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Complete Protection of Antisense Oligonucleotides against Serum Nuclease Degradation by an Avidin-Biotin System Ruben J. Boado’ and William M. Pardridge Department of Medicine, Division of Endocrinology, and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90024. Received June 18, 1992
It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUTl glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUTl biotinylated oligonucleotide-avidin construct showing complete protection against serum 3’-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUTl glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5’- or near 3’-biotinylated oligonucleotide (bio-DNA), and 5’- and 3’-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5’-32Pl-5’-bio-DNA(>95% at 30 min). Avidin partially protected this construct (-31% of intact 21-mer oligo remained a t 1h). Similar results were obtained with the [3’-32Pl-5’-bio-DNA;however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3’-exonuclease. Incubation of the [5’-32Pl-3’-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1p 13min). Conversely, avidin totally protected this construct against the serum 3’-exonuclease. In conclusion, avidin fully protects antisense oligonucleotidesbiotinylated at the near 3’-terminus against serum 3’-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.
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INTRODUCTION
The glucose transporter type I (GLUTl), a member of the Na+-independentglucose transporter supergenefamily (1-9), is overexpressed in malignancy and by cellular transformation and oncogenes ( I , 10-12). Therefore, inhibition of the GLUTl gene in cancer cells may result in glucose deprivation to the malignancy and potential inhibition of cell division. Antisense oligonucleotides are potential specific chemotherapeutic agents for the treatment of cancer, viral infections, and other disorders (1315). However, major problems limiting the therapeutic efficacy of these agents in vivo are the minimal transcapillary transport or cellular uptake of the highly charged oligonucleotide and the rapid degradation by serum nucleases (16,17),which have recently been shown to be predominantly 3’-exonucleases (I7). Owing to these two problems, antisense oligonucleotides must be added to cells in tissue culture in high concentrations ranging from 10-100 pM to achieve biological effects (18-22). Even higher concentrations will be required in vivo, where capillary barriers retard the delivery of oligonucleotides to cells. In particular, the delivery of these therapeutics to brain is impaired by the poor transport of these molecules through the brain capillary wall, which makes up the blood-brain barrier (BBB) in vivo (23). Although recent advances have shown that the use of methylphosphonate or phophorothioate linkages protect antisense oligonucleotides against serum nuclease degradation, this protection was shown to be partial (18, 19,241. Given these considerations with regard to poor transport and rapid serum metabolism of antisense oligonucleotides, it would be advantageous to conjugate the antisense
* Author to whom correspondenceshould be addressed Ruben J. Boado, Ph.D., Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90024. Phone: (310) 825-8858. FAX: (310) 206-5163.
oligonucleotide to a transport vector via high-affinity binding that is stable in the circulation but is labile in cells. A single solution to these multiple problems is potentially offered by the avidin-biotin system. Avidin binds biotin with very high affinity (i.e., KD = 10-15M and dissociation tl 2 = 89 days) (25), and the avidin-biotin reaction is stable in the circulation but is labile in cells (26). Finally, avidin may act as a transport vector for mediating the uptake of biotinylated antisense oligonucleotide or biotinylated peptide by tissues in vivo. Avidin is a polycationic protein and recent studies have shown that avidin, like other polycationic proteins such as cationized albumin (27), is taken up by cells in vitro via