Covalent Protein−Oligonucleotide Conjugates for Efficient Delivery of

Bioconjugate Chem. 1997, 8, 935−940

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Covalent Protein-Oligonucleotide Conjugates for Efficient Delivery of Antisense Molecules S. B. Rajur, C. M. Roth, J. R. Morgan, and M. L. Yarmush* Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Shriners Burns Institute, and Harvard Medical School, Boston, Massachusetts 02114. Received February 11, 1997X

Antisense oligonucleotides have been covalently attached to asialoglycoprotein (ASGP) via disulfide bond conjugation chemistry. These conjugates were characterized extensively by an array of chemical, chromatographic, and spectroscopic means. Multiple (approximately six) oligonucleotides can be conjugated to each ASGP molecule. The molecular conjugates were used to deliver antisense oligonucleotides complementary to the mRNA of the interleukin 6 signal transduction protein (gp130) to modulate the acute phase response of hepatoma (HepG2) cells in vitro. These conjugates were biologically active, as measured by inhibition of the cytokine-stimulated up-regulation of the acute phase protein haptoglobin. The level of inhibition was comparable to that found with previous technology featuring noncovalent complexes of ASGP-poly(L-lysine) and oligonucleotide. Because of the ability to control the stoichiometry of the conjugate and its unimolecular nature (as opposed to bimolecular for the noncovalent conjugates), this methodology holds great promise for further development and application.

INTRODUCTION

Antisense oligonucleotides have great potential as therapeutic agents. Natural and synthetic analogues of these oligonucleotides have been shown to inhibit gene expression in a variety of in vitro and in vivo studies. Targets for antisense molecules in clinical and preclinical investigations include cancer, viral diseases, inflammation, and organ transplant rejection (Heidenreich et al., 1995; Agrawal, 1996; Le Doux et al., 1996). Recently, it has been reported that antisense molecules can downregulate cytokines as well as cytokine receptor expression (Miyajima et al., 1992). For example, cytokines [interleukin 1R and interleukin 6 (IL-61)] and receptors for IL6, nerve growth factor, and epidermal growth factor have been successfully inhibited by antisense oligonucleotides (Maier et al., 1990; Sariola et al., 1991; Schwab et al., 1991; Keller et al., 1995; Wang et al., 1995; Roth et al., 1997). Given an efficacious oligonucleotide sequence, the limiting factor in the development of antisense therapeutics becomes delivering the antisense molecule to its target in a particular tissue with satisfactory efficiency, selectivity, and stability. At physiological pH, natural oligonucleotides exist as polyanions and hence do not readily penetrate cell membranes, making it difficult to achieve high intracellular concentrations. Oligonucleotides must therefore be packaged into a vehicle capable of efficient entry into cells. Various methods have been developed to address this problem. Particularly for liver specific delivery, Wu and Wu (1992) have developed a soluble DNA carrier system that * Address correspondence to this author at the Center for Engineering in Medicine, Massachusetts General Hospital, Bigelow 1401, Boston, MA 02114 [telephone (617) 726-3474; fax (617) 726-7458]. X Abstract published in Advance ACS Abstracts, November 1, 1997. 1 Abbreviations: IL-6, interleukin 6; ASGP, asialoglycoprotein; PLL, poly(L-lysine); HPLC, high-performance liquid chromatography; SPDP, succinimidyl 6-[3′-(2-pyridylthio)propionamido]hexanoate; 2-PT, 2-pyridinethione; DTT, dithiothreitol; AS, antisense oligonucleotide; ASGP-S-S-AS, asialoglycoproteinantisense disulfide-containing conjugate.

