Rhys D. Fogarty, Sandra C. McKean, Paul J. White, Lynne M. Atley, George A. Werther, Christopher J. Wraight. 2002. Sequence Dependence of C5-Propynyl-dU ...
ANTISENSE RESEARCH AND DEVELOPMENT 4:67-69 Mary Ann Liebert, Inc., Publishers
(1994)
Editorial Problems in Interpretation of Data Derived from in Vitro and in Vivo Use of Antisense Oligodeoxynucleotides
Thepression (for
polyanions include rsCD4 (Yakubov et al., 1993), HIV-I transcriptase (RT; Majumdar et al., 1989), gpl20 (Stein et al., 1993), and the protein kinase C (PKC) , ß1? and isoforms (Rideout et al., 1994). In addition, PS oligomers have been shown to activate a nuclear transcription factor (Sp-1 like) in a sequence-independent manner. This induction was similar to that obtained by treatment with the polyanion suramin (Perez
oligodeoxynucleotides to inhibit genetic ex¬ sequence-specific manner has been well doc¬
fated
ability of
in
a
reverse
a review, see Stein and Cheng, 1993). Because of umented their potential for exquisite specificity, oligodeoxynucleotides (oligos) have been proposed as therapeutic agents for a variety of human diseases, including cancer, viral infections, and con¬ nective tissue disorders. However, it has long been recognized that phosphodiester (PO) oligos will not be suitable as thera¬ peutic agents because of their rapid rate of digestion by nucleases (Wickstrom, 1986; Dagle et al., 1991). This has driven the development of oligos with modified backbones, such as the nuclease-resistant phosphorothioate (PS) oligos, which are now easily synthesized and in common use (for a review, see Stein et al., 1991). However, there are several crucial problems with the in vitro and in vivo use of PO, PS, or other charged oligos as antisense agents that we believe have not been sufficiently ad¬ dressed. This editorial seeks to define these problems, as well as to present a statement of the policy of this journal with re¬ spect to the interpretation of data obtained in antisense experi¬ ments. We believe that several critical points must be consid¬ ered.
et al., 1994). There can be little doubt that this short list repre¬ sents a mere fraction of the total number of cellular proteins that can interact with PS oligos. These interactions are not observed to the same extent with PO oligos, possibly either because the dissociation constants are several orders of magnitude higher for PO vs. PS oligos, or, when this is not the case, because the
off-rate of the PO oligo from the protein is relatively fast. However, the interactions that give rise to either of these two scenarios are not well understood on a molecular level. 2. Phosphorothioate oligos may exhibit nonantisense effects that may be sequence selective. A nonantisense effect of PS oli¬ gos that is increasingly recognized as creating problems with data interpretation involves oligos that contain four contiguous guanosine residues (the G-quartet). Oligos that contain the Gquartet may be antiproliferative in a sequence-independent manner (Yaswen et al., 1992). This means that if the control (sense or scrambled) oligo does not contain the G-quartet se¬ quence, artifactual "antisense" efficacy may be observed. However, it should not be concluded, ipso facto, that all oligos with four contiguous guanosine residues cannot be sequencespecific antisense agents. For example, an antisense PS oligo against the murine rei A NF- transcription factor contains the G-quartet at its 5' terminus, and has been shown to exhibit sequence-specific antiproliferative effects in mice (Higgins et al., 1993). A control, the human sequence (which was, how¬ ever, three bases shorter), but which contained a G-quintet at the 5' terminus, was ineffective. It is also possible that some biological effects observed with PS oligos result from combinations of sequence- and non-sequence-specific mechanisms. This must be considered when mechanistic claims are made and each oligo in every system must be examined individually, always employing the proper controls. Nonspecific effects can also be observed with PO oligos. PO oligo palindromes of six or more bases can induce interferon and production (Yamamoto et al., 1992), which may have un¬ predictable effects on measured experimental end points. These palindromes can also enhance natural killer (NK) cell activity in mouse spleen cells (Kuramoto et al., 1992). Moreover, palin-
1. Charged oligodeoxynucleotides are polyanions. This fact is often ignored in the literature. Polyanions, such as the natu¬ rally occurring sulfated glycosaminoglycans heparin and heparan, dermatan, and chondroitin sulfates, play many extremely important physiological roles. For example, these compounds bind and sequester (heparin-binding) growth factors to the basement membrane. Other synthetic sulfated carbohydrate polyanions, such as polyxylyl hydrogen sulfate (pentosan polysulfate), can mimic the effects of heparin by binding to several heparin-binding growth factors, including basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Wellstein et al., 1991; Zugmaier et al., 1993). This blocks the binding of the growth factor to its cell surface recep¬ tor. Like heparin, PS oligos, such as SdC28, appear to block the binding of both bFGF (50% inhibitory concentration [IC50] 5 µ ) and PDGF (IC50 200 nM) to receptors on the surface of 3T3 fibroblasts and other cells (M. Guvakova and C. Stein, un¬ published observations, 1994). Another shared property of he¬ parin and SdC28 is that both cause the release of bFGF bound to extracellular matrix, at concentrations frequently used in an¬ tisense experiments (I. Vlodavsky and C. Stein, unpublished observations, 1994). Shorter oligos (18-mers) in general block binding and cause release less efficiently. Other proteins that bind non-sequence specifically to both charged oligos and sul¬ 67
68 dromes with flanking oligo-dG regions were the strongest en¬ hancers. A. Krieg and co-workers (unpublished results, 1994) have shown that treatment of or NK cells with PO oligos as short as 8 bases containing a CpG dinucleotide can stimulate cellular activity. Furthermore, as eloquently pointed out by Milligan et al. (1993), the mononucleotide degradation prod¬ ucts of PO oligos, particularly dAMP and dGMP, can have pro¬ nounced cytotoxic or cytostatic effects on cells, particularly those of hematopoietic origin. Furthermore, the degradation products of an oligo complementary to one that is rich in A and G would be rich in C and T, and thus would not be a proper ex¬ perimental control. Experiments using T-rich PO oligos that re¬ quire measurement of [3H]thymidine uptake must also be care¬ fully controlled, owing to the potential suppression of uptake by the nuclease digestion products of the oligo (Matson and Krieg,
1992).
Given all of these problems, and the others that undoubtedly will appear as more investigators use antisense oligos, how can true antisense
sequence-specific inhibition of genetic expres¬ sion be demonstrated? Because of the increase in our recogni¬ tion of potential protein targets for charged oligos, it may be ex¬ tremely difficult if not impossible, at this point, to prove that Watson-Crick base pairing