INTRODUCTION

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The conceptual simplicity of antisense design, its high theoretical specificity, the affinity of the antisense oligonucleotides for their targets, the ease of chemical synthesis, and the low systemic toxicity have endowed the antisense approach with considerable therapeutic potential, especially in the treatment of diseases such as cancers and viral infections (44). Antisense oligonucleotides are particularly attractive as antiviral agents as they can be designed to block viral replication within infected cells without affecting the metabolism of the host cells (2). However, prominent among the problems that limit the success of the antisense approach are (i) nuclease sensitivity of the oligonucleotide molecules and (ii) inefficient uptake by cells. While the problem of nuclease sensitivity of normal phosphodiester (PO) oligonucleotides has been circumvented to some extent by modifying the phosphate backbone of the oligonucleotides, as for analogs such as phosphorothioate (PS) and methylphosphonate oligonucleotides, the poor cellular uptake of these molecules remains a severe limitation (17).

The PO and PS oligonucleotides cannot passively diffuse across the cell membrane because of their polyanionic nature. Thus the processes of absorptive endocytosis and fluid phase endocytosis appear to be the principal routes of entry of these molecules into the cells (28, 48). However, there are reports suggesting the presence of cell surface receptors for oligonucleotides (5, 6, 21, 24, 39, 51). The putative cell membrane proteins vary in molecular size, exhibit moderate affinities, and bind to oligonucleotides in a specific as well as nonspecific fashion. These unrelated proteins have not been characterized fully, and hence their structural and functional significance is not readily apparent. At any rate the oligonucleotide delivery to cells remains a persistent problem considering that only 1 to 2% of the oligonucleotides become cell associated when added directly to cells in culture (40).

Current approaches to overcome the problem of poor cellular uptake of oligonucleotides include microinjection of oligonucleotides (41), delivery by the calcium phosphate precipitation method (47), and the use of cationic lipids such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-tetramethylammonium methyl sulfate and cytofectin (27). Lipid-oligonucleotide conjugates (42) and cholesteryl-oligonucleotide conjugates (26), antisense molecules conjugated either to fusogenic peptides (9) or poly(L-lysine) (25), have been shown to be bioeffective at concentrations at which the free oligonucleotides are ineffective. Liposome-mediated delivery (52) and receptor-mediated delivery of antisense oligonucleotides to cells have also been reported (14, 15, 49, 50).

Targeting of antisense oligonucleotides by using endocytic receptor systems specific to a given cell type is an attractive strategy for circumventing the intrinsic problems of nonpermeability and nonspecific cellular uptake of oligonucleotides. This strategy has been employed for delivery of antisense oligonucleotides with the asialoglycoprotein receptor (50), transferrin receptor (49), and epidermal growth factor receptor (15). In most of these cases, the oligonucleotide molecules are complexed to a poly(L-lysine)-ligand conjugate for delivery to cells expressing the particular receptor. The poly(L-lysine) is conjugated to the carrier molecule through a heterobifunctional cross-linker, and the conjugate is incubated with the oligonucleotide to allow for noncovalent association of the oligonucleotide with the poly(L-lysine). The resultant coacervates of receptor-recognizable macromolecules consist of chemically heterogeneous complex mixtures which cannot be easily formulated into pharmaceutical preparations for therapeutic applications. Thus antisense delivery strategies involving direct chemical manipulation of oligonucleotides and yielding a homogeneous product should be preferred over those that generate mixtures of covalently and/or noncovalently associated molecules (17).

In this study we report a simple strategy for designing antisense oligonucleotides for macrophage targeting. We show that the presence of a short poly(G) tail at the 3' end of the antisense sequence is sufficient for scavenger receptor (SCR)-mediated targeting of oligonucleotides to macrophages. In a model system, we demonstrate the enhanced cellular uptake and bioefficacy of a vesicular stomatitis virus (VSV)-specific antisense oligonucleotide in VSV-infected J774E cells and CHO transfectants expressing SCR.

