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Attempt for liver-targeted delivery of antisense oligonucleotides by cholesterol modification and oral administration
Hepatology Research 13 (1999) 252 – 258
Preliminary note
Attempt for liver-targeted delivery of antisense oligonucleotides by cholesterol modification and oral administration Yasuyuki Okamoto *, Hiroshi Nakano Department of Clinico-Laboratory Diagnostics, Nara Medical Uni6ersity, Shijo-Cho 840, Kashihara, Nara 634, Japan Received 20 May 1998; received in revised form 3 September 1998; accepted 17 September 1998
Abstract Antisense oligonucleotides (ODNs) are promising anti-viral and anti-tumor therapeutical agents. Liver-targeted delivery of ODNs was studied by oral administration of cholesterolmodified phosphorothioate ODNs (Chol-S-ODNs). Cholesterol modification predominantly accelerated the uptake of ODNs by the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5. Pravastatin and chylomicron enhanced the uptake by PLC/PRF/5 cells. Antisense Chol-S-ODNs complementary to the murine retinoblastoma protein (pRB) mRNA were orally administered to 6-week-old male Balb C mice. After 24 h, the levels of pRB protein expression were diminished in the liver, not affected in the kidney, and markedly reduced in the spleen. However, ODNs without cholesterol modification showed little effect. Chol-S-ODNs have been suggested to be nuclease-resistant and taken up into the circulation efficiently enough to be effective in the liver and the spleen. Although further drug designs are needed for liver-specificity, oral administration could be practical for future antisense ODNs therapy. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chylomicron; Gene therapy; Mouse; Retinoblastoma protein
* Corresponding author. Tel.: + 81 744 223051 ext. 3471; fax: + 81 744 245739; e-mail: [email protected] 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00093-X
Y. Okamoto, H. Nakano / Hepatology Research 13 (1999) 252–258
253
1. Introduction Antisense oligonucleotides (ODNs), synthetic single-stranded DNA molecules which inhibit gene expression in a sequence-specific manner, are promising anti-viral and anti-tumor therapeutical agents [1,2]. Appropriate modifications of ODNs may improve their stability, cellular uptake and specific delivery. Cholesterol- and phosphorothioate-modified ODNs (Chol-S-ODNs) have been shown to be resistant to nucleases, to have accelerated cellular internalization and increased efficacy [3]. For the final purpose of liver-specific delivery of ODNs, we examined internal use of Chol-S-ODNs, since they may also be resistant to nucleases in the intestine and efficiently taken up by intestinal epithelial cells with minimal loss and incorporated into chylomicrons, which have a role in liver-specific delivery.
2. Materials and methods
2.1. Cells and mice The rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5 were cultured in Dulbecco’s minimal essential medium (MEM) and Eagle’s MEM supplemented with 10% fetal bovine serum (FBS), respectively. These media and serum were obtained from ICN Biomedicals, Aurora, Ohio. The cells were seeded at a density of 2 ×104/cm2 in 96-well Falcon plates and cultured under 5% CO2 and 95% humidity at 37°C. Six-week-old Balb C mice (Japan SLC, Shizuoka, Japan), bred with free access to food and water, were orally administered ODNs.
2.2. ODNs All ODNs were synthesized and modified on an Applied Biosystems 394 DNA synthesizer, and purified by chromatography. Reagents for modifications including Cholesterol-ON™ Phosphoramidite were purchased from CLONTECH Laboratories, Palo Alto, CA. For cellular uptake experiments, biotin-labeled ODNs with and without cholesterol modification were synthesized as 5%-TGATGTAATGTTCTCCAT-3% complementary to the first six codons of the mRNA encoding the hepatitis B surface antigen (HBsAg) secreted by PLC/PRF/5 cells. These ODNs were dissolved at 1 mM in appropriate culture medium without serum for use. Alternatively, ODNs were dissolved at the same concentration in medium containing 20% chylomicron-rich serum and used after incubation at 37°C. The chylomicron-rich fraction of serum obtained from a patient with chylomicronemia was isolated by centrifugation at 12000 rpm for 30 min at 4°C. For oral administration to mice, fully phosphorothioated ODNs with and without cholesterol modification (Chol-S-ODNs and S-ODNs) were synthesized as 5%-CGGGGCTTTGGGCGGCAT-3% complementary to the first six codons of murine pRB mRNA. Chol-S-ODNs with the sense-oriented sequence as 5%-ATGC-
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CGCCCAAAGCCCCG-3% were used for control. These ODNs were dissolved at 10 mg/10 ml of water for use.
