Depleting regulatory T cells with arginine-rich, cell-penetrating, peptide-conjugated morpholino oligomer targeting FOXP3 inhibits regulatory T-cell function.

CD4+CD25+regulatory T cells (T(reg)) impair anti-tumor and anti-viral immunity. As there are higher T(reg) levels in cancer patients compared with healthy individuals, there is considerable interest in eliminating them or altering their function as part of cancer or viral immunotherapy strategies. The scurfin transcriptional regulator encoded by the member of the forkhead winged helix protein family (FOXP3) is critical for maintaining the functions of T(reg). We hypothesized that targeting FOXP3 expression with a novel arginine-rich, cell-penetrating, peptide-conjugated phosphorodiamidate morpholino (PPMO) based antisense would eliminate T(reg) and enhance the induction of effector T-cell responses. We observed that the PPMO was taken up by activated T cells in vitro and could downregulate FOXP3 expression, which otherwise increases during antigen-specific T-cell activation. Generation of antigen-specific T cells in response to peptide stimulation was enhanced by pre-treatment of peripheral blood mononuclear cells with the FOXP3-targeted PPMO. In summary, modulation of T(reg) levels using the FOXP3 PPMO antisense-based genomic strategy has the potential to optimize immunotherapy strategies in cancer and viral immunotherapy.

Inhibition of p53 expression by peptide-conjugated phosphorodiamidate morpholino oligomers sensitizes human cancer cells to chemotherapeutic drugs.

The p53 tumor suppressor gene encodes a transcription factor that is commonly mutated in cancer. Tumors arise when premalignant cells are unable to undergo p53-dependent apoptosis, cell cycle arrest or DNA repair. The p53-signaling pathway affects not only tumor development, but also the response of tumors to chemotherapeutic drugs. In this study, we use cell penetrating peptide conjugates of phosphorodiamidate morpholino oligomers (PPMOs) to inhibit p53 expression. We examine the functional properties of endogenous p53 isoforms that are produced upon PPMO-mediated inhibition of p53 translation and splicing, and report that loss of N-terminal or C-terminal sequences interferes with the transcriptional activity of p53. Importantly, we report that PPMO-mediated inhibition of p53 expression sensitizes human cancer cells with wild-type p53 to chemotherapeutic drugs.

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.

Cell-penetrating-peptide-based delivery of oligonucleotides: an overview.

Cationic CPPs (cell-penetrating peptides) have been used largely for intracellular delivery of low-molecular-mass drugs, biomolecules and particles. Most cationic CPPs bind to cell-associated glycosaminoglycans and are internalized by endocytosis, although the detailed mechanisms involved remain controversial. Sequestration and degradation in endocytic vesicles severely limits the efficiency of cytoplasmic and/or nuclear delivery of CPP-conjugated material. Re-routing the splicing machinery by using steric-block ON (oligonucleotide) analogues, such as PNAs (peptide nucleic acids) or PMOs (phosphorodiamidate morpholino oligomers), has consequently been inefficient when ONs are conjugated with standard CPPs such as Tat (transactivator of transcription), R(9) (nona-arginine), K(8) (octalysine) or penetratin in the absence of endosomolytic agents. New arginine-rich CPPs such as (R-Ahx-R)(4) (6-aminohexanoic acid-spaced oligo-arginine) or R(6) (hexa-arginine)-penetratin conjugated to PMO or PNA resulted in efficient splicing correction at non-cytotoxic doses in the absence of chloroquine. SAR (structure-activity relationship) analyses are underway to optimize these peptide delivery vectors and to understand their mechanisms of cellular internalization.

Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo.

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

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.

Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD.

Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2'-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.

Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers.

Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response. A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs. In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63%. A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response. The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO. Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase. We conclude that the outer membrane of E. coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.

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.

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.

Transdermal delivery of antisense oligonucleotides can induce changes in gene expression in vivo.

The potential for using antisense compounds as therapeutic agents has generated great enthusiasm. Strategies for delivery of these compounds are, therefore, of great interest. Transdermal iontophoresis has been used successfully as an enhancement technique for the transdermal delivery of these compounds in vitro. The effectiveness of using percutaneous penetration as a means to deliver therapeutic levels of these compounds in vivo, however, remains to be demonstrated. The purpose of this work was to demonstrate the ability of iontophoretically delivered compounds to alter enzyme levels in the intact rat. A C5 propyne-modified phosphorothioate oligonucleotide (PS-ODN) targeted to the cytochrome p450-3A2 (CYP3A2) mRNA translational start site and the reverse sequence, used as a control, were synthesized. A patch containing either an oligonucleotide or a buffer control was placed on the animal's back, and an iontophoretic current of 0.5 mA/cm2 was applied for 3.5 hours. Twenty-four hours later, CYP3A2 levels were measured noninvasively using the midazolam-induced sleeping rat model. Liver and small intestinal microsomes were made after completion of sleep studies and assayed for CYP3A2, CYP1A1/2, CYP2B1/2, and CYP2E1. Midozolam-treated animals with antisense to CYP3A2 slept significantly longer than did the controls (p < 0.05). CYP3A2 levels were significantly lower in liver microsomes from antisense-treated animals than in either buffer control (p < 0.001) or reverse sequence animals (p < 0.05). The reverse sequence was also significantly different from the buffer control (p < 0.01), indicating a nonspecific effect of the PS background. Nontarget cytochrome levels were not altered by treatment. There were no significant differences in small intestine CYP3A2 levels between treatment groups. These data demonstrate that transdermally delivered PS-ODN can reach concentrations sufficient to induce changes in specific target enzymes in vivo. Further studies are warranted to investigate potential uses for these molecules.