	MATERIALS AND METHODS

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Cell lines and virus. J774E, a murine monocyte/macrophage cell line, was a gift from P. Stahl, Washington University, St. Louis, Mo. The Chinese hamster ovary (CHO) transfectant PJA28.C5 expressing the type I SCR and the parent CHO cell line were obtained from M. Krieger, Massachusetts Institute of Technology. All cell lines were cultured in medium A (RPMI 1640 containing 10% fetal bovine serum [FBS] and gentamicin [50 mg/liter]) at 37°C in a 5% CO₂-95% air atmosphere. The transfectant was cultured in medium containing Geneticin (400 µg/ml) as a selection marker. VSV (Indiana strain) was a kind gift from Ranjit Ray (St. Louis University, St. Louis, Mo.). VSV was grown to high titers in J774E cells cultured in medium B (RPMI containing 2% FBS and 50 mg of gentamicin/ml).

Oligonucleotides. PS and chimeric antisense oligodeoxyribonucleotides were purchased from Biosynthesis Inc., Louisville, Tex. The PO oligonucleotides were purchased from Rama Biotechnologies, New Delhi, India. The oligonucleotide samples were radiolabeled, and their purity was determined by polyacrylamide gel electrophoresis. The commercially procured oligonucleotides were purified by ion exchange chromatography using standard procedures.

CD spectroscopy. Circular dichroism (CD) spectra were recorded on a Jasco-J710 instrument equipped with a Peltier-type constant-temperature cell holder (PTC-348W). The instrument was calibrated with (+)-10-camphorsulfonic acid. The spectra of oligonucleotides were recorded at 37°C, and data were presented as mean residue ellipticity expressed in units of degrees times centimeters squared divided by decimoles. The spectra were smoothed with the built-in algorithm of the Jasco program.

Radiolabeling. Oligonucleotides were end labeled with a T4 polynucleotide kinase kit (Promega, Madison, Wis.). The kination reaction was carried out on 10 pmol of the oligonucleotide at 37°C for 1 h in a total volume of 10 µl containing 10 U of the enzyme, 1 µl of 10× polynucleotide kinase buffer, and 30 µCi of [-³²P]ATP. The labeled oligonucleotides were purified on a DE52 cellulose matrix, and specific activity was determined.

Binding assays. Cells (0.5 × 10⁶ cells/well) were plated in six-well culture dishes in medium A and incubated for 18 h at 37°C in a humidified 5% CO₂-95% air atmosphere. The growth medium was replaced with 1 ml of ice-cold medium C (RPMI 1640 containing BSA [1 mg/ml]) containing various concentrations of ¹²⁵I-MBSA, and the cells were incubated at 4°C for 2 h. The cells were then washed three times with ice-cold PBS containing BSA (5 mg/ml), followed by three washes with ice-cold PBS to remove unbound radioactivity. The cells were then lysed in 0.1 N NaOH (1 ml per well), and cell-associated radioactivity was determined with a gamma counter.

Degradation assays. The assays were carried out as described previously (19). Briefly, cells were plated as described above. Each well received 1 ml of prewarmed medium C containing ¹²⁵I-MBSA alone or with putative competitors, and the cells were incubated at 37°C for 5 h. The supernatant (500 µl) from each well was collected, 200 µl of 50% trichloroacetic acid (TCA) was added, and the solutions were placed at 4°C overnight. The supernatants were spun at 1,000 × g for 10 min to pellet out the precipitated protein. To 400 µl of the TCA-soluble supernatant, 7 µl of 40% KI and 15 µl of 30% H₂O₂ were added, and the mixture was vortexed and kept at room temperature for 10 min. To each sample, 1 ml of chloroform was added, and the samples were vortexed and kept at room temperature for 10 min to allow for extraction of free iodine. The ¹²⁵I content in the aqueous phase was determined with a gamma counter. The TCA-soluble radioactivity obtained from the "no cell blank" was subtracted from the experimental readings to calculate cell-specific degradation, the results being expressed as nanograms of ¹²⁵I-MBSA degraded per milligram of cell protein.

Uptake assays. Cells were plated as described above. Each well received 1 ml of medium C containing [-³²P]ATP-labeled oligonucleotide alone or with putative competitors, and the cells were incubated for different periods of time. After this, the cells were processed as described for binding assays and cell-associated radioactivity was determined.