2.3. Cellular uptake of ODNs Cellular uptake of biotin-labeled ODNs was determined by colorimetric assay with peroxidase-conjugated streptavidin. Medium was replaced by new medium containing ODNs following 24 h of culture. After incubation for 10 min, cells were washed with Dulbecco’s Ca2 + - and Mg2 + -free PBS (PBS) three times, fixed with acetate:ethanol (1:3) for 30 min, and washed again with PBS three times. Bovine serum albumin (Sigma, St. Louis, MO) at 5% in PBS was used for blocking for 30 min and then changed to streptavidin-peroxidase conjugates (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) in PBS. After 30 min, cells were washed with PBS containing 0.1% Tween 20 three times, and 3,3%,5,5%-tetramethylbenzidine in buffer (Moss, Pasadena, MD) was added for 20 min. Uptake of ODNs was determined as optical density (OD) at 450 nm with reference to that at 570 nm. Medium without ODNs and solid-phase ODNs prepared by CHEMICOAT (Medicine and Applied Sciences, Sterling, VA) were used to determine the background OD and the 100% OD, respectively. These values were 0.05 and 0.16, respectively. Data are expressed as percent uptakes with reference to 100% OD. Pravastatin (kindly provided by Sankyo Pharmaceutical, Tokyo, Japan) was dissolved at 10 mM in medium containing FBS, and PLC/PRF/5 cells were treated with this for 2 h before addition of ODNs.
2.4. Oral administration of ODNs Chol-S-ODNs or S-ODNs without cholesterol modification were orally administered to mice at a dose of 10 mg in 10 ml water/g body weight (180–200 ml) using flexible thin tips. After 24 h, mice were anesthetized with ether and killed by cutting the abdominal aorta. The liver, kidney and spleen were isolated and homogenized in cold lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulphate (SDS), 0.1% sodium deoxycholate, 1 mM EDTA and 10 mg/ml aprotinin). The lysates were centrifuged at 10000× g for 30 min. Protein contents in the supernatants were determined using BCA* Protein Assay Reagent (PIERCE, Rockford, IL). Ten micrograms of proteins from the supernatant were separated on 4 – 15% linear gradient polyacrylamide gels containing 0.1% SDS and transferred onto PVDF membranes for immunoblotting. Membranes were immunoblotted with an antibody to pRB (C-15)-G (Santa Cruz Biotechnology, Santa Cruz, CA) or to p27Kip1 (PharMingen, San Diego, CA), followed by a peroxidaseconjugated secondary antibody. Antibody-associated pRB or p27Kip1 was visualized by ECL (Amersham LIFE SCIENCE). The same blots were also stained with 0.1% Amido Black 10B in 45% methanol and 10% acetate to evaluate background protein content.