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takes advantage of receptor-mediated endocytosis to achieve internalization. They targeted hepatocytes by complexing oligomeric DNA with asialoglycoproteinpoly(L-lysine) (ASGP-PLL) conjugates. In this system, the negatively charged DNA complexes with positively charged polylysine to form a noncovalent complex, and the asialoglycoprotein, by means of its terminal galactose moieties, augments uptake by binding to a receptor uniquely expressed by hepatocytes, including the transformed cell line HepG2. By utilizing this ASGP-PLL delivery system, we were able to target the mRNA of the IL-6 signal transduction protein (gp130) and modulate the acute phase response of hepatoma (HepG2) cells in vitro (Roth et al., 1997). Addition of ASGP-PLL reduced the effective dose of antisense oligonucleotides relative to unconjugated antisense by 10-fold; furthermore, we found that the specificity of action is improved by using lower doses. However, we found that further improvements using the ASGP-PLL conjugates were limited by the physical equilibrium between ASGP-PLL and oligonucleotides and by the toxicity of the ASGP-PLL moiety (Bunnell et al., 1992; C. M. Roth, unpublished data). In this paper, we describe the covalent conjugation of antisense oligonucleotides to ASGP using the heterobifunctional cross-linker sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP). We describe the synthesis and characterization of these new conjugates as well as their biological activity in an in vitro system (Roth et al., 1997). This methodology is generalizable to other antisense sequences and also to other carrier molecules, including alternative ligands for the asialoglycoprotein and ligand/receptor systems for other cell types. EXPERIMENTAL PROCEDURES

Materials for Oligonucleotide Synthesis and Purification. Unless otherwise stated, all solvents and chemicals were of the highest quality commercially available. Oligonucleotides were synthesized using 2-deoxynucleotide phosphoramidites on a Cyclone Plus DNA synthesizer (Millipore, Bedford, MA) or were obtained from Oligos, Etc. (Wilsonville, OR). The four fully © 1997 American Chemical Society

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protected common 2′-deoxynucleoside phosphoramidites containing aryl- or isobutyrylamides and the tritylprotected thiol linker phosphoramidite, 6-(tritylthio)hexanol/2-cyanoethyl N,N-diisopropylphosphoramidite, were purchased from Cruachem (Sterling, VA). Highperformance liquid chromatography was performed on a system equipped with a dual-wavelength detector. Both purchased and in-house oligonucleotides were isolated on a 9.4 × 250 mm reversed-phase column of ODS-Hypersil at a flow rate of 3.0 mL/min in buffer A: 50 mM TEAA (pH 7.2) with a gradient of acetonitrile (20-80% over 40 min). The purity of the oligonucleotides was evaluated by chromatography on a 250 × 4.6 mm reversed-phase column of ODS-Hypersil at a flow rate of 1 mL/min in buffer B: 20 mM potassium phosphate (pH 5.5) with a gradient of methanol (0-100% over 60 min). Purity of the protein-oligonucleotide conjugates was assessed on a size exclusion column (TSK 3000) at a flow rate of 1 mL/min in buffer C: 0.1 M sodium phosphate/0.1 M sodium sulfate (pH 6.4). Cell Culture. The human hepatoma cell line HepG2 (ATCC, HB 8065) was maintained in Modified Eagle’s Medium, supplemented with 10% fetal bovine serum, penicillin-streptomycin solution (200 units/mL penicillin G activity and 200 µg/mL streptomycin activity), and sodium pyruvate (1 mM) in a humidified atmosphere of 5% CO2 at 37 °C. The medium was changed every 2 days. Preparation of N-{6-[3′-(2-Pyridyldithio)propionamido]hexanoyl}-ASGP (ASGP-PDP). ASGP was prepared by digesting human R1-acid glycoprotein with neuraminidase according to the published procedure (Lu et al., 1994). In brief, human R1-acid glycoprotein was dissolved in PBS, neuraminidase (1 mg/mL in PBS) was added to it, and the mixture was incubated at 37 °C for 1 h. The solution was dialyzed twice against PBS at 4 °C (MW cutoff ) 8000). To 40.56 mg (0.994 mM) of ASGP in 1 mL of PBS (1×, pH 7.4) was added 6.42 mg (12.17 mM, 13-fold excess) of sulfo-LC-SPDP (Pierce, Rockford, IL). The mixture was stirred gently for 1 h at ambient temperature. The crude product mixture was then passed through a PD-10 desalting column with PBS elution to remove the byproduct N-hydroxysuccinimide and unreacted sulfo-LC-SPDP. The macromolecular fraction was further dialyzed against 1× PBS (2 × 4 L) through a membrane with 12 00014 000 MW exclusion limits to remove any remaining N-hydroxysuccinimide or sulfo-LC-SPDP. The purity of the conjugates was assessed by running small aliquots through the size exclusion column TSK 3000 with buffer C. To estimate the number of modified groups, an aliquot of the purified product was treated with DTT to reduce the disulfide bond between the ASGP-PDP moiety and produce 2-pyridine thione 5, which has a characteristic absorbance with a maximum of 343 nm. One molecule of ASGP-PDP contained about 4 residues of 2-pyridyl disulfide derived from sulfo-LC-SPDP, on the basis of the extinction coefficient of 2-PT (Carlsson et al., 1978). Synthesis of 5′-Thiol Oligonucleotide. Oligonucleotide 1 was synthesized using standard β-cyanoethyl phosphoramidite chemistry at the 1 µmol scale. A sixcarbon (C6) thiol linker phosphoramidite (25 mg) was dissolved in 1 mL of acetonitrile and attached to a separate port on the DNA synthesizer. After synthesis of the 15-base sequence, the amidite of the thiol modifier was applied to the reaction column. After completion of the coupling, the column was removed from the synthesizer, washed thoroughly with acetonitrile, oxidized with iodine (0.05 M in THF/pyridine/water 7:1:2) for 30 s using a syringe, and finally washed with acetonitrile and dried