Virus replication inhibition assay. Cells (0.125 × 10⁶ cells/well) were plated in a 24-well culture plate and were incubated at 37°C for 6 to 8 h. The cells were washed in serum-free RPMI 1640 and incubated with the desired concentration of oligonucleotide in 250 µl of serum-free RPMI 1640 at 37°C for 4 h. The oligonucleotide-containing medium was then removed, and the cells were infected with VSV at a multiplicity of infection (MOI) of 0.5 for 30 min in 300 µl of serum-free RPMI 1640. The cells were then washed three times with serum-free RPMI 1640, and the oligonucleotide-containing medium was readded along with 50 µl of 10% FBS. The cells were now incubated at 37°C for 10 h. The cell supernatants were collected, and viral titers were determined by plaque assays. The assays were carried out on CHO cell monolayers by the agar overlay method. Plates were flushed with 0.07% neutral red solution to help count the plaques, and viral titers were calculated.

	RESULTS

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Design of oligonucleotides for targeted delivery to macrophages. VSV, which infects a variety of cell types including macrophages, was chosen as a candidate virus for the study. The translation initiation region of the mRNA corresponding to the N protein of VSV was selected as a target sequence since the antisense oligonucleotide sequence (5'CATTTTGATTACTGT3') directed against this site has been earlier shown to inhibit viral replication by an antisense-specific mechanism in VSV-infected L929 cells (25).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   CD spectra of poly(G) antisense oligonucleotides. The respective oligonucleotides were dissolved in water at a nucleotide residue concentration of about 60 µM, and the spectra were recorded with a 10-mm cell at 37°C. Each spectrum represents an average of five scans. ANS with 4-mer poly(G) at the 3' end (----), ANS with 7-mer poly(G) (-), and ANS with 10-mer poly(G) () are shown.

Recognition of oligonucleotide sequences by SCR. The specificity of recognition of the oligonucleotide-poly(G) constructs by SCR was examined with J774E, a murine monocyte/macrophage cell line. For this purpose, first the status of SCR on these cells was assessed. The binding of ¹²⁵I-MBSA to J774E cells at 4°C exhibited saturation kinetics with a half-maximal binding of 2 µg/ml (data not shown). The degradation of ¹²⁵I-MBSA by J774E cells in the presence or absence of different polyanionic molecules as competitors exhibited the characteristic SCR profile; cells that received 2 µg of ¹²⁵I-MBSA/ml alone degraded 333 ± 9.2 ng of ¹²⁵I-MBSA/mg of cell protein in 5 h. This value fell to 71 ± 11 ng in the presence of unlabeled MBSA (40 µg/ml), 90 ± 8 ng in the presence of fucoidan (20 µg/ml), and 33 ± 5 ng in the presence of poly(G) (2 µg/ml). Fetuin (20 µg/ml) and poly(C) (2 µg/ml) did not compete for the degradation of ¹²⁵I-MBSA. These results suggest the presence of normal SCR activity on J774E cells.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Recognition of poly(G) constructs by SCR; competition for degradation of 125I-MBSA. Cells (0.5 × 106 cells/well) were plated in six-well culture vessels and incubated for 18 h at 37°C. Each cell monolayer received 1 ml of RPMI 1640 containing BSA (1 mg/ml) with 2 µg of 125I-MBSA (specific activity: 100 cpm/ng)/ml alone or along with different competitors and was incubated at 37°C. After 5 h of incubation, the medium in each well was processed to determine the amount of 125I-MBSA degraded, as described in Materials and Methods. The molar ratio of 125I-MBSA to different oligonucleotides was 1:20. Unlabeled MBSA was used as a competitor at a concentration of 2 µg/ml. Both poly(G) and poly(C) were 10-nucleotide-long molecules. Results shown are means ± standard errors of three independent determinations.