absorptive-mediated endocytosis through a reaction that is inhibited by other polycationic proteins such as protamine (28). In addition, avidin enhances the organ delivery of biotinylated antisense oligonucleotides or peptides in vivo (29). In addition to catalyzing the endocytosis of biotinylated antisense oligonucleotides, it is possible that the avidinbiotin system may protect the oligonucleotide from serum 3’-exonuclease activity by selectively biotinylating the oligonucleotide at the 3’4erminus. Data reported here demonstrate that avidin fully protects antisense oligonucleotides biotinylated a t the near 3’-terminus against serum nuclease-mediated degradation. EXPERIMENTAL PROCEDURES
Materials. [~-3~PIAdenosine 5’4riphosphate (ATP) (3000 Ci/mmol), [c~-~*PIdeoxyadenosine 5’-triphosphate (dATP) (3000 Ci/mmol), and Crones lighting plus intensifying screens were purchased from Du Pont-NEN (Boston,MA). T4 polynucleotidekinase was obtained from Promega Corp. (Madison, WI), and terminal deoxynucleotidyl transferase was purchased from BRL (Gaithersburg, MD). Acrylamide, N,”-methylenebisacrylamide, urea, and ammonium persulfate were purchased from 0 1992 American Chemical Society
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I NEAR 5’-BIOTINYLATED, 3‘-LABELED ANTISENSE OLIGONUCLEOTIDE 1
I NEAR 5’-BIOTINYLATED, 5’-LABELED ANTISENSE OLIGONUCLEOTIDE I A
5’-[32P]-G-G-U-G-G-G-C-T-C-C-A-T-G-G-C-C-G-C-G-C-T-3’ I
[biotin
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5f-G-G-U-G-G-G-C-T-C-C-A-T-G-G-C-C-G-C-G-C-T-[32P]-A-[ -j2P]-A-3’ I biotin]
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I - I + I + I+ I + I + I -
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100 80]
.-c
I
I
I
I
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30
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1 time 60 (min)
Figure 1. Avidin protection of near 5’-biotinylated [5’-32p]antisense oligonucleotide in serum. (A) A 21-mer antisense oligonucleotide complementary to nucleotides 162-182 of the bovine GLUTl glucose transporter mRNA was synthesized with an extended primary amine group by replacement of the deoxythymidinethat corresponds to position 180 with 6-aminodeoxyuridine and biotinylated with NHS-biotin. (B)Effect of avidin on the stability of the near 5’-biotinylated oligonucleotide in serum. Six femtomole (26 400 dpm; specific activity = 2.0 pCi/pmol) of [5’-32P]-5’-bio-oligonucleotide or [5’J2P] -5’-biooligonucleotide/avidincomplex was incubated with buffer or rat serum at 37 “C for the time indicated in the figure. Samples were resolved in a 155% polyacrylamide/7M urea sequencinggel. The autoradiogram of the gel is shown in part B of the figure. (C) Quantitation of the autoradiogram by laser scanning densitometry. Results expressed as percent 21-mer [32P]oligonucleotide/X, [32P]oligonucleotides(degradationproducts). There is a consistent slightly lower migration of labeled product through the gel in the lanes containing serum.
Schwartz/Mann Biotech (Cleveland, OH). N-Hydroxysuccinimideester of biotin (NHS-biotin)was obtained fiom Pierce Chemical Co. (Rockford, IL). Avidin and all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Customized oligonucleotides were obtained from GenosysBiotechnologies,Inc. (The Woodlands,TX). Synthesis of Biotinylated Oligonucleotides. Two 21-mer antisense oligonucleotides complementary to nucleotides 162-182 (Figure 1A) and 161-181 (Figure 3A) of the bovine GLUTl glucose transporter mRNA (30) were synthesized with an extended primary amino group by replacement of the deoxythymidine at position 3 and 20, respectively, with 6-aminodeoxyuridine (aU) (Genosys Biotechnologies). T h i s yielded t h e following oligonucleotides: 5’-GGaUGGGCTCCATGGCCGCGCT3‘ (3-amino oligonucleotide) and 5’-GTGGGCTCCATGGCCGCGCaUG-3’ (20-amino oligonucleotide). The size of these oligonucleotides was determined by the manufacturer by comparing migration distances with that of a parallel oligonucleotide standard using a 20 % polyacrylamide/7 M urea gel electrophoresis. The gel-purified 3-amino oligonucleotide was biotinylated to yield the 5’-biotinyl oligonucleotide (5’-bio-