Rajur et al.

under a stream of argon. The modified sequence was deprotected (concentrated ammonia for 6 h at 50 °C), isolated on a semipreparative column using buffer A, evaporated, and stored at -20 °C in ddH2O. Preparation of ASGP-Antisense Conjugates 6 (ASGP-S-S-AS). The trityl group present on the C6 linker of antisense was deprotected according to standard protocols (Figure 1). In summary, the trityl on antisense 1 (216.3 mM, 36.3 OD units) was dissolved in 0.1 M TEAA (360 mL), 1 M silver nitrate (54 mL) was added, and the mixture was vortexed and allowed to react for 30 min. Next, 1 M DTT (84 mL) was added, and the reaction mixture was vortexed and allowed to stand for an additional 20 min. The yellow mixture was microfuged for 5 min and the supernatant collected. The precipitated silver salt was washed twice with 0.1 M TEAA and microfuged, and the supernatants were pooled and stored under argon atmosphere. Excess DTT was then removed by extractions with 2 mL of ethyl acetate (saturated with water and outgassed by bubbling of nitrogen). The resulting oligonucleotide 2 containing a free sulfhydryl group was immediately added to the ASGP-PDP conjugate 3 (36.05 nM, 42.7 mL) in PBS (pH 7.4). The reaction was conducted for 12 h at 20 °C under nitrogen. The disulfide exchange reaction (conjugation) was monitored by an increase in optical absorbance at 343 nm due to 2-PT. The molecular conjugates thus obtained were dialyzed against PBS (4 × 2 L) through a membrane with 12 000-14 000 MW exclusion limits. The purity of the conjugates was assessed by running analytical aliquots over the size exclusion column (TSK 3000) with buffer C. The amounts of oligonucleotide and protein coligo and cASGP-SPDP in the purified product were determined spectroscopically from the absorbances of the conjugates at 215 and 260 nm, after measuring that of ASGP-SPDP and oligonucleotide individually. That is, the equations conj ASGP-SPDP ASGP-SPDP oligo A215nm ) 215nm c + 215nm coligo

(1)

conj ASGP-SPDP ASGP-SPDP oligo c + 260nm coligo ) 260nm A260nm

(2)

and

(where A represents the solvent-corrected absorbance of the indicated compound at the indicated wavelength and  represents the extinction coefficient of the compound) were solved simultaneously for the concentrations of ASGP-SPDP and oligonucleotide. RESULTS