Enhanced SCR-mediated uptake of oligonucleotides. Next we examined the uptake of oligonucleotides with or without poly(G) tails or containing poly(C) tails to assess the extent of SCR-mediated facilitated delivery to the J774E cell line. The data shown in Fig. 3A indicate that uptake of the oligonucleotide constructs with a poly(G) tail was 8- 12-fold higher than uptake of constructs without the poly(G) extension. ANS-G, cANS-G, sANS-G, and sense-G were taken up with comparable efficiencies, whereas the uptake of ANS-C was around the same level as that of ANS. This is consistent with the results of the recognition studies described earlier in that poly(G)-containing oligonucleotide sequences competed for the degradation of ¹²⁵I-MBSA to similar extents while ANS and ANS-C did not.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Enhanced uptake of poly(G) constructs by J774E cells through the SCR. (A) Cells (0.5 × 106 cells/well) were plated in six-well culture vessels and were incubated for 18 h at 37°C. Each cell monolayer received 1 ml of RPMI 1640 containing BSA (1 mg/ml), with 1 µM 32P-labeled oligonucleotide and was incubated at 37°C for the indicated periods of time. The cells were then processed as described in Materials and Methods, and cell-associated radioactivity was determined. Results are means ± standard errors of three independent determinations. (B) The assay was set up as described above. Cells received 1 µM 32P-labeled ANS-G (specific activity: 555 cpm/pmol) either alone or along with different polyanionic macromolecules. The competitors used were MBSA (500 µg/ml), fetuin (100 µg/ml), fucoidan (100 µg/ml), poly(G) (7 µg/ml), and poly(C) (7 µg/ml). After incubation for 4 h at 37°C, cell-associated radioactivity was determined. Uptake of ANS-G in the control cells was 43.63 ± 1.5 pmol/mg of cell protein. Results are expressed as means ± standard errors of three independent determinations.

Bioefficacy of the poly(G)-containing oligonucleotides in VSV-infected J774E cells. In order to test if the SCR-mediated uptake led to enhanced bioefficacy, the ability of the antisense oligonucleotide to inhibit VSV replication in SCR-bearing J774E cells was investigated. VSV infected the J774E cell line, and the virus was able to replicate in this cell line. The viral titer generated 10 h postinfection, with a MOI of 0.5, was 15 × 10⁶ PFU/ml. Cells were treated with increasing concentrations of the ANS-G before being infected with VSV at a MOI of 0.5 as described in Materials and Methods. Under these conditions significant inhibition of VSV replication was not observed up to a concentration of 11 µM ANS-G. At 15 µM, the ANS-G treatment brought about 80% inhibition of VSV replication (Fig. 4). The cANS-G treatment yielded a similar profile of inhibition of VSV replication. The antisense effects due to ANS-G and cANS-G were not further enhanced when the concentration was increased to 30 µM. In contrast, the free antisense oligonucleotide (ANS), under assay conditions similar to those employed for the ANS-G treatment, did not significantly inhibit the VSV replication up to a concentration of 30 µM. Representative data in Fig. 5A show that the control oligonucleotides, sense-G, scram-G, and ANS-C, exhibited behavior similar to that of ANS. The carrier molecule [10-mer poly(G)] itself did not interfere with the viral replication.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Dose dependence of ANS-G on inhibition of VSV replication. Cells (0.125 × 106 cells/well) were treated with various concentrations of ANS-G in 250 µl of serum-free RPMI 1640 for 4 h at 37°C. The cells were then infected with VSV at a MOI of 0.5 for 30 min after which they were given three rapid washes. The cells were incubated at 37°C for 10 h postinfection in medium containing the oligonucleotides with 2% FBS. Culture supernatants were collected, and viral titers were estimated by plaque assays of CHO cell monolayers. Results shown are means ± standard errors of three independent determinations.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Inhibition of VSV replication in J774E cells by different oligonucleotide constructs. (A) Cells (0.125 × 106 cells/well) were treated with 15 µM concentrations of oligonucleotides, and assays were carried out as described in the legend for Fig. 4. Results shown are means ± standard errors of three independent determinations. (B) Reversal of antisense effects by competing polyanionic macromolecules. The assay was performed as described above. Cells received, in addition to ANS-G, 7 µg of either poly(G) or poly(C) as competitors. The viral titer in the control group was 16 × 106 PFU/ml. Results shown are means ± standard errors of three independent determinations.