I
II5 io 4; 80 ): : ; Figure 2. Avidin protection of the near 5’-biotinylated [3’-32p]labeled oligonucleotide in serum. (A) The 21-mer oligonucleotide described in Figure 1was labeled a t the 3’-terminus with [ c ~ - ~ ~ P ] dATP and terminal deoxynucleotidyl transferase and purified by gel electrophoresis. (Band C) The experiment was performed under identical conditions as those described in Figure 1 using 6 fmol of [3’-32P]-5’-bio-oligonucleotide (29 OOO dpm; specific activity = 2.2 pCi/pmol). Since no degradation products of varying size were seen in the [3’-32P]-labeled experiment (B) due to action of 3’-exonucleases, results were expressed as integrated area/are%, where are% = area a t time zero. The initial labeled oligonucleotidemainly contains 1and 2 molecules of [32P]dATP and trace amounts containing up to 5 dATP molecules/molecule of oligonucleotide. A consistent slightly slower migration of labeled product through the gel was observed in the lanes containing serum. 0
oligonucleotide) by incubation with a 10.6 M excess ratio of NHS-biotin (31)to oligonucleotide in 0.125 M NaHC03 at pH = 9 for 16 h at 22 OC. Following biotinylation, the excess of NHS-biotin was removed from the 5’-biooligonucleotide by Sephadex G-25 spin-column chromatography (32) (Boehringer Mannheim, Indianapolis, IN). The 20-amino oligonucleotidewas similarly biotinylated with NHS-XX-biotin (X = 6-aminohexanoyl; Glen Research, Hemdon, VA) to yield the 3’-bio-oligonucleotide. The 3’-bio-oligonucleotide was purified by high-pressure liquid chromatography using a 5-pm C18 reverse-phase column (10 X 250 mm) with a 300-A pore size. The liquid phases were A, 0.1 M triethylammonium acetate in water, pH = 7, and B, 100% acetonitrile. The gradient was 3038 % B over 40 min. Under these conditions, biotinylation produced a 5-min shift in the retention of the oligonucleotide. Tubes corresponding to the biotinylated oligonucleotide were pooled and concentrated. The extinction coefficient(c) was calculated as described previously (32), and it was found to be 207 mM-l. The concentration of c] oligonucleotides [equal to optical density ( O D ) ~ ~ /was determined in all samples after measuring ODaa in a Beckmann 34 UV spectrophotometer (Imine, CA). Preparation of [3zP]-LabeledOligonucleotides. Aliquots of 15 pmol of 5’- and 3’-bio-oligonucleotide were
Antkense Oligonucleotides Serum Protection
Bioconjcllgate Chem., Vol. 3, No. 6, 1992 521
all the free [32P]dATPand -ATP, filters were individually washed three times in 50 mL of 0.5 M phosphate buffer, pH = 7, and one time in 70% ethanol. Filters were then dried, and the radioactivity was determined in a Packard scintillation counter Tri-carb 4530 (Downers Grove, IL) -G-T-G-G-G-C-T-C-C-A-T-G-G-C-C-G-C-G-CU - G -3’ using SCINT-A XF scintillation cocktail (Packard). The percentage of incorporation was determined as (dpm in washed filters/dpm in unwashed filters) X 100. The specific activity of the [32P]-labeled oligonucleotides, ]Avidin I I + I calculated as percentage of incorporation X amount of radioactivity used (pCi)/massof oligonucleotide (15pmol) x 100, was 2.0 and 2.2 pCi/pmol for the 5’- and 3’-labeling reaction, respectively. The purity of the [32P]-labeled oligonucleotides was determined after the purification 21 mersteps (G-25 spin column for the T4 kinase reaction and polyacrylamide gel electrophoresis for the terminal transferase reaction) by adsorption to the DE-81 filters as described above and was more than 99% pure. For the serum protection studies, aliquots of 12.8 pmol Time(min) 0 0 5 I 5 3060 60 0 0 5 15 3060 60 of [32P]-5’-or -3’-bio-DNA were incubated with 140 pg of Serum - + + + + + - - + + + + + avidin in 100 pL of PBST (PBST = 10 mM phosphate buffer, pH = 7.5;0.15 M NaCl; 0.1 % bovine serum albumin; 500 pg tRNA/mL) at room temperature for 15 min. The avidin/ [32P] bio-oligonucleotidecomplex was purified from unbound [32P]-labeledoligonucleotide,if any, by Sephadex G 7 5 spin-columnchromatographyprior to the experiment. Serum Protection Studies. The effect