Synthesis of Compounds. We desired to construct a molecular conjugate of ASGP and antisense oligonucleotides that provides for efficient, tissue-selective cellular uptake and subsequently for release of the oligonucleotide from ASGP for hybridization to its target. For these reasons, we employed SPDP as a heterobifunctional cross-linker that provides a spacer arm that should provide the conjugate sufficient conformational flexibility for the terminal galactose residues to bind to the ASGP receptor and a disulfide linkage that should be reduced in the intracellular endosome (Feener et al., 1990). We employed an antisense oligonucleotide that we have previously found effective in inhibiting the response of HepG2 cells to the cytokine interleukin 6 (IL-6) (Roth et al., 1997). First, the oligonucleotide was synthesized using standard solid phase synthesis by incorporating the tritylprotected C6-thiol linker, 6-(tritylthio)hexanol/2-cyano-

Asialoglycoprotein−Antisense Conjugates

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Figure 1. Preparation of ASGP-antisense molecular conjugates (ASGP-S-S-AS). Asialoglycoprotein (R1-acid glycoprotein previously treated with neuraminidase) was reacted with sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) to produce ASGP-PDP 3. Antisense oligonucleotide carrying a six-carbon (C6) thiol modifier on its 5′ end 1 was synthesized by utilizing 6-(tritylthio)hexanol/2-cyanoethyl N,N-diisopropylphosphoramidite. The trityl protecting group of the antisense was deprotected with AgNO3/DTT to form 2, which was immediately reacted with ASGP-PDP conjugate 3 to obtain the ASGP-S-S-AS molecular conjugate 4.

Figure 2. RP-HPLC analysis of the modified antisense oligonucleotide 1 and the deprotected derivative 2. Analytical aliquots of 1 and 2 were analyzed by using a 250 × 4.6 mm reversed-phase column of ODS-Hypersil at a flow rate of 1 mL/ min in buffer B 20 mM potassium phosphate (pH 5.5) with gradient of methanol (0-100% over 60 min).

ethyl N,N-diisopropylphosphoramidite, at the 5′ end of the sequence. Reversed-phase HPLC analysis of the protected C6-thiol-modified oligonucleotide demonstrated an increased retention time due to the presence of the hydrophobic trityl group and the six-carbon aliphatic chain (Figure 2). The trityl group was removed by treatment with silver nitrate, and the product was stored under nitrogen in a solution containing DTT. The deprotection was further confirmed by reversed-phase HPLC. As shown in Figure 2, the trityl-deprotected sequence 2 eluted before its precursor 1.

The oligonucleotide 2 with a free thiol group was coupled to ASGP bearing pyridyldithiohexanoyl groups introduced by using sulfo-LC-SPDP (Figure 1). The 5′thiol function reacted with ASGP-PDP 3 bearing dithiopyridinyl groups, leading to conjugates with about six antisense oligonucleotides per ASGP molecule. Conjugation of oligonucleotide to ASGP was monitored by a combination of size exclusion chromatography (SEC) and UV-vis absorption spectroscopy. The purity of the conjugate and the nature of the linkage between ASGP and antisense were evaluated by SEC before and after reduction with DTT. Due to their significant size difference, ASGP and free oligonucleotide are easily distinguished by SEC (Figure 3A,B). This difference was used in the characterization of conjugates. A chromatogram of the crude product mixture before purification showed three peaks at approximately 9.4, 11.6, and 13.8 min (Figure 3C), corresponding to conjugate, free oligonucleotide, and low molecular weight species (probably 2-PT), respectively. After dialysis (12 000-14 000 MW cutoff), only the conjugate ASGP-S-S-AS remained (Figure 3D), eluting with a retention time roughly equal to that of free ASGP. At this point, UV-vis spectroscopy was performed as described below. Upon treatment of the ASGP-S-S-AS with DTT, a new peak eluting at a retention time of 11.6 min, due to free oligonucleotide, was observed (Figure 3E). This clearly shows that the antisense was linked to ASGP by a disulfide bridge sensitive to DTT.

