinocytes indicate diffuse cytoplasmic and nuclear localization. The ability of these molecules to enter cells was linearly related to size. Cellular uptake data were inversely correlated with previously reported steady-state transport levels of oligonucleotides that had been transdermally delivered by iontophoresis across hairless mouse skin. Oligonucleotides that readily entered keratinocytes had a decreased ability to penetrate skin under iontophoretic conditions. The results indicate that oligonucleotide sequences may be designed for treating skin diseases (high uptake, low transport) or systemic disorders (low uptake, high transport).

Tumor cell growth is inhibited by suppressing metallothionein-I synthesis.

The effect of metallothionein (MT)-I antisense oligodeoxynucleotide (ODN) on the growth of three kinds of tumor cells was studied, since MTs may be involved in cell growth. When MT-I antisense ODN was added to leukemia P388 cells, cell growth was inhibited in a manner dependent on the dose and incubation time. MT-I antisense ODN was also inhibitory for other tumor cell lines, i.e. Ehrlich carcinoma and sarcoma 180. A significant decrease in the level of MT, but not of Zn, was observed in MT-I antisense ODN-treated cells. On the other hand, control ODN did not inhibit the cell growth appreciably. These results indicate that MT-I expression may be necessary for the growth and survival of these tumor cells.

Induction of cytochromes P450IA1 and P450IA2 as determined by solution hybridization.

Two oligodeoxyribonucleotides were synthesized that were specific for the messenger RNAs for the polycyclic hydrocarbon-inducible cytochromes P450IA1 and P450IA2. The solution hybridization technique was modified for the use of these oligodeoxyribonucleotide probes so as to increase the sensitivity and specificity of this method. Using this technique, the steady-state levels of the mRNAs for cytochromes P450IA1 and P450IA2 in control rat liver were determined to be less than 3 and 6 molecules/cell, and 1.8 and 4.0 attomol/micrograms poly (A)+ RNA, respectively. At 15 hr after induction with 3-methylcholanthrene, the steady-state levels of the mRNAs for P450IA1 and P450IA2 were 68 and 200 molecules/cell, and 41.6 and 123 attomol/micrograms poly (A)+ RNA.

Synergy of phenobarbital and 3-methylcholanthrene in "superinduction" of cytochrome P-450c mRNA but not enzyme activity.

The combination of phenobarbital and 3-methylcholanthrene in the inductive process of the rat hepatic cytochrome P-450c gene was evaluated. Daily injections of phenobarbital (80 mg/kg, i.p.) had little or no effect on the amount of poly (A)+ RNA encoding cytochrome P-450c, whereas a single injection of 3-methylcholanthrene (25 mg/kg, i.p.) produced a significant accumulation at 15 hr in cytosolic mRNA coding for cytochrome P-450c. Four daily injections of phenobarbital followed by a single dose of 3-methylcholanthrene produced 5-24 times more poly (A)+ RNA coding for P-450c than 3-methylcholanthrene treatment alone. This superinduction of RNA transcripts was also observed for a species coding for cytochrome P-450d, which was increased 3-6 times over 3-methylcholanthrene treatment alone. However, the elevated concentration of transcripts for both the P-450d and P-450c RNA species did not result in an increase in the marker enzyme activity for cytochrome P-450c, 7-ethoxyresorufin O-deethylase. These data implicate a regulatory step in the induction of cytochrome P-450c enzyme activity which must occur at a level beyond transcription.

Phosphorodiamidate morpholino oligomers: favorable properties for sequence-specific gene inactivation.

The phosphorodiamidate morpholino oligomers (PMOs) are a distinct class of oligonucleotide analogs. They bind to RNA and efficiently interfere with gene expression in a sequence-specific manner. Their non-ionic character combined with resistance to degradation in the body offer efficacy with minimal toxicity. Hence, the PMOs are emerging as an excellent approach to therapeutics that can be rationally designed based on target gene sequence data.