of avidin on the stability of biotinylated oligonucleotide in serum was Avidin 0 studied in 4 pL of PBST containing 6 fmol (26 400 and I I I I I 29 OOO dpm for 5’- and 3’-labeled oligonucleotide, respec0 15 30 45 60 Time (mid tively, calculated using the specific activity and pCi = 2.2 Figure 3. Avidin protection of near 3’-biotinylated [5’-32P]- X lo6 dpm) of [32P]bio-DNA or [32P]bio-DNA/avidin antisense oligonucleotide in serum. (A) A 21-mer antisense complex, which were incubated with PBST or rat serum oligonucleotide complementary to nucleotides 161-181 of the pool (16 pL) at 37 “C for 0,5,15,30, or 60 min. The final bovine GLUT1 glucose transporter mRNA was synthesized with concentration of avidin in the reaction tube was 3.5 pg/ an extended primary amine group at position 20 and biotinylated mL. The reaction was stopped by transferring tubes onto with NHS-XX-biotin. (B and C) The experiment was performed an ice bath and adding 2 volumes of 8M urea/ 10% glycerol. under identical conditions as those described in Figure 1 using Samples were heated for 5 min at 95 “Cand incubated for 6 fmol of [5’-32P]-3’-bio-oligonucleotide (26 400 dpm, specific activity = 2.0 &i/pmol). The initial labeled oligonucleotide is 5 min on ice immediately before resolving them in a 0.4 80 76 21-mer and 20 76 20-mer [32P]oligonucleotide,respectively, mm 15% polyacrylamide/7 M urea sequencing gel (32). as determined by scanninglaser densitometry (methods). There The gel was fixed for 10 min in 5% methanol/lO% acetic is a consistentslightly slower migration of labeled product through acid, washed for 10 min in H20, and dried on Whatman the gel in the lanes containing serum. 3 MM using a gel drier (Bio-Rad 583, Richmond, CA). labeled at the 5’-end with 50 pCi of [y32P]ATP and 8 Autoradiography was performed at -70 “C with intensiunits of T4 polynucleotide kinase in a 20-pL reaction fying screens for 1-3 days. Results were quantified by containing 50 mM TRIS at pH = 7.6,lO mM MgC12,5 mM laser scanning densitometry (LKB Model 2202 ultrascan DDT, 0.1 mM spermidineOHC1, and 0.1 mM EDTA for 45 laser densitometer, Bromma, Sweden) of the autoradiomin at 37 “C. The labeled product was purified through gram, which yielded the area of each radioactive peak. In a Sephadex G-25 spin column (32). The 5’-bio-oligonuFigures 1 and 3, the area of the individual bands correcleotide (15 pmol) was also labeled at the 3’-end with 100 sponding to labeled oligonucleotides of varying size was T P 15 units of terminal deoxynupCi of [ Q - ~ T I ~ Aand determined and expressed as percent 21-mer [32P]01igocleotidyl transferase (33) in a 10-pL reaction containing nucleotide/Xn, where Xn = the sum of all [32P]oligonu0.1 M potassium cacodylate (pH = 7.2), 2 mM CaC12, and cleotides (degradation products). Since no degradation 0.2 mM DTT for 2 h at 37 “C. The [3’-32P]-labeled5’products of varying size were seen in the [3’J2P]-labeled bio-oligonucleotidewas purified by electrophoresis on a experiment (Figure 2) due to action of 3’-exonucleases, 15% denaturing polyacrylamide/7 M urea gel (32). The the results were expressed as integrated area for each peak/ labeled oligonucleotide, mainly containing 1 and 2 molmew, where are@ is the area at zero time. ecules of [32P]dATP/molecule of oligonucleotide, was The rat serum pool was obtained from anesthetized identified by autoradiography and eluted from gel slices animals killed by decapitation. Blood was collected by in 0.5 M NH4 acetate/l mM EDTA for 16 h at 22 “C, draining the trunk and serum obtained by centrifugation. concentrated in a vacuum centrifuge evaporator (Savant), The serum pool was kept at -20 “C,and aliquots were and suspended in H20. thawed once. The efficiencyof the labeling procedure using either T d polynucleotide kinase or terminal deoxynucleotidyltransRESULTS ferase was determined by adsorption to DE-81 filters (Whatman International, Ltd., Maidstone, England) as The protective effect of avidin from serum nuclease described previously (32). Briefly, aliquots of the labeled degradation of biotinylated oligonucleotides is shown in reaction were applied to the positively charged DE-81 Figures 1-3. Incubation of the unprotected (i.e., no avidin) filters and dried at room temperature. In order to remove [5’-32P]-5’-bio-antisense oligonucleotide (Figure 1) with
