Cell-type specific and ligand specific enhancement of cellular uptake of oligodeoxynucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3.

A novel, structurally defined, and homogeneous oligodeoxynucleoside methylphosphonate (oligo-MP) neoglycopeptide conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, has been synthesized. The linkage between the carbohydrate ligand and the oligo-MP is a metabolically stable thioether. Experiments establish that uptake of this conjugate by human hepatocellular carcinoma (Hep G2) is cell-type specific when compared with its uptake by human fibrosarcoma (HT 1080) and human promyleocytic leukemia (HL-60). Uptake of the conjugate with Hep G2 cells can be totally inhibited by the addition of a 100-fold excess of free YEE(ah-GalNAc)3 in the culture medium indicating the observed cell uptake is ligand specific. The conjugate is rapidly taken in by Hep G2 cells in a linear fashion reaching a saturation plateau of 26 pmol per 10(6) cells after 24 h. Conjugation of oligo-MPs to ligands for hepatic carbohydrate receptors, such as YEE(ah-GalNAc)3, represents an efficient and ligand-specific method for the intracellular delivery of oligo-MPs.

Cellular uptake of oligodeoxyribonucleoside methylphosphonates.

The cellular uptake of oligodeoxyribonucleoside methylphosphonates has been evaluated using three radiolabeled oligomers. Oligomers I and II ([3H]-T8 and [3H]-T16, respectively) are nonionic methylphosphonate oligomers labeled with tritium on the phosphonate internucleotide linkage. EDA-III contains a single phosphodiester linkage, a [32P]-label and an ethylenediamine conjugate at the [32P]-5'-end. All three oligomers are stable in cells. At a 1 microM concentration, oligomer I is not taken up by human erythrocytes. The octanol/DPBS partition coefficients for oligomers I and II (1.5 x 10(-4) and 4.2 x 10(-4), respectively) further indicate that these molecules should not diffuse across cell membranes at appreciable rates. Oligomer I is taken up by HL-60 cells, although at a slower rate than the uptake of the fluid-phase marker sucrose. The cell-associated levels of oligomer II in K-562 cells following incubation of cells with the oligomer for 2 days is independent of concentration and nonsaturable, suggesting a mechanism of uptake independent of receptor. Finally, the initial uptake rate of EDA-III in mouse L cells is greater than the uptake of two oligodeoxyribonucleotides (T8, T16), reaching a plateau after 3 hours incubation with cells. These observations should aid in the elucidation of the mechanism by which this class of antisense agents enters the intracellular environment.

Interactions of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with vesicular stomatitis virus messenger RNA.

The ability of oligonucleotides to interact selectively with their targets is an important consideration in the design of antisense oligonucleotides. This is especially important in the case of antisense oligomers, such as psoralen-derivatized oligomers, which can irreversibly bind to their targets. We have studied the interactions of a series of psoralen-derivatized antisense oligonucleoside methylphosphonates with the mRNAs of vesicular stomatitis virus (VSV), mRNAs that have a high degree of sequence homology. Cross-linking reactions were carried out under conditions of low ionic strength in order to reduce mRNA secondary structure. A 12-mer, whose sequence was complementary to VSV M-mRNA and partially complementary to sequences found in N, NS, and G mRNA cross-linked extensively to N-message. On the other hand, 16-mers whose sequences were uniquely complementary to binding sites on N- or M-mRNA specifically and efficiently cross-linked to their targeted mRNAs over the temperature range 0 degree to 37 degrees C. A reverse transcriptase-catalyzed primer extension assay was used to show that one of the N-specific oligomers cross-linked at the expected site on N-mRNA and to estimate the extent of cross-linking. The results demonstrate that psoralen-derivatized oligonucleoside methylphosphonates can cross-link in a sequence-specific manner if the sequences of these oligomers are chosen carefully so as to avoid extensive partial complementarity with other mRNA sequences.

Properties of exonuclease-resistant, psoralen-conjugated oligodeoxyribonucleotides in vitro and in cell culture.

We have prepared oligodeoxyribonucleotides that are modified at the 3'-terminal with N4-(4-aminobutyl)deoxycytidine and derivatized at the 5'-end with a 4'-([N-(aminoethyl)amino]methyl)-4,5',8-trimethylpsoralen, (ae)AMT, and whose sequences are complementary to vesicular stomatitis virus (VSV), N-protein mRNA, (ae)AMT-II, or VSV M-protein mRNA, (ae)AMT-III. (ae)AMT-II cross-links exclusively to VSV N-mRNA when a mixture of the oligomer and poly(A+) RNA from VSV-infected cells is irradiated in vitro with long wavelength UV light at either 20 degrees or 37 degrees C. N4-(4-Aminobutyl)deoxycytidine at the 3'-end of (ae)AMT-II does not appear to affect the binding or cross-linking of the oligomer to its target RNA. Oligomer (ae)AMT-II is completely resistant to hydrolysis by the 3'-5'-exonuclease activity found in fetal calf serum whereas a similar oligomer, (ae)AMT-I, which contains a 3'-terminal deoxycytidine, is hydrolyzed within 30 min when incubated at 37 degrees C. Intact (ae)AMT-II was found in both the cell lysate and cell culture medium after 12 hr of incubation with mouse L-cells along with d-(ae)AMTpT, which appears to result from endonuclease degradation of the oligomer. In contrast no intact (ae)AMT-I was found in either the cell lysate or the culture medium after 1 hr incubation. Although 10 microM (ae)AMT-II had no effect on VSV-protein synthesis in either unirradiated or UV-irradiated VSV-infected mouse L-cells, 10 microM (ae)AMT-III inhibited VSV protein synthesis 30% in irradiated cells. These results show that introduction of a N4-(4-aminobutyl)deoxycytidine at the 3'-end of an oligodeoxyribonucleotide significantly increases the resistance of the oligomer to degradation by 3'-5'-exonucleases but does not interfere with its ability to bind selectively to complementary RNA. Further derivatization with psoralen creates an oligomer that can be triggered to cross-link with RNA in a sequence-specific manner, is taken up intact by mammalian cells in culture, and exhibits biological activity. In combination, these two modifications endow the oligodeoxyribonucleotide with novel properties that could be exploited in the design of antisense or antigene reagents for use in controlling gene expression in mammalian cells.