Redirection of drug metabolism using antisense technology.

The cytochrome P450 (CYP) family is the most catalytically versatile component of the phase I oxidation metabolic pathway and participates in the metabolism of a large majority of drugs used in clinical practice. The inhibition of specific enzymes of this family can significantly alter the disposition and toxicity of substrate drugs by reducing and/or redirecting their metabolism. This review discusses the approaches available for CYP inhibition, with particular emphasis on the potential use of antisense phosphorodiamidate morpholino oligonucleotide strategies to inhibit human CYP3A4. Inter-individual variations of 10- to 50-fold have been reported in the activity of CYP3A4 enzyme, which contributes to the metabolism of more than half of all clinically relevant drugs. The application of antisense technology for inhibition of specific CYP enzymes can provide significant therapeutic benefits, including: (i) reduction of first-pass drug metabolism; (ii) reduction in drug dosage; (iii) selective reduction of toxic metabolites; and (iv) increased oral/topical drug bioavailability. The use of antisense morpholino oligonucleotide strategies to target CYP enzymes may result in safer and more uniform therapeutic applications.

Regulation of zinc metallothionein II mRNA level in rat brain.

Metallothionein isoforms I and II (MTI and MTII) have been identified in the rat brain, monkey brain, bovine retina, pineal gland and hippocampus, and in the neuroblastoma IMR 32. Since intraperitoneally administered zinc passes across the blood-brain barrier slowly, the rat brain metallothionein can be induced in a time- and dose-dependent fashion only following intracerebroventricularly (i.c.v.) administered zinc sulfate at a rate of 0.20 ?mol/?l/h for 24 h using an Alzet minipump. The zinc-induced proteins, incorporate large quantities of [(35)S]cysteine, bind (65)Zn and produce two isoforms which contain 17 and 18 cysteine residues, respectively, but lack aromatic amino acids or histidine. In this communication, we report that i.c.v.-administered zinc in a bolus of 0.1 and 0.5 ?mol increased the synthesis of poly A(+) RNA from 6.6 to 8.0 and 9.6 ?g/g brain tissue, respectively. Furthermore, we probed the poly A(+) RNA with (32)P-labeled 180 base pair BamH1/PvuII restriction fragment containing the cDNA for human MTII from the phMT-II(3) plasmid. Slot blot analysis of poly A(+) RNA revealed a dose-dependent increase in brain MTII hybridizable mRNA. Northern blot analysis of poly A(+) RNA extracted from the rat liver and brain using (32)P-labeled cDNA exhibited a major band of hybridization at 700 bases in both tissues. These data provide evidence that the zinc-induced MT synthesis in the brain is associated with an accumulation of mRNA which is analogous to the zinc-induced synthesis of hepatic MT mRNA. However, other evidence indicates that the factors regulating the synthesis of the brain and the hepatic metallothioneins are not identical.

Expression and regulation of brain metallothionein.