Near 3’-Biotinylated, 5’- Labeled Antisense Oligonucleotide
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serum showed a rapid conversion to degradation products (20-16-mer) (Figure 1, B and C). In the absence of avidin, more than 95% of the labeled oligomer was degraded during the first 30 min of incubation. Incubation with avidin partially protected the near 5‘-biotinylated [5’-32Ploligomer against degradation by the serum-mediated nucleases, as 31% of intact 21-mer labeled oligonucleotide was left at the end of the 1-h incubation period (Figure 1,B and C). The degradation of the [5’-32P]-5’-bio-o1igonucleotide in serum was sequential (Figure 1); this observation, in conjunction with the absence of small degradation products, suggests the participation of a serum exonuclease. To test this hypothesis, the serum protection experiment was repeated with the near 5’-biotinylated oligonucleotide labeled with [ c Y - ~ ~ P I ~ at A Tthe P 3’-end with terminal transferase (Figure 2). The labeled product mainly contains 1 and 2 molecules of [32P]dATP and traces containing up to 5 dATPs/molecule of oligonucleotide (Figure 2B). Avidin also protected the [3’-32Pl-5’-biooligomer partially and in a similar manner as it protected (shown in Figure 1).In the [5’-32P]-5’-bio-oligonucleotide these studies,no degradation products of varying molecular size were observed in the presence of absence of avidin, confirming that the degradation of oligonucleotides in the present study was mainly mediated by a serum 3’exonuclease. Since avidin partially protects the Y-bio-DNA against the serum 3‘-exonuclease degradation, its effect was tested on the second oligomer which is biotinylated near the 3‘end (Figure 3). The labeled oligonucleotide was 80% 21mer and 20% 20-mer [32Ploligonucleotide. Incubation of the [5’-32P]-3’-bio-oligonucleotide with serum without avidin showed a rapid conversion to degradation products (20- and 19-mer) with a half-life ( t l p )of approximately 13 min. Conversely, incubation with avidin totally proagainst degratected the [5’-32Pl-3’-bio-oligonucleotide dation by the 3’-exonuclease in serum during the incubation period (Figure 3). DISCUSSION
The present study demonstrates that biotinylation of the oligonucleotide near the 3‘-end (at nucleotide 20 of the 21-mer, Figure 3A) and avidin binding fully protects the oligomer against serum 3‘-exonuclease degradation (Figure 3). On the other hand, when the oligonucleotide was biotinylated near the 5’-terminus (Figure 1) and incubated in serum, avidin partially protected the oligomer and degradation products of varying size (from 20- to 16mer) were observed. Interestingly, the avidin/3’-biotinylated oligomer conjugate was totally resistant to the serum nuclease action, whereas when incubated in the absence of avidin, a rapid conversion of the 3’-biotinylated 21-mer/20-mer mixture to a 20-mer/l9-mer mixture was observed, indicating that biotinylation, per se, at the 3’terminus but not the 5’-terminus (Figure 11, protects against 3’-exonuclease-mediated degradation. This represents an important characteristic of 3’-biotinylated oligonucleotides,and suggests that after the biotin-avidin bond is cleaved in the cell, the 3‘-bio-antisense oligonucleotide may still be stable to cellular 3’-exonuclease. The model antisense oligonucleotide therapeutic employed in these studies was designed to contain only a single primary amino group available for biotinylation. The presence of multiple biotin groups on the therapeutic compound would cause the formation of high molecular weight aggregates owing to the multivalency of avidin binding of biotin (25). Although the GLUTl antisense
