Proof Of Mechanism

3.1 Factors That May Influence Experimental Interpretations

Clearly, the ultimate biological effect of an oli-gonucleotide will be influenced by the local concentration of the oligonucleotide at the target RNA, the concentration of the RNA, the rates of synthesis and degradation of the RNA, type of terminating mechanism, and the rates of the events that result in termination of the RNA's activity. At present, we understand essentially nothing about the interplay of these factors.

3.1.1 Oligonucleotide Purity. Currently, phosphorothioate oligonucleotides can be pre-

pared consistently and with excellent purity

(79). However, this has been the case for only the past several years. Before that time, synthetic methods were evolving and analytical methods were inadequate. In fact, our laboratory reported that different synthetic and purification procedures resulted in oligonucleo-tides that varied in cellular toxicity (72) and that potency varied from batch to batch. Although there are no longer synthetic problems with phosphorothioates, undoubtedly they complicated earlier studies. More important, with each new analog class, new synthetic, purification, and analytical challenges are encountered.

3.1.2 Oligonucleotide Structure. Antisense oligonucleotides are designed to be single stranded. We now understand that certain sequences (e.g., stretches of guanosine residues) are prone to adopt more complex structures

(80).The potential to form secondary and tertiary structures also varies as a function of the chemical class. For example, higher affinity 2'-modified oligonucleotides have a greater tendency to self-hybridize, resulting in more stable oligonucleotide duplexes than would be expected based on rules derived from work with oligodeoxynucleotides (Freier, unpublished results, 1990).

3.1.3 RNA Structure. RNA is structured. The structure of the RNA has a profound influence on the affinity of the oligonucleotide and on the rate of binding of the oligonucleotide to its RNA target (81,82). Moreover, RNA structure produces asymmetrical binding sites that then result in very divergent affinity constants, depending on the position of oligonucleotide in that structure (82-84). This in turn influences the optimal length of an oligonucleotide needed to achieve maximal affinity because in structured RNA the optimal affinity is determined by the difference between the binding energies required for a nucleotide to invade a duplex and those gained per nucleotide by binding of the oligonucleotide. Furthermore, this is only a fraction of the story, given the numerous proteins that interact with RNA that undoubtedly influence binding, and very little is understood about these ternary interactions.

3.1.4 Variations in In Vitro Cellular Uptake and Distribution. Studies in several laboratories have clearly demonstrated that cells in tissue culture may take up phosphorothioate oligonucleotides through an active process, and that the uptake of these oligonucleotides is highly variable, depending on many conditions (72, 85). Cell type has a dramatic effect on total uptake, kinetics of uptake, and pattern of subcellular distribution. At present, there is no unifying hypothesis to explain these differences. Tissue culture conditions, such as the type of medium, degree of confluence, and the presence of serum, can all have enormous effects on uptake (85). The oligonu-cleotide chemical class obviously influences the characteristics of uptake as well as the mechanism of uptake. Within the phosphoro-thioate class of oligonucleotides, uptake varies as a function of length, but not linearly. Uptake varies as a function of sequence, and stability in cells is also influenced by sequence (85, 86).

Given the foregoing, it is obvious that conclusions about in uitro uptake must be very carefully made and generalizations are virtually impossible. Thus, before an oligonucleotide could be said to be inactive in vitro, it should be studied in several cell lines. Furthermore, although it may be absolutely correct that receptor-mediated endocytosis is a mechanism of uptake of phosphorothioate oli-gonucleotides (87), it is obvious that a generalization that all phosphorothioates are taken up by all cells in uitro primarily by receptor-mediated endocytosis is simply unwarranted.

Finally, extrapolations from in uitro uptake studies to predictions about in uiuo pharmaco-kinetic behavior are entirely inappropriate and, in fact, there are now several lines of evidence in animals and humans that, even after careful consideration of all in uitro uptake data, one cannot predict in vivo pharmacokinetics of the compounds (85, 88-90).

3.1.5 Binding and Effects of Binding to Non-nucleic Acid Targets. Phosphorothioate oligonucleotides tend to bind to many proteins and those interactions are influenced by many factors. The effects of binding can influence cell uptake, distribution, metabolism, and excretion. They may induce non-antisense effects that can be mistakenly interpreted as antisense or complicate the evaluation of whether the pharmaceutical effect is the consequence of an antisense mechanism. By inhibiting RNase H, protein binding may inhibit the antisense activity of some oligonucleo-tides. Finally, binding to proteins can certainly have toxicological consequences.

Oligonucleotides may interact not only with proteins but also with other biological molecules, such as lipids or carbohydrates, and such interactions like those with proteins will be influenced by the chemical class of oli-gonucleotide studied. Unfortunately, essentially no data bearing on such interactions are currently available.

An especially complicated experimental situation is encountered in many in vitro antiviral assays. In these assays, high concentrations of drugs, viruses, and cells are often coincubated. The sensitivity of each virus to non-antisense effects of oligonucleotides varies, depending on the nature of the virion proteins and the characteristics of the oligonucleotides (91, 92). This has resulted in considerable confusion. In particular, for HIV, herpes simplex viruses, cytomegaloviruses, and influenza virus, the non-antisense effects have been so dominant that identifying oligonucle-otides that work through an antisense mechanism has been difficult. Given the artificial character of such assays, it is difficult to know whether non-antisense mechanisms would be as dominant in vivo or result in antiviral activity.

3.1.6 TerminatingMechanisms. It has been amply demonstrated that oligonucleotides may employ several terminating mechanisms. The predominant terminating mechanism is influenced by RNA receptor site, oligonucleo-tide chemical class, cell type, and probably many other factors (93). Obviously, because variations in terminating mechanism may result in significant changes in antisense potency and studies have shown significant variations from cell type to cell type in vitro, it is essential that the terminating mechanism be well understood. Unfortunately, at present, our understanding of terminating mechanisms remains rudimentary.

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