Bioefficacy in CHO transfectants. One of the enduring problems faced in antisense research has been the variability of antisense effects in different cell lines against the same target. Therefore, it was pertinent to examine the bioefficacy of the SCR-mediated delivery of antisense oligonucleotides in other SCR-bearing cell lines. CHO cells that were stably transfected with the SCR type I (PJA28.C5 cells) were chosen for this purpose. The PJA28.C5 cells bound ¹²⁵I-MBSA at 4°C with saturation kinetics (data not shown). Also, degradation of ¹²⁵I-MBSA by these cells (64 ± 1.47 ng/mg of cell protein in 5 h) was effectively competed by MBSA and poly(G) but not by poly(C) or fetuin, indicating expression of characteristic SCR activity. However, in comparison to that for the J774E cell line, the level of degradation of ¹²⁵I-MBSA in PJA28.C5 was about four- to fivefold lower.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Enhanced uptake of ANS-G by parent CHO cells and PJA28.C5 cells expressing SCR. (A) The assay was performed as described for Fig. 3A. Results shown are means ± standard errors for three independent determinations. ODN, oligonucleotide. (B) Assay was carried out as described for Fig. 3B. Cells received 1 µM 32P-labeled ANS-G (specific activity: 390 cpm/pmol) either alone or along with different competitors, viz., MBSA (500 µg/ml), poly(G) (7 µg/ml), fucoidan (100 µg/ml), poly(C) (7 µg/ml), and fetuin (100 µg/ml). Results shown are means ± standard errors of three independent determinations.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Inhibition of VSV replication in PJA28.C5 cells treated with ANS-G. The experiment was performed as described for Fig. 4. All oligonucleotides were used at a concentration of 15 µM. Results shown are means ± standard errors of three independent determinations.

	DISCUSSION

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The poor cellular uptake of oligonucleotides remains a major stumbling block in the development of antisense therapy. Although the problem has been circumvented to some extent by utilizing cationic and other lipids (27, 42) and certain receptor systems (15, 49, 50), most of these delivery systems are complicated by multistep preparation procedures yielding poorly defined heterogeneous mixtures. Here we report a simple approach to augment the antisense oligonucleotide uptake in macrophages that express SCR. We have exploited the innate tetraplex-forming propensity of poly(G) sequences and the ability of SCR to recognize this feature of the poly(G) conformation for targeting antisense oligonucleotides to macrophages. We show that oligonucleotides containing 10-mer poly(G) stretches at the 3' termini are recognized and efficiently taken up by SCR and that this receptor-mediated uptake of antisense oligonucleotides directed against the N protein mRNA of VSV leads to inhibition of VSV replication in J774E macrophage cells as well as in CHO cells transfected with SCR.

In designing the poly(G)-antisense oligonucleotide constructs for SCR-mediated targeted delivery to cells, the nuclease sensitivity of antisense molecules was kept in mind. Thus antisense molecules using PS chemistry (sANS) were synthesized. However, in standard trypan blue exclusion studies, sANS was found to be extremely toxic to cells at concentrations of 15 µM; nearly 90 to 95% of the cells became permeable to the dye after incubation with sANS for 5 h at 37°C. However, chimeric molecules (cANS) in which only the first and last internucleotide linkages were modified by PS chemistry were not toxic to cells at concentrations as high as 100 µM. Nonetheless, the poly(G) constructs of PO oligonucleotides (ANS-G) were as bioeffective as chimeric oligonucleotides (cANS-G). The ability of ANS-G to inhibit VSV replication with efficiency almost identical to that of the cANS-G is an interesting result that suggests longer intracellular survival of the poly(G)-tethered antisense oligonucleotides. The reasons for the enhancement in the stability of oligonucleotides delivered through the SCR pathway are not readily apparent. The trafficking into specific intracellular compartments, which ensures a longer half-life of the antisense molecules, may be one of the reasons. It is also likely that the quadruplex structures acquired by the poly(G) tail may offer resistance to degradation and increase the half-life of the molecule. If this be so, then the fact that we have used antisense oligonucleotides with poly(G) tails at the 3' ends may be important because the degradation of oligonucleotides is believed to occur predominantly due to 3'-exonuclease activity (37). It would also mean that using poly(G) as a carrier for PO antisense oligonucleotides may increase their stability due to the intrinsic properties of the carrier molecule. These results are important in the light of several recent reports questioning the use of PS oligonucleotides (43), which are now known to have serious drawbacks which include non-sequence-specific effects (37, 38), inhibition of important cellular enzymes such as human DNA polymerases and RNase H (16), and binding to several cytosolic proteins leading to cellular toxicity (10, 21).