oligonucleotide was selected as a model therapeutic, since the GLUTl glucose transporter gene is overexpressed in malignancy ( I , IO), this system is applicable to other antisense oligonucleotides. Whether biotinylated antisense oligonucleotides still bound to avidin retain their ability to hybridize to specific targets is at present unknown. However, the release of the biotinylated oligonucleotide from the avidin carrier may be facilitated by employing sulfosuccinimidyl2-[2- (biotinamido)ethyl]1,3’-dithiopropionate (NHS-SS-biotin),a disulfide-based biotinylation analogue that has been used previously to biotinylate oligonucleotides (34) or peptides (28,29). Overall, the present study shows that biotinylation of antisense oligonucleotides at the 3’4erminus and avidin binding provides complete protection against serum nuclease degradation. This property, in conjunction with previous studies showing endocytosis of avidin-drug conjugates (28, 29),may be useful for drug delivery of antisense oligonucleotides to tissues in vivo or to cultured cells in vitro using the avidin-biotin system. ACKNOWLEDGMENT
The authors are indebted to Sherri J. Chien and Sara Morimoto for skillful preparation of the manuscript. This work was supported by NIH Grant R01-AI28760. LITERATURE CITED (1) Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M.,Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Sequence and Structure of a Human Glucose Transporter. Science 229, 941-945. (2) Birnbaum, M. J., Haspel, H. C., and Rosen, 0. M. (1986) Cloning and Characterization of a cDNA Encoding the Rat Brain Glucose-Transporter Protein. Proc. Natl. Acad. Sci. U.S.A. 83,5784-5788. (3) Thorens, B., Sarkar, H. K., Kaback, H. R., and Lodish, H. F. (1988) Cloning and Functional Expression in Bacteria of a Novel Glucose Transporter Present in Liver, Intestine, Kidney, and &Pancreatic Islet Cells. Cell 55, 281-290. (4) Fukumoto, H., Seino, S., Imura, H., Seino, Y., Eddy, R. L., Fukushima, Y., Byers, M. G., Shows, T. B., and Bell, G. I. (1988) Sequence, Tissue Distribution, and Chromosomal Localization of mRNA Encoding a Human Glucose Transporter-Like Protein. Proc. Natl. Acad. Sci. U.S.A.85,54345438. (5) Kayano, T., Fukumoto, H., Eddy, R. L., Fan, Y.-S., Byers, M. G., Shows, T. B., and Bell, G. I. (1988) Evidence for a Family of Human Glucose Transporter-Like Proteins. Sequence and Gene Localization of a Protein Expressed in Fetal Skeletal Muscle and Other Tissues. J.Biol. Chem. 263,15245-15248. (6) James, D. E., Strube, M., and Mueckler, M. J. (1989) Molecular Cloning and Characterization of an Insulin-Regulatable Glucose Transporter. Nature 338, 83-87. (7) Birnbaum, M. J. (1989) Identification of a Novel Gene Encoding an Insulin-Responsive Glucose Transporter Protein. Cell 57, 305-315. (8) Charron, M. J., Brosius, F. C., 111, Alper, S. L., and Lodish, H. F. (1989) A Glucose Transport Protein Expressed Predominantly in Insulin-Responsive Tissues. Proc. Natl. Acad. Sci. U.S.A. 86, 2535-2539. (9) Kayano,T., Burant, C. F., Fukumoto, H., Gould, G. W., Fan, Y.-S., Eddy, R. L., Byers, M. G., Shows, T. B., Seino, S., and Bell, G. I. (1990) Human Facilitative Glucose Transporters. Isolation, Functional Characterization, and Gene Localization of cDNAs Encoding an Isoform (GLUT5) Expressed in Small Intestine, Kidney, Muscle, and Adipose Tissue and an Unusual Glucose Transporter Pseudogene-Like Sequence (GLUT6). J. Biol. Chem. 265, 13276-13282. (10)Yamamoto, T., Seino, Y., Fukumoto, H., Koh, G., Yano, H., Ingaki, N., Yamada, Y., Inoue, K., Manabe, T., and Imura, H. (1990) Over-Expression of Facilitative Glucose Transporter
Antisense Ollgonucleotldes Serum Protection
Genes in Human Cancer. Biochem. Biophys. Res. Commun. 170, 223-230. (11) Birnbaum, M. J., Haspel, H. C., and Rosen, 0. M. (1987)
Transformation of Rat Fibroblasts by FSV Rapidly Increases Glucose Transporter Gene Transcription. Science 235,14951498. (12) Flier, J. S., Mueckler, M. M., Usher, P., and Lodish, H. F. (1987) Elevated Levels of Glucose Transport and Transporter