Many, but not all, zinc-containing neurons in the brain are a subclass of the glutamatergic neurons, and they are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These "vesicular" pools of zinc are viewed as endogenous modulators of ligand- and voltage-gated ion channels. Metallothioneins (MTs) are low molecular weight zinc-binding proteins consisting of 25-30% cysteine, with no aromatic amino acids or disulfide bonds. The areas of the brain containing high contents of zinc such as the retina, the pineal gland, and the hippocampus synthesize unique isoforms of MT on a continuous basis. The four MT isoforms are thought to provide the neurons and glial elements with mechanisms to distribute, donate, and sequester zinc at presynaptic terminals; or buffer the excess zinc at synaptic junctions. In this cause, glutathione disulfide may participate in releasing zinc from MT. A similar nucleotide and amino acid sequence has made it difficult to obtain cDNA probes and antibodies capable of distinguishing indisputably among MT isoforms. MT-I and MT-II isoforms are found in the brain and in the peripheral tissues; MT-III isoform, possessing an additional seven amino acids, is expressed mostly in the brain and to a very minute extent in the intestine and pancreas; whereas MT-IV isoform is found in tissues containing stratified squamous epithelial cells. Since MTs are expressed in neurons that sequester zinc in their synaptic vesicles, the regulation of the expression of MT isoforms is extremely important in terms of maintaining the steady-state level of zinc and controlling redox potentials. The concentration of zinc has been shown to be altered in an extensive number of disorders of the central nervous system, including alcoholism. Alzheimer-type dementia, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Friedreich's ataxia, Guillaine-Barré syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson's disease, Pick's disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wernicke-Korsakoff syndrome. The status of MT isoforms and other low molecular weight zinc-binding proteins in these conditions, diseases, disorders, or syndromes is being delineated at this time. Since several of these disorders, such as amyotrophic lateral sclerosis, are associated with oxidative stress, and since MT is able to prevent the formation of free radicals, it is believed that cytokine-induced induction of MT provides a long-lasting protection to avert oxidative damage.

Preparation and partial characterization of highly purified primary cultures of neurons and non-neuronal (glial) cells from embryonic chick cerebral hemispheres and several other regions of the nervous system.

Purified cultures of neurons and non-neuronal (glial) cells were prepared from the cerebral hemispheres of 10-day chick embryos by a method previously used for embryonic chick sympathetic ganglia 16. This technique separates these cell types on the basis of both: (1) differences in the adhesiveness of neurons and non-neuronal cells to a collagen substrate; and (2) the capacity of neurons to form homotypic aggregates. Purity of the cerebral non-neuronal cultures was determined to be greater than or equal to 99.5% by microscopic examination, while that of the cerebral neuronal cultures was only 92%. Modification of the technique by periodic redissociation of the neuronal aggregates during cell separation increased the purity of the neuronal cultures to greater than or equal to 97% as determined both by microscopic examination and by measurement of levels of butyrylcholinesterase, an enzyme present in the non-neuronal cells. Highly purified cultures of neurons were also prepared from the optic lobes of 10-day chick embryos (greater than or equal to 98%), but attempts to obtain non-neuronal cultures of reasonable density from this tissue were unsuccessful. In addition, highly purified non-neuronal cultures (greater than or equal to 99.5%) were prepared from the dorsal root ganglia of 12-day chick embryos, but cultures enriched with dorsal root neurons could only be partially purified (82%). Specific activity of butyrylcholinesterase in cerebral non-neuronal cells was found to vary inversely with the density of non-neuronal cells.

Neuronal stimulation of non-neuronal (glial) cell proliferation: lack of specificity between different regions of the nervous system.

Purified and recombined primary cultures of neurons and non-neuronal (glial) cells were prepared from the cerebral hemispheres of 10-day chick embryos. Addition of cerebral neurons to homologous non-neuronal cultures stimulated incorporation of [3H]thymidine by 2.8-fold and increased the frequency of labelling of the non-neuronal cells by 3.5-fold as visualized by autoradiography. In contrast, cerebral neurons did not stimulate the proliferation of either embryonic chick fibroblasts or leptomeningeal cells. Furthermore, addition of either fibroblasts or extra non-neuronal cells did not stimulate non-neuronal cel proliferation. These data demonstrate for the first time that neurons isolated from the central nervous system can selectively stimulate the proliferation of homologous non-neuronal cells. Cell proliferation was also studied in cultures containing both homologous and heterologous combinations of neuronal and non-neuronal cells prepared from several different portions of the nervous system (cerebral hemispheres, optic lobes, sympathetic ganglia, and sensory ganglia). Addition of embryonic neurons stimulated non-neuronal cell proliferation in all cell combinations. Thus, neurons isolated from one region of the nervous system can stimulate the proliferation of non-neuronal cells isolated from other neural regions.