Inhibition of Vesivirus infections in mammalian tissue culture with antisense morpholino oligomers.

Caliciviruses infect and cause disease in animals and humans. They are nonenveloped, positive-stranded RNA viruses with a genome of approximately 7.5 kb that encodes viral proteins in three open reading frames (ORF). Antisense oligomers targeting one of the three ORF of caliciviruses of the genus Vesivirus significantly inhibit viral replication in tissue culture. Porcine kidney and African green monkey kidney cells were infected with Vesivirus isolates SMSV-13 and PCV Pan-1. Phosphorodiamidate morpholino oligomers (PMO) with sequence complementary to the AUG translation start site regions of ORF1, ORF2, and ORF3 were evaluated for their effect on viral titer. Scrape-loading delivered PMO to 50%-70% of the cells of the two cell lines, as measured by fluorescence microscopy and flow cytometry. A PMO targeting ORF3 caused a significant increase in viral titer. A PMO targeting ORF2, a scrambled PMO control sequence, and an unrelated PMO antisense sequence did not alter viral titer. Various PMO sequences antisense to an upstream region of ORF1 were effective in reducing viral titer up to 80% in a dose-dependent and sequence-specific manner. The extent of viral titer reduction was proportional to the delivery of PMO to cells. These observations demonstrate that antisense PMO can disrupt caliciviral gene function in a nucleic acid sequence-specific manner and are potentially effective antiviral agents.

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.

Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers.

Antisense oligonucleotides inhibit gene expression by interfering with transcription, translation, or splicing. They show great potential as gene-specific, nontoxic therapy for a wide variety of diseases. They are also powerful tools to study gene function as well as for validation of therapeutic targets. Even with compelling evidence of activity in vivo, the majority of cell types in culture require technologies capable of efficiently delivering antisense oligonucleotides into the cytosolic/nuclear compartment of the cells in culture. Phosphorodiamidate morpholino oligomers (PMO) are a new generation of antisense oligomers with high specificity and efficacy. They inhibit translation of targeted mRNA by steric blockade. Different methods were evaluated for efficient delivery of PMO into the cells in culture. Efficacy was compared using the PMO targeted to the 5'-untranslated region (5'-UTR) of alpha-globin-luciferase reporter fusion gene mRNA. A functional assay based on the lunciferase reporter system was used to measure efficacy. The fluorescence-activated cell sorting (FACS) method was used for quantitative determination of PMO uptake into the cells. Physical methods, such as scrape-loading, syringe-loading, and osmotic-loading, provided efficient transfer of PMO into the cells, which resulted in higher efficacy. These procedures caused minimal damage to the cells. Cell permeabilization with streptolysin O did not improve the cell uptake of PMO. Complexation with cationic lipids, Lipofectin and Lipofectamine (GIBCO-BRL, Gaithersburg, MD) also failed to enhance the uptake of PMO. We conclude that physical methods are optimal for the delivery of neutrally charged PMO into cells in culture. Further, these methods do not leave residual material that may interfere with the interpretation of targeted gene function.

Evidence of enhanced iron excretion during systemic phosphorothioate oligodeoxynucleotide treatment.

BACKGROUND: Phosphorothioate oligonucleotides, in general, possess properties that could be utilized in the development of therapeutic heavy metal chelators. METHODS: Iron excretion was measured in 16 patients participating in studies to test the safety of OL(1)p53, a 20-mer phosphorothioate oligonucleotide complementary to p53 mRNA. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for 10 days by continuous intravenous infusion. Urine was collected during the study and analyzed for iron, copper, cadmium, and zinc. RESULTS: We found that phosphorothioate oligonucleotides have a high affinity for iron as well as several other clinically relevant toxic metals. Analysis of patient urine following administration of OL(1)p53 reveals a 7.5-fold increase in iron excretion at low doses (0.05 mg/kg/h). CONCLUSIONS: Phosphorothioate oligonucleotides may have therapeutic potential as heavy metal chelators. Low doses of phosphorothioate oligonucleotide facilitated the excretion of iron. Renal clearance of iron-phosphorothioate oligonucleotide complexes most likely involves secretion into proximal tubules.

Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc.

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.

Pharmacokinetics and in vivo effects of a six-base phosphorothioate oligodeoxynucleotide with anticancer and hematopoietic activities in swine.