Messenger RNA are Induced by ras and src Oncogenes. Science 235, 1492-1495. (13) Weintraub, H., Izant, J. G., and Harland, R. M. (1985) Antisense RNA as a Molecular Tool for Genetic Analysis. Trends Gene. 1, 22-25. (14) Dolnick, B. J. (1991) Antisense Agents in Cancer Research and Therapeutics. Cancer Znuest. 9, 185-194. (15) Cohen, J. S. (1991) Antisense Oligodeoxynucleotides as Antiviral Agents. Antiviral Res. 16, 121-133. (16) Wickstrom, E. (1986) Oligodeoxynucleotide Stability in SubcellularExtracts and Culture Media. J . Biochem. Biophys. Methods 13,97-102. (17) Tidd, D. M., and Varenius, H. M. (1989) Partial Protection of Oncogene, Anti-SenseOligodeoxynucleotidesAgainst Serum Nuclease Degradation Using Terminal Methylphosphonate Groups. Br. J. Cancer 60, 343-350. (18) Smith, C. C., Aurelian, L., Reddy, M. P., Miller, P. S., and Ts’o, P. 0. P. (1986) Antiviral Effect of an Oligo(nuc1eoside methylphosphonate) Complementary to the Splice Junction of Herpes Simplex Virus Type 1Immediate Early Pre-mRNAs 4 and 5. Proc. Natl. Acad. Sci. U.S.A. 83, 2787-2791. (19) Marcus-Sekura, C. J., Woerner, A. M., Shinozuka, K., Zon, G., and Quinnan, G. V. Jr., (1987) Comparative Inhibition of Chloramphenicol Acetyltransferase Gene Expression by Antisense Oligonucleotide Analogues Having Alkyl Phosphotriester, Methylphosphonate and Phosphorothioate Linkages. Nucleic Acids Res. 15, 5749-5763. (20) Letsinger, R. L., Zhang, G. R., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-Conjugated Oligonucleotides: Synthesis,Properties, and Activityas Inhibitors of Replication of Human Immunodeficiency Virus incellculture. Proc.Natl. Acad. Sci. U.S.A. 86,6553-6556. (21) Leonetti, J. P., Machy, P., Degols, G., Delbeu, B., and Leserman,L. (1990) Antibody-TargetedLiposomes Containing Oligodeoxyribonucleotides Complementary to Viral RNA Selectivity Inhibit Viral Replication. Proc. Natl. Acad. Sci. U.S.A. 87, 2448-2451. (22) Morrison, R. S. (1991) Suppression of Basic Fibroblast Growth Factor Expressionby Antisense Oligodeoxynucleotides
Bloconlugere Chem., Voi. 3, No. 8, 1992 529
Inhibits the Growth of Transformed Human Astrocytes. J. Biol. Chem. 266, 728-734. (23) Pardridge, W. M. (1991)Peptide DrugDeliuery to the Brain, Raven Press, New York, NY, pp 1-356. (24) Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J. S., and Broder, S. (1987) Phosphorothioate Analogs of Oligodeoxynucleotides: Inhibitors of Replication and Cytopathic Effects of Human Immunodeficiency Virus. Proc. Natl. Acad. Sci. U.S.A. 84, 7706-7710. (25) Green, N. M. (1975) Avidin. Adv. Protein Chem. 29,85133. (26) Wei, R. D., KOOU, D. H., and Hoo, S. L. (1970) Dissociation of Avidin-Biotin Complex in vivo. Experientia 27, 366-368. (27) Kumagai, A. K., Eisenberg, J., and Pardridge, W. M. (1987)
Absorptive-MediatedEndocytosis of CationizedAlbumin and a 8-Endorphin-Cationized Albumin Chimeric Peptide by Isolated Brian Capillaries. Model System of Blood-Brain Barrier Transport. J . Biol. Chem. 262, 15214-15219. (28) Pardridge, W. M., and Boado,R. J. (1991) Enhanced Cellular Uptake of Biotinylated Antisense Oligonucleotideor Peptide Mediated by Avidin, a Cationic Protein. FEBS Lett. 288,3032. (29) Pardridge, W. M., Boado, R. J., and Buciak, J. L. (1992)
Drug Delivery of Antisense Oligonucleotides or Peptides to Tissues in Vivo Using an Avidin-Biotin System. Drug Targeting Delivery. In press. (30) Boado, R. J., and Pardridge, W. M. (1990) Molecular Cloning of the Bovine Blood-BrainBarrier Glucose Transporter cDNA and Demonstration of Phylogenetic Conservation of the 5’Untranslated Region. Mol. Cell. Neurosci. 1, 224-232. (31) Langer, P. R., Waldrop, A. A., and Ward, D. C. (1981) EnzymaticSynthesisof Biotin-LabeledPolynucleotides: Novel Nucleic Acid Affinity Probes. Proc. Natl. Acad. Sci. U.S.A. 78, 6633-6637. (32) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. (33) Collins, M. L., and Hunsaker, W. R. (1985) Improved Hybridization Assays Employing Tailed Oligonucleotide Probes: A Direct Comparison with 5’-end labeled Oligonucleotide Probes and Nick-Translated Plasmid Probes. Anal. Biochem. 151,211-224. (34) Shimkus, M., Levy, J., and Herman, T. (1985) AChemically Cleavable Biotinylated Nucleotide: Usefulness in the Recovery of Protein-DNA Complexes From Avidin Affinity Columns. Proc. Natl. Sci. U.S.A. 82, 2593-2597.