Selective cytotoxicity to human leukemic myeloblasts produced by oligodeoxyribonucleotide phosphorothioates complementary to p53 nucleotide sequences.

Cells were treated in vitro with oligodeoxyribonucleotide phosphorothioates (ODNs) complementary to sites common to both wild-type and mutant p53 nucleotide sequences. Acute myelogenous leukemia (AML) blasts from peripheral blood were exposed to four different p53 ODNs and showed anti-leukemic effects in suspension culture. This effect continued after removal of the ODN from the medium. Blocking of self-renewal of the leukemic blast stem cells in secondary plating of cells from cloning assays by two of the p53 ODNs was also observed. Control ODNs had no effect on leukemic blasts. Treatment of normal bone marrow cells with the four p53 ODNs did not influence their growth, nor was there any effect by the p53 ODNs on the leukemic cell-line, HL60, that does not express p53. These data suggest that p53 ODNs are selectively toxic to primary myelogenous blasts and may be therapeutically useful in AML.

Oligonucleotide enhanced cytotoxicity of Idarubicin for lymphoma cells.

Oligonucleotides offer the potential to manipulate gene expression in targeted cells which might be exploitable for therapeutic benefit. The effects of combining a phosphorothioate oligonucleotide OL(1) p53, which transiently down-regulates p53 levels, with an anthracycline, Idarubicin, on the growth of wild-type p53 WMN gene-expressing lymphoma cells was evaluated. Fluorescent OL(1) p53, was used to demonstrate oligonucleotide uptake and retention by the WMN cells. Uptake was maximal at 24 hours and compared to baseline (0 hours) increasing apoptotic cells were evident in WMN cells treated with OL(1) (1 microM) alone and in combination with Idarubicin (0.2 nM) for 24 to 48 hours. In cells treated with OL(1) p53 and Idarubicin, truncated p53 message of a predicted 201 base pair length based on RNAase H cleavage of the OL(1) p53-p53 mRNA heteroduplex was detected after 7 hours of incubation. The message for p53 was transiently downregulated as detected by RT-PCR analysis at 24 hours, and protein levels transiently reduced at 36 hours, as shown by a quantitative Western blot. Corresponding to these events, the growth of WMN cells ceased after 48 hours in the concurrent presence of OL(1) p53 and Idarubicin and, the lymphoma cells were dead after 72 hours. No reduction in hematopoietic colony forming cell capacity of similarly treated hematopoietic progenitor cells harvested from cytokine-mobilized blood by apheresis was observed. Therefore, synergistic cytotoxicity of Idarubicin for lymphoma cells treated with an oligonucleotide targeting p53 message was demonstrated at oligonucleotide and Idarubicin concentrations which were minimally toxic to hematopoietic progenitor cells. This approach offers new opportunities for purging of lymphoma cells from hematopoietic harvests and systemic lymphoma therapy.

Effects of BCR-ABL antisense oligonucleotides (AS-ODN) on human chronic myeloid leukemic cells: AS-ODN as effective purging agents.