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Figure 4. UV-vis spectra of ASGP-PDP (3), unconjugated oligonucleotide (AS), and ASGP-S-S-AS (4). UV-vis absorption spectra in which the conjugate ASGP-S-S-AS 4 exhibits spectral features of both AS and ASGP-PDP 3.

Figure 3. HPLC analysis. Samples were analyzed by size exclusion chromatography on a TSK 3000 column (buffer C): (A) ASGP alone; (B) antisense oligonucleotide (AS130) alone; (C) crude product mixture showing ASGP-S-S-AS 4 (retention time ) 9.4 min), unconjugated oligonucleotide (11.6 min), and low molecular weight species (13.7 min); (D) purified conjugate following dialysis of the crude reaction mixture; (E) purified conjugate after 4 h of reduction with DTT.

Because size exclusion HPLC is not sensitive to chemical composition or to minor differences in molecular weight, we performed absorption spectroscopy to ensure that our purified high molecular weight conjugate indeed contained oligonucleotide. Furthermore, spectroscopy presents a means for quantitation. Figure 4 shows the UV-vis absorption spectra of ASGP-PDP 3, the 15-mer antisense oligonucleotide (AS130) corresponding to compound 2 (compound 2 itself is not stable in water and could not be tested directly), and purified ASGP-S-SAS 4. The conjugate possesses strong absorption at 260 nm due to the presence of oligonucleotide. Furthermore, the presence of protein is indicated by the wavelength shift and increased magnitude of the peak around 210 nm relative to those for oligonucleotide alone. Because of the distinct nature of the spectroscopic transitions for each species, these spectra can be used to quantitate the loading of oligonucleotide per ASGP. The extinction coefficients of ASGP-SPDP and of unconjugated oligonucleotides were determined at 215 and 260 nm. Assuming that the contributions of each molecule to the conjugate add linearly, the absorbances of the purified conjugate at 215 and 260 nm then allow determination of the amount of protein and oligonucleotide contained within them (eqs 1 and 2). We confirmed the amount of protein determined using this method by BCA protein assay. The stoichiometries determined with each method were the samesabout six oligonucleotides per ASGP. The batch to batch variation was small; in seven independent batches of conjugates prepared over an 8 month period, the average ratio of oligonucleotide to ASGP was 5.9 ( 1.0, with a maximum of 7.5 and a minimum of 4.6. In Vitro Evaluation. We tested the bioactivity of these conjugates using a system that has been the focus of recent investigation in our laboratory (Roth et al., 1997). The synthesized antisense oligonucleotide (AS130) used in our conjugates is complementary to the mRNA encoding gp130, a signal transduction protein central to the response of cells to the inflammatory cytokine, IL-6. Stimulation of HepG2 cells results in the up-regulation of a number of acute phase proteins, including haptoglobin. These acute phase proteins are secreted by hepatocytes into their external environment. We have previously shown that AS130 substantially inhibits the upregulation of haptoglobin in HepG2 cells (Roth et al., 1997). To test these new conjugates, HepG2 cells were treated with various concentrations of the ASGP-S-S-AS conjugates (with a stoichiometry of six oligonucleotides per ASGP) for 8 h, after which time the cells were stimulated with IL-6 (5 ng/mL) for 24 h. The medium

Asialoglycoprotein−Antisense Conjugates

Figure 5. Inhibition of IL-6-mediated acute phase protein production by ASGP-S-S-AS targeted antisense (4). Confluent monolayers of HepG2 cells were incubated with various concentrations of unconjugated antisense oligonucleotide (AS), antisense oligonucleotide with 5 µM ASGP-PLL (ASGP-PLL + AS), covalent antisense conjugate (ASGP-S-S-AS), or covalent nonsense conjugate (ASGP-S-S-NS). After this time, the cells were stimulated with IL-6 for 24 h, after which time the medium was collected and assayed for haptoglobin by ELISA. Results are normalized to control cells that received neither IL-6 nor other treatment.