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3. Results and discussion Cellular uptake of biotin-labeled ODNs with and without cholesterol modification was determined in vitro using two cell lines, the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5, which were used to test intestinal uptake and uptake by cells from the target organ, respectively. After the addition of 1 nmol of ODNs in serum-free medium to 105 cells for 10 min, ODNs uptake was determined by colorimetric assay as described in Section 2. Rapid uptake of the cholesterol-modified ODNs (Chol-ODNs) was observed in both IEC-6 and PLC/PRF/5 cells, while no significant uptake of the unmodified ODNs was seen (Fig. 1). Since Chol-ODNs taken up by the intestine may be incorporated into chylomicrons, the lipoproteins which carry absorbed lipids through the intestinal epithelium and are specifically internalized by the liver, and Chol-ODNs themselves are known to be partially taken up through LDL receptors [3], the effects of pravastatin and chylomicrons on the uptake by PLC/PRF/5 were also tested. Pretreatment of PLC/PRF/5 cells with pravastatin, which is known to increase low density lipoprotein (LDL) receptor expression, at 10 mM for 2 h increased the uptake of Chol-ODNs. When Chol-ODNs in medium containing chylomicrons were used after incubation for 2 h, no further increase of uptake was observed. However, pravastatin treatment seemed to increase the uptake more in the presence than in the absence of chylomicrons.
Fig. 1. Cellular uptake of biotin-labeled oligonucleotides (ODNs) by the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5. Cholesterol: cholesterol-modified ODNs; chylomicron: ODNs incubated in solution containing chylomicrons for 2 h before addition; pravastatin: ODNs were added to cells treated with pravastatin for 2 h. * P B0.05, ** PB 0.01. Data are represented as means 9 S.D. of eight wells in two experiments. Cellular uptake was determined after addition of 1 nmol of ODNs to 105 cells for 10 min as described in Section 2.
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Fig. 2. Effects of oral administration of ODNs corresponding to pRB on the expression of pRB in the mouse liver, kidney and spleen. ODNs( − ): ODNs not administered; S-ODNs: phosphorothioatemodified ODNs; Chol-S-ODNs: cholesterol- and phosphorothioate-modified ODNs; sense: sense-oriented Chol-S-ODNs. L: liver; K: kidney; S: spleen. Back: background proteins on the blot stained with Amido Black. (A) Chol-S-ODNs but not S-ODNs suppressed pRB expression in the liver and the spleen 24 h after oral administration at the dose of 10 mg in 10 ml water/g body weight. Background proteins in each tissue show similar patterns. The expression of p27Kip1 protein as an internal control was not affected. (B) Sense-oriented Chol-S-ODNs showed no significant effect.
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To test the efficacy of oral administration of Chol-S-ODNs, we decided to target retinoblastoma protein (pRB), a representative tumor suppressor. pRB is one of the suitable proteins to test the efficacy comparatively because pRB expresses in any tissue and is not secreted to have an indirect effect on other cells, and its half life (10 h [4]) is appropriate to evaluate changes in the protein level 24 h after administration of Chol-S-ODNs. Antisense Chol-S-ODNs complementary to the first six codons of murine pRB mRNA or S-ODNs without cholesterol modification were orally administered to 6-week-old male Balb C mice (10 mg in 10 ml of water/g body weight). After 24 h, the liver, kidney and spleen were isolated and used for immunoblotting of pRB. In Chol-S-ODNs administered mice, the levels of pRB expression were diminished in the liver, not affected in the kidney and markedly reduced in the spleen (Fig. 2A). However, ODNs without cholesterol modification showed little effect. The expression of p27Kip1 protein, another tumor suppressor, as an internal control was not affected in the liver, kidney or spleen. Sense-oriented Chol-S-ODNs showed no significant effect (Fig. 2B). These results indicate that the effect of Chol-S-ODNs was sequence-specific. In this study, we demonstrated that ODNs, even by oral administration, can inhibit protein expression in internal organs such as the liver and spleen. We also showed that Chol-S-ODNs have an advantage for this purpose, because they had more consistent effects than S-ODNs. Considering the findings in a previous report concerning mucosal uptake of ODNs [1,5] and our in vitro study using rat intestinal epithelial cells, S-ODNs are suggested to be nuclease-resistant but uptake is too inconsistent to show a final effect, whereas Chol-S-ODNs are suggested to be taken up into the circulation efficiently enough to be effective in the liver and the spleen. However, liver-specific delivery, our primary aim, is not yet sufficient, since delivery to the spleen could not be mediated by a liver-specific carrier like chylomicron. It is suggested that Chol-S-ODNs in the present form failed to be incorporated into chylomicron. To increase the specific uptake, further modifications or drug design are needed. Our results provide a new experimental animal model for gene targeting. Furthermore, the accumulation of Chol-S-ODNs in the circulation may be therapeutically useful for several human hematological disorders such as lymphoma and HIV infection. For these diseases, clinical trials of antisense ODN therapy were performed by intravenous or subcutaneous infusion [1,2]. As described previously [6,7], doses similar to that used in our study have lower toxicity even by intravenous infusion. In addition, oral administration is practical for long-term treatment of chronic diseases, since medical restrictions on patients can be largely eliminated. Thus, oral administration may become practical in the field of antisense ODN therapy. References [1] Agrawal S. Antisense oligonucleotides: towards clinical trials. Trends Biotechnol 1996;14:376 – 387. [2] Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 1997;349:1137–1141.