A short phosphorothioate oligodeoxynucleotide telomere mimic with the sequence 5'-d(TTAGGG)-3', TAG-6, has been shown to inhibit telomerase activity and have antineoplastic and hematopoietic stimulatory properties. In this study, three immature male domestic swine (weighing approximately 40 kg) were administered 200 mg/m2 of TAG-6 by continuous intravascular infusion at rates of 0.48 +/- 0.07 mg/hr for 14 days to evaluate the pharmacokinetics, toxicity, and tissue distribution. There was considerable variability (both within each animal and across animals) observed in the pharmacokinetic data. The plasma half-life (t1/2 appeared to be short enough that it could be assumed that steady state was attained by at least 96 h after the start of the infusion. The t1/2 estimates for the three pigs were 8.96, 109, and 1.97 h (the long t1/2 for pig 2 may be explained by poor parameter estimation due to the variability). The volume of distribution ranged from 9.80 to 51.8 L (0.3-1.4 L/kg), and plasma clearance estimates ranged from 0.33 to 3.46 L/h (5.5-57.7 ml/min). The average plasma concentrations at steady state were 0.845, 0.933, and 0.178 microg/ml (0.44, 0.49, and 0.093 microM) for the three animals. Nearly 30% of the administered dose was cleared through renal excretion by day 7 postinfusion. The distribution of TAG-6 was primarily to the liver and kidney, but the spleen and thyroid accumulated relatively high concentrations of TAG-6. TAG-6 was metabolized to apparently higher molecular weight products, which were observed in the urine. The size periodicity of these apparently higher molecular weight products was in 6-base intervals, which is consistent with the actions of telomerase. The infusion did not produce significant changes in serum chemistry or circulating blood cells, but a decrease in colony-forming unit-granulocyte-monocyte (CFU-GM) colony formation from BM was observed. These data suggest that TAG-6 may be a very specific pharmacophore.

c-Myc antisense limits rat liver regeneration and indicates role for c-Myc in regulating cytochrome P-450 3A activity.

Expression of c-myc protein is associated with cell proliferation. The present study uses antisense oligomers to inhibit c-myc expression in the regenerating rat liver after 70% partial hepatectomy (PH). Antisense phosphorodiamidate morpholino oligomers (novel DNA analogs) were administered i.p. immediately after surgery to block expression of c-myc within the first 24 h after PH. A 20-mer PMO complimentary to the c-myc mRNA at the translation start site was an effective sequence (AVI-4126, 5'-ACGTTGAGGGGCATCGTCGC-3'). A single i.p. dose of 0.5 mg/kg AVI-4126 caused reduction of the regenerating liver c-myc protein in a sequence-specific and dose-dependent manner. Inhibition of c-myc expression resulted in reduction of proliferating cell nuclear antigen and arrested cells in the G(0)/G(1) phase of the cell cycle. The ratio of G(2):G(0) cell populations in the regenerating liver 24 h after PH dropped from 29.1 in saline vehicle-treated rats to 18.0 in rats treated with 2.5 mg/kg AVI-4126. The expression of cell cycle checkpoint protein p53 was inhibited with increasing doses of AVI-4126, but expression of p21(waf-1) was unaffected. The activity of cytochrome P-450 3A2 (CYP3A2) was evaluated by immunoblot analysis and erythromycin N-demethylation. AVI-4126 did not alter CYP3A activity in nonhepatectamized animals but showed a dose-dependent decrease in PH rats. We conclude that AVI-4126, antisense oligomer to c-myc, can reduce cell proliferation in the regenerating rat liver. Furthermore, inhibition of c-myc may indirectly influence the expression of CYP3A.

Antisense oligonucleotides targeted to the p53 gene modulate liver regeneration in vivo.

The rapidly proliferating cells of the regenerating liver after partial hepatectomy (PH) present a reproducible in vivo model to study the functional role of the tumor suppressor gene p53. The present study uses the rat 70% PH model along with systemic administration of three different structural types of antisense oligonucleotides (ODNs) designed to suppress p53 expression. We tested the hypothesis that antisense ODNs can inhibit the expression of p53, resulting in the loss of the G(1)-S cell cycle checkpoint and an altered pattern of liver regeneration. Intraperitoneal administration of 5 mg/kg/day antisense phosphorothioate ODN after 70% PH resulted in reduced expression of the p53 protein in the regenerating liver. There were concomitant increases in weight gain of remnant-regenerating liver and expression of proliferating cell nuclear antigen and p21(waf-1) compared with either saline or 5 mg/kg/day mispaired phosphorothioate ODN treatment. Flow cytometric analysis of DNA content of isolated hepatocytes revealed a reduction in the G(0)/G(1) cell population and accumulation of cells with more than 4n DNA in antisense-treated rats. The regenerating livers had significantly diminished cytochrome P-450 (CYP) enzyme activities. Rats treated with p53 antisense ODNs, but not saline or mispair ODN controls, had significantly elevated CYP activities. These observations functionally link the expression of p53 with diminished expression of several CYP isoforms in the liver regeneration model.