was harvested, and the haptoglobin secreted over the 24 h time period was quantitated by an ELISA (Roth et al., 1997). The conjugates inhibited the up-regulation of haptoglobin in a dose-dependent manner (Figure 5). Cells receiving no treatment other than IL-6 secreted ∼5 times as much haptoglobin as basal (unstimulated with IL-6) cells. The cells treated with antisense conjugatess either the complex of ASGP-PLL or the covalent conjugate ASGP-S-S-ASsexhibited less up-regulation of haptoglobin than those that received no oligonucleotide treatment. The sequence of oligonucleotide is important, as conjugates containing a nonsense sequence (same base order as AS130 in scrambled order) demonstrated no inhibition. Furthermore, the presence of a conjugate is required, as equivalent amounts of oligonucleotide to those contained in the conjugates effected no inhibition in the unconjugated form. Therefore, the conjugation to ASGP produced an effective antisense oligonucleotide that inhibits the response to IL-6 in a sequence-specific manner. DISCUSSION

A number of researchers have recognized the importance of targeted delivery to increase the effectiveness of antisense oligonucleotides. For in vivo applications, the delivery of antisense molecules to the target organ is likely to be a critical factor in their effectiveness. A molecular conjugate developed for the delivery of both oligonucleotides and plasmid DNA to hepatocytes is the asialoglycoprotein-poly(L-lysine) (ASGP-PLL) molecule, which provides a ligand (ASGP) for a liver-specific receptor (the asialoglycoprotein receptor) and a positively charged moiety (PLL), which is intended to condense the polyanionic DNA and overcome its electrostatic repulsion from negatively charged cell membranes (Wu and Wu, 1992; Bunnell et al., 1992; Lu et al., 1994). These conjugates have proven biologically effective in several in vitro systems and have provided enhanced uptake of oligonucleotides by the liver in vivo (Wu and Wu, 1992; Bunnell et al., 1992; Lu et al., 1994; Roth et al., 1997). Although the ASGP-PLL delivery system has proven effective for in vitro studies, it has several drawbacks. For example, polylysine itself has potent toxicity. There-

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fore, the quantity of ASGP-PLL that can be used is constrained. Low concentrations of ASGP-PLL were ineffective at condensing DNA and producing biological effect in our assay with IL-6-stimulated HepG2 cells. Moreover, we have found that higher doses of ASGPPLLs∼25 µMscause nonspecific reductions in protein secretion independent of the effects of oligonucleotides (Rajur et al., unpublished data). The ASGP-PLL/oligonucleotide complexes, held together by noncovalent electrostatic interactions, are unstable due to the instability of the electrostatic interaction between the carrier system and the oligonucleotides in ionic media such as blood (Lu et al., 1994). Furthermore, in these ionic conjugates, we have little control over the number of antisense molecules complexed with the carrier system and/or delivered to the target site because it is dictated by a physical equilibrium between the cationic ASGP-PLL and the anionic oligonucleotide. To increase the quantity of complexed oligonucleotide, a much higher quantity of ASGP-PLL is required, as this equilibrium requires ∼10-20 ASGP-PLL molecules per single oligonucleotide (Bunnell et al., 1992; Rajur et al., unpublished data). However, ASGP-PLL that is not complexed with oligonucleotide will compete with the complexed ASGP-PLL for binding sites (Kato et al., 1996). To overcome these difficulties, we have developed a method in which we have eliminated the use of polylysine and covalently conjugated antisense oligonucleotides to asialoglycoprotein by a stable disulfide linkage. The advantage of this method is that the disulfide linkage is stable in the extracellular oxidizing environments typically found in vitro and in vivo and has the added advantage of undergoing reduction when it reaches the intracellular environment, permitting the release of free antisense within the cytoplasm (Feener et al., 1990). Approximately six oligonucleotides were conjugated per ASGP molecule using the quantities of reagents described here. This is somewhat greater than the calculated number of PDP groups attached to each ASGP (∼4). However, the latter estimate is inexact since it is based on low relative absorbance values (A343 ∼0.02) and a published extinction coefficient (8080 M-1 cm-1; Carlsson et al., 1978) that may depend on the exact buffer conditions. An alternative explanation is that some oligonucleotide binds noncovalently to ASGP. Repeated dialysis against PBS buffer using a 12 000-14 000 MW cutoff membrane did not change the loading ratio of oligonucleotides onto ASGP, so any noncovalent binding was irreversible. In our experiments, we demonstrated that these conjugates are active in vitro, increasing the efficiency of antisense oligonucleotides at inhibiting the acute phase response in a cell culture model. The key advantages of the covalent conjugate relative to ASGP-PLL conjugates are the ability to alter loading via reaction stoichiometry and the inherent stability of the covalent linkage. Furthermore, the amount of ASGP required to target was reduced using the covalent chemistry. Using ASGP-PLL physically complexed to antisense, 5 µM was required to target the 10 µM oligonucleotide sufficiently to produce an effective dose. With the covalent conjugation, ∼1.6 µM of ASGP was required due to the high loading of oligonucleotide per ASGP (approximately 6:1). It is noteworthy that neither conjugate was able to provide 100% inhibition, even at higher and repeated doses than those shown here. There are several possible reasons for this behavior, two of which are most plausible. First, the relationship between bound receptors and biological response is nonlinear, and examples are known where only a small fraction of receptors bound results in