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[3] Krieg AM, Tonkinson J, Matson S, et al. Modification of antisense phosphodiester oligonucleotides by a 5% cholesteryl moiety increases cellular association and improves efficacy. Proc Natl Acad Sci USA 1993;90:1048–1052. [4] Mihara K, Cao XR, Yen A, et al. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 1989;246:1300– 1303. [5] Vlassov VV, Karamyshev VN, Yakubov LA. Penetration of oligonucleotides into mouse organism through mucosa and skin. FEBS Lett 1993;327:271 – 274. [6] Galbraith WM, Hobson WC, Giclas PC, Schechter PJ, Agrawal S. Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey. Antisense Res Dev 1994;4:201 – 206. [7] Bishop MR, Iversen PL, Bayever E, et al. Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies. J Clin Oncol 1996;14:1320 – 1326.
.
Preliminary note
Attempt for liver-targeted delivery of antisense oligonucleotides by cholesterol modification and oral administration Yasuyuki Okamoto *, Hiroshi Nakano Department of Clinico-Laboratory Diagnostics, Nara Medical Uni6ersity, Shijo-Cho 840, Kashihara, Nara 634, Japan Received 20 May 1998; received in revised form 3 September 1998; accepted 17 September 1998
Abstract Antisense oligonucleotides (ODNs) are promising anti-viral and anti-tumor therapeutical agents. Liver-targeted delivery of ODNs was studied by oral administration of cholesterolmodified phosphorothioate ODNs (Chol-S-ODNs). Cholesterol modification predominantly accelerated the uptake of ODNs by the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5. Pravastatin and chylomicron enhanced the uptake by PLC/PRF/5 cells. Antisense Chol-S-ODNs complementary to the murine retinoblastoma protein (pRB) mRNA were orally administered to 6-week-old male Balb C mice. After 24 h, the levels of pRB protein expression were diminished in the liver, not affected in the kidney, and markedly reduced in the spleen. However, ODNs without cholesterol modification showed little effect. Chol-S-ODNs have been suggested to be nuclease-resistant and taken up into the circulation efficiently enough to be effective in the liver and the spleen. Although further drug designs are needed for liver-specificity, oral administration could be practical for future antisense ODNs therapy. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chylomicron; Gene therapy; Mouse; Retinoblastoma protein
* Corresponding author. Tel.: + 81 744 223051 ext. 3471; fax: + 81 744 245739; e-mail: [email protected] 1386-6346/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00093-X
Y. Okamoto, H. Nakano / Hepatology Research 13 (1999) 252–258
253
1. Introduction Antisense oligonucleotides (ODNs), synthetic single-stranded DNA molecules which inhibit gene expression in a sequence-specific manner, are promising anti-viral and anti-tumor therapeutical agents [1,2]. Appropriate modifications of ODNs may improve their stability, cellular uptake and specific delivery. Cholesterol- and phosphorothioate-modified ODNs (Chol-S-ODNs) have been shown to be resistant to nucleases, to have accelerated cellular internalization and increased efficacy [3]. For the final purpose of liver-specific delivery of ODNs, we examined internal use of Chol-S-ODNs, since they may also be resistant to nucleases in the intestine and efficiently taken up by intestinal epithelial cells with minimal loss and incorporated into chylomicrons, which have a role in liver-specific delivery.
2. Materials and methods
2.1. Cells and mice The rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5 were cultured in Dulbecco’s minimal essential medium (MEM) and Eagle’s MEM supplemented with 10% fetal bovine serum (FBS), respectively. These media and serum were obtained from ICN Biomedicals, Aurora, Ohio. The cells were seeded at a density of 2 ×104/cm2 in 96-well Falcon plates and cultured under 5% CO2 and 95% humidity at 37°C. Six-week-old Balb C mice (Japan SLC, Shizuoka, Japan), bred with free access to food and water, were orally administered ODNs.
2.2. ODNs All ODNs were synthesized and modified on an Applied Biosystems 394 DNA synthesizer, and purified by chromatography. Reagents for modifications including Cholesterol-ON™ Phosphoramidite were purchased from CLONTECH Laboratories, Palo Alto, CA. For cellular uptake experiments, biotin-labeled ODNs with and without cholesterol modification were synthesized as 5%-TGATGTAATGTTCTCCAT-3% complementary to the first six codons of the mRNA encoding the hepatitis B surface antigen (HBsAg) secreted by PLC/PRF/5 cells. These ODNs were dissolved at 1 mM in appropriate culture medium without serum for use. Alternatively, ODNs were dissolved at the same concentration in medium containing 20% chylomicron-rich serum and used after incubation at 37°C. The chylomicron-rich fraction of serum obtained from a patient with chylomicronemia was isolated by centrifugation at 12000 rpm for 30 min at 4°C. For oral administration to mice, fully phosphorothioated ODNs with and without cholesterol modification (Chol-S-ODNs and S-ODNs) were synthesized as 5%-CGGGGCTTTGGGCGGCAT-3% complementary to the first six codons of murine pRB mRNA. Chol-S-ODNs with the sense-oriented sequence as 5%-ATGC-
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CGCCCAAAGCCCCG-3% were used for control. These ODNs were dissolved at 10 mg/10 ml of water for use.
2.3. Cellular uptake of ODNs Cellular uptake of biotin-labeled ODNs was determined by colorimetric assay with peroxidase-conjugated streptavidin. Medium was replaced by new medium containing ODNs following 24 h of culture. After incubation for 10 min, cells were washed with Dulbecco’s Ca2 + - and Mg2 + -free PBS (PBS) three times, fixed with acetate:ethanol (1:3) for 30 min, and washed again with PBS three times. Bovine serum albumin (Sigma, St. Louis, MO) at 5% in PBS was used for blocking for 30 min and then changed to streptavidin-peroxidase conjugates (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) in PBS. After 30 min, cells were washed with PBS containing 0.1% Tween 20 three times, and 3,3%,5,5%-tetramethylbenzidine in buffer (Moss, Pasadena, MD) was added for 20 min. Uptake of ODNs was determined as optical density (OD) at 450 nm with reference to that at 570 nm. Medium without ODNs and solid-phase ODNs prepared by CHEMICOAT (Medicine and Applied Sciences, Sterling, VA) were used to determine the background OD and the 100% OD, respectively. These values were 0.05 and 0.16, respectively. Data are expressed as percent uptakes with reference to 100% OD. Pravastatin (kindly provided by Sankyo Pharmaceutical, Tokyo, Japan) was dissolved at 10 mM in medium containing FBS, and PLC/PRF/5 cells were treated with this for 2 h before addition of ODNs.