We examined phosphorothioate oligodeoxyribonucleotides (ODNs) directed against bcr in exon 3 or exon 2, which are rearranged with exon 2 of abl (B3A2 and B2A2) at t(9;22) of chronic myelogenous leukemia (CML). Since these ODNs are designed to be CML cell specific, we studied their effects on the human CML cell line K562, which is known to have B3A2 rearrangement, and leukemic cells from patients, as well as normal hematopoietic stem cells in vitro. In vitro experiments were performed to determine a potential role of these two ODNs as ex vivo purging agents. Incubation of B3A2 antisense at 40, 80, and 120 micrograms/ml with K562 CML cells for 72 hours at 37 degrees C resulted in 44%, 56%, and 63% reduction of CFU-L as compared to controls. In contrast, B3A2 sense and B2A2 antisense had no significant growth inhibitory effect on K562 cells. Incubation of B3A2 and B2A2 antisense ODNs at concentration of 80 micrograms/ml at 37 degrees C for 36 hours with normal peripheral blood stem/progenitor cells (PBSC) resulted in 124% and 98% CFU-GM formation as compared to untreated controls, respectively. However, incubation of PBSC with B3A2 and B2A2 sense-ODNs resulted in a 22% and 44% reduction in CFU-GM, respectively. In order to determine the ex vivo purging effects of bcr-abl ODNs, the K562 cells were mixed with PBSC from normal donors at a ratio of 1:20 (CML:PBSC). The mixture of cells was then incubated with B3A2 antisense at 80 micrograms/ml for 36 hrs at 37 degrees C. After incubation, no CML cells were detected by fluorescence in situ hybridization (FISH) as compared to untreated controls. These results were confirmed by RT-PCR using bcr-abl primers and mRNA isolated from the mixture of cells. Further, these results support the hypothesis that bcr-abl antisense ODNs are potentially effective agents for ex vivo purging of autologous stem cells before transplantation to eliminate/reduce the burden of leukemic cells. No significant toxicity to normal hematopoietic stem/progenitor cell population by the bcr-abl antisense ODNs was observed. Although unanticipated reductions in normal hematopoietic progenitor cells (CFU-GM) were observed with sense ODNs, no reduction in CFU-GM was observed with unrelated phosphorothioate ODN controls.

Pharmacology and toxicology of phosphorothioate oligonucleotides in the mouse, rat, monkey and man.

Phosphorothioate oligonucleotides (PS-ODN) designed to temporarily modulate selected gene expression have made the journey from bench top to beside in a remarkably short period of time. A PS-ODN with sequence complementary to the p53 mRNA was administered to mice (4 mg/kg subcutaneously), rats (3-300 mg/kg intravenously), monkeys (intravenous infusions for up to 15 days) and humans (up to 0.25 mg/kg/h intravenous infusions for 10 days). These studies demonstrate the PS-ODN provides feasible pharmacokinetic parameters and minimal toxicity.

Effects of size and sequence on the iontophoretic delivery of oligonucleotides.

Adequate cellular availability of synthetic oligonucleotides is crucial to their success as therapeutic agents. These compounds, however, are not expected to be orally active. This has led to interest in a variety of alternate drug delivery methods, including iontophoretically enhanced transdermal delivery. The purpose of this work is to begin characterizing the structure-activity relationship for iontophoresis of oligonucleotides through the skin. The in vitro permeation of 16 biologically relevant phosphorothioate oligonucleotides across hairless mouse skin was studied. Oligonucleotides with less than 20 bases (n = 10) had a wide range of steady-state flux levels (2.1-26.2 pmol/ cm2 h). A lower flux differential was observed for compounds ranging from 20 to 40 bases long (1.2-2.2 pmol/cm2 h). For the smaller compounds, transport, in general, decreased with increasing size; however, there were several oligonucleotides that did not follow this pattern. These data indicate that factors other than size influence transport and that the impact is greater at shorter lengths. Differential penetration between equal sized oligonucleotides synthesized with identical bases in reversed order indicates that sequences and not simply base composition affects steady-state flux across skin. Molecular structure, therefore, is a key contributor to iontophoretically assisted transport. Further studies are necessary to develop more precise predictions about the relationship between oligonucleotide structure and transdermal delivery.

Continuous infusion of antisense phosphorothioate therapeutics.

Current therapy for acute myelogenous leukemia (AML) includes induction with Ara-C and an anthracycline, such as daunorubicin, idarubicin, or mitoxantrone. Unfortunately, most patients relapse from initial remission. Nearly one-fifth of early relapses experience treatment-related deaths. In addition, patients refractory to Ara-C die within months. Hence, new therapeutic agents must be identified capable of enhanced remission rates, diminished treatment-related mortality, or that can achieve remissions in refractory patients.