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maximal biological response (Lodish et al., 1995). Therefore, it is possible that the majority of gp130 (which is a secondary, high-affinity receptor for IL-6, associating with IL-6/gp80 complexes) molecules can be downregulated before any functional inhibition is observed, and significant levels of haptoglobin up-regulation may be observed even with a small fraction of normal expression of gp130. The second factor is that the oligonucleotides are likely degraded by nucleases during the time course of the experiment, and this may allow sufficient time for the down-regulated gp130 to be newly synthesized, bound, and induce production and secretion of haptoglobin. A similar approach to ours was developed by Bonfils et al. (1992) for the delivery of oligonucleotides to cells via a mannosylated BSA ligand that binds to membrane lectins. The oligonucleotides were synthesized on a 3′disulfide derivatized support and conjugated to bovine serum albumin that had been previously 6-phosphomannosylated (to provide a lectin binding ligand) and derivatized with SPDP. Increased cellular uptake of the conjugates was observed in both macrophages and baby hamster kidney cells as compared to unconjugated oligonucleotide. These conjugates were also found to be stable in culture medium. However, the ability of the conjugated oligonucleotides to act as antisense molecules was not investigated. Our results demonstrate the following: (1) the generalizability of disulfide proteinoligonucleotides to another ligand and cell type and to conjugation via the 5′ end of the oligonucleotide; (2) the ability to achieve higher loadings of oligonucleotides (5-6 as compared to 1-2) per protein molecule; and (3) biological activity of oligonucleotide conjugated in this way. These results provide a basis for optimization of oligonucleotide loading for maximum effectiveness and a stable molecule that can be tested in vivo. In summary, we have prepared a molecular conjugate of ASGP and a 15-mer antisense oligonucleotide using disulfide chemistry designed to enhance the delivery of the oligonucleotides to hepatocytes. These conjugates were found to significantly increase the effectiveness of antisense oligonucleotides in an in vitro model. This chemistry provides a means to produce stable conjugates with a desired loading of oligonucleotide and to reduce the amount of antisense and carrier required for effective delivery. Further development of these conjugates will involve investigation of their biodistribution, stability, and bioactivity in vivo. ACKNOWLEDGMENT

This work was supported by a grant from the Shriners Hospital for Children (SHCC 8610). C.M.R. was supported by a NIH postdoctoral fellowship (GM-07035). LITERATURE CITED Agrawal, S. (1996) Antisense oligonucleotides: towards clinical trials. Trends Biotechnol. 14, 376-387. Bonfils, E., Depierreux, C., Midoux, P., Thuong, N. T., Monsigny, M., and Roche, A. C. (1992) Drug targeting: synthesis and

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