2.4. Oral administration of ODNs Chol-S-ODNs or S-ODNs without cholesterol modification were orally administered to mice at a dose of 10 mg in 10 ml water/g body weight (180–200 ml) using flexible thin tips. After 24 h, mice were anesthetized with ether and killed by cutting the abdominal aorta. The liver, kidney and spleen were isolated and homogenized in cold lysis buffer (10 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulphate (SDS), 0.1% sodium deoxycholate, 1 mM EDTA and 10 mg/ml aprotinin). The lysates were centrifuged at 10000× g for 30 min. Protein contents in the supernatants were determined using BCA* Protein Assay Reagent (PIERCE, Rockford, IL). Ten micrograms of proteins from the supernatant were separated on 4 – 15% linear gradient polyacrylamide gels containing 0.1% SDS and transferred onto PVDF membranes for immunoblotting. Membranes were immunoblotted with an antibody to pRB (C-15)-G (Santa Cruz Biotechnology, Santa Cruz, CA) or to p27Kip1 (PharMingen, San Diego, CA), followed by a peroxidaseconjugated secondary antibody. Antibody-associated pRB or p27Kip1 was visualized by ECL (Amersham LIFE SCIENCE). The same blots were also stained with 0.1% Amido Black 10B in 45% methanol and 10% acetate to evaluate background protein content.
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3. Results and discussion Cellular uptake of biotin-labeled ODNs with and without cholesterol modification was determined in vitro using two cell lines, the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5, which were used to test intestinal uptake and uptake by cells from the target organ, respectively. After the addition of 1 nmol of ODNs in serum-free medium to 105 cells for 10 min, ODNs uptake was determined by colorimetric assay as described in Section 2. Rapid uptake of the cholesterol-modified ODNs (Chol-ODNs) was observed in both IEC-6 and PLC/PRF/5 cells, while no significant uptake of the unmodified ODNs was seen (Fig. 1). Since Chol-ODNs taken up by the intestine may be incorporated into chylomicrons, the lipoproteins which carry absorbed lipids through the intestinal epithelium and are specifically internalized by the liver, and Chol-ODNs themselves are known to be partially taken up through LDL receptors [3], the effects of pravastatin and chylomicrons on the uptake by PLC/PRF/5 were also tested. Pretreatment of PLC/PRF/5 cells with pravastatin, which is known to increase low density lipoprotein (LDL) receptor expression, at 10 mM for 2 h increased the uptake of Chol-ODNs. When Chol-ODNs in medium containing chylomicrons were used after incubation for 2 h, no further increase of uptake was observed. However, pravastatin treatment seemed to increase the uptake more in the presence than in the absence of chylomicrons.
Fig. 1. Cellular uptake of biotin-labeled oligonucleotides (ODNs) by the rat intestine epithelial cell line IEC-6 and the human hepatoma cell line PLC/PRF/5. Cholesterol: cholesterol-modified ODNs; chylomicron: ODNs incubated in solution containing chylomicrons for 2 h before addition; pravastatin: ODNs were added to cells treated with pravastatin for 2 h. * P B0.05, ** PB 0.01. Data are represented as means 9 S.D. of eight wells in two experiments. Cellular uptake was determined after addition of 1 nmol of ODNs to 105 cells for 10 min as described in Section 2.
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Fig. 2. Effects of oral administration of ODNs corresponding to pRB on the expression of pRB in the mouse liver, kidney and spleen. ODNs( − ): ODNs not administered; S-ODNs: phosphorothioatemodified ODNs; Chol-S-ODNs: cholesterol- and phosphorothioate-modified ODNs; sense: sense-oriented Chol-S-ODNs. L: liver; K: kidney; S: spleen. Back: background proteins on the blot stained with Amido Black. (A) Chol-S-ODNs but not S-ODNs suppressed pRB expression in the liver and the spleen 24 h after oral administration at the dose of 10 mg in 10 ml water/g body weight. Background proteins in each tissue show similar patterns. The expression of p27Kip1 protein as an internal control was not affected. (B) Sense-oriented Chol-S-ODNs showed no significant effect.