The optimum concentration (15 µM) required to achieve 80% inhibition of VSV replication in J774E-infected cells compares favorably with the previously published data. Agris et al. (3) used methylphosphonate oligonucleotides at 150 µM for inhibiting VSV replication in L929 cells, while 50 µM was required for in vitro specific inhibition of hepatitis B viral gene expression by asialoglycoprotein-mediated delivery of antisense compounds to virus-infected HepG2 cells that possess the asialoglycoprotein receptors (50). Thus the results of the present study are significant in the light of the above observations. However, oligonucleotides are known to effect the inhibition of replication of certain viruses even at nanomolar concentrations. The use of a relatively higher oligonucleotide concentration for VSV in the present study may be related to the intrinsic high growth rate of this virus. In a recent study, Tackas and Banerjee (45) could demonstrate the inhibition of VSV in cells constitutively expressing an antisense RNA targeted against the virus L protein gene only at a very low MOI of 0.01 to 0.1 and concluded that the "robust growth rate of VSV eventually overwhelms the available antisense RNA and leads to delayed cell death."

MBSA, a ligand of the SCR, has been used to target a variety of chemotherapeutic agents (14, 30-34) to macrophage cells. Chaudhury (14) has used liposome coated with MBSA to deliver antisense oligonucleotides to macrophage cells. We have also noted SCR-mediated enhanced uptake of oligonucleotides conjugated to MBSA by macrophages (data not shown). However, the immunogenicity of maleylated proteins (1) and the multistep, complex procedure for preparation of conjugates of an antisense oligonucleotide with the maleylated protein complicate its utility as a carrier of oligonucleotides to macrophages. In contrast, poly(G) molecules are not likely to induce an immune response and using poly(G) as a carrier eliminates the need for complex conjugation procedures because the addition of a poly(G) tail to the antisense sequence is very much a part of the solid-phase synthesis of oligonucleotides. However, there have been reports suggesting that the presence of G tetrads comprising both PS and PO oligonucleotides does cause sequence-independent effects (43); some examples include inhibition of human immunodeficiency virus (35, 46) and the biological activity of RelA (7) and c-myb (12). Binding of G tetrads to fibroblast growth factor has also been reported (21). Nonetheless, we did not observe any effect of 10-mer poly(G) on cell proliferation and viability up to concentrations of 100 µM. Poly(G) also did not elicit antiviral activity in VSV-infected J774E cells. However, VSV replication was marginally inhibited in CHO transfectants, suggesting that the antiviral activity of poly(G) might be intrinsic to the nature of the cell lines.

In some recent studies, SCR has been suggested to mediate oligonucleotide binding. SCR present on endothelial liver cells are implicated in liver uptake of oligonucleotides (8). Also Kimura et al. (24) reported the blockage of oligomer induction of interferon production on NK cells by ligands of SCR and suggested that SCR bound to oligonucleotides. However, these results are not in agreement with the findings of Benimetskaya et al. (7), who showed that binding of oligonucleotides to macrophages and microglia cells was unaffected by ligands of the SCR. The findings of Benimetskaya et al. are consistent with our results in that only those oligonucleotides that are compatible with the SCR ligand specificity are recognized. Furthermore, SCR recognition of poly(G) constructs and bioefficacy are consistent with the results of Chaudhury (14), who showed enhanced efficacy of SCR-mediated delivery of antileishmanial oligonucleotides encapsulated in MBSA-coated liposome.

Macrophages are an important component of the cellular arm of the immune system and are infected by many viral pathogens including human immunodeficiency virus and dengue virus. The idea of targeting oligonucleotides specifically to macrophages is, therefore, of considerable therapeutic significance. The SCR system lends itself admirably to this purpose, as the SCR receptors are predominantly expressed on cells of the macrophage lineage. Further, the levels of expression are high and the receptors are not down regulated on association with ligands and are recycled rapidly back to the surface after internalization (4). The SCR system has been used extensively for the targeted delivery of a variety of chemotherapeutic agents to macrophages to eliminate intracellular infectious agents as well as certain forms of cancer involving macrophage lineage cells both in vitro and in vivo (30-34). The present studies describing a simple strategy of SCR-mediated targeting of antisense oligonucleotides to macrophages is likely to facilitate the antisense-mediated therapy of macrophage-related intracellular infections of viral, bacterial, or protozoal etiology and would also serve as a facile way for modulating or controlling certain metabolic responses of macrophages that might have therapeutic implications. Furthermore, it would be of interest to explore if poly(G)-mediated targeting could be adapted for delivery of genes to SCR-bearing cells.