Y. Okamoto, H. Nakano / Hepatology Research 13 (1999) 252–258
257
To test the efficacy of oral administration of Chol-S-ODNs, we decided to target retinoblastoma protein (pRB), a representative tumor suppressor. pRB is one of the suitable proteins to test the efficacy comparatively because pRB expresses in any tissue and is not secreted to have an indirect effect on other cells, and its half life (10 h [4]) is appropriate to evaluate changes in the protein level 24 h after administration of Chol-S-ODNs. Antisense Chol-S-ODNs complementary to the first six codons of murine pRB mRNA or S-ODNs without cholesterol modification were orally administered to 6-week-old male Balb C mice (10 mg in 10 ml of water/g body weight). After 24 h, the liver, kidney and spleen were isolated and used for immunoblotting of pRB. In Chol-S-ODNs administered mice, the levels of pRB expression were diminished in the liver, not affected in the kidney and markedly reduced in the spleen (Fig. 2A). However, ODNs without cholesterol modification showed little effect. The expression of p27Kip1 protein, another tumor suppressor, as an internal control was not affected in the liver, kidney or spleen. Sense-oriented Chol-S-ODNs showed no significant effect (Fig. 2B). These results indicate that the effect of Chol-S-ODNs was sequence-specific. In this study, we demonstrated that ODNs, even by oral administration, can inhibit protein expression in internal organs such as the liver and spleen. We also showed that Chol-S-ODNs have an advantage for this purpose, because they had more consistent effects than S-ODNs. Considering the findings in a previous report concerning mucosal uptake of ODNs [1,5] and our in vitro study using rat intestinal epithelial cells, S-ODNs are suggested to be nuclease-resistant but uptake is too inconsistent to show a final effect, whereas Chol-S-ODNs are suggested to be taken up into the circulation efficiently enough to be effective in the liver and the spleen. However, liver-specific delivery, our primary aim, is not yet sufficient, since delivery to the spleen could not be mediated by a liver-specific carrier like chylomicron. It is suggested that Chol-S-ODNs in the present form failed to be incorporated into chylomicron. To increase the specific uptake, further modifications or drug design are needed. Our results provide a new experimental animal model for gene targeting. Furthermore, the accumulation of Chol-S-ODNs in the circulation may be therapeutically useful for several human hematological disorders such as lymphoma and HIV infection. For these diseases, clinical trials of antisense ODN therapy were performed by intravenous or subcutaneous infusion [1,2]. As described previously [6,7], doses similar to that used in our study have lower toxicity even by intravenous infusion. In addition, oral administration is practical for long-term treatment of chronic diseases, since medical restrictions on patients can be largely eliminated. Thus, oral administration may become practical in the field of antisense ODN therapy. References [1] Agrawal S. Antisense oligonucleotides: towards clinical trials. Trends Biotechnol 1996;14:376 – 387. [2] Webb A, Cunningham D, Cotter F, et al. BCL-2 antisense therapy in patients with non-Hodgkin lymphoma. Lancet 1997;349:1137–1141.
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[3] Krieg AM, Tonkinson J, Matson S, et al. Modification of antisense phosphodiester oligonucleotides by a 5% cholesteryl moiety increases cellular association and improves efficacy. Proc Natl Acad Sci USA 1993;90:1048–1052. [4] Mihara K, Cao XR, Yen A, et al. Cell cycle-dependent regulation of phosphorylation of the human retinoblastoma gene product. Science 1989;246:1300– 1303. [5] Vlassov VV, Karamyshev VN, Yakubov LA. Penetration of oligonucleotides into mouse organism through mucosa and skin. FEBS Lett 1993;327:271 – 274. [6] Galbraith WM, Hobson WC, Giclas PC, Schechter PJ, Agrawal S. Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey. Antisense Res Dev 1994;4:201 – 206. [7] Bishop MR, Iversen PL, Bayever E, et al. Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies. J Clin Oncol 1996;14:1320 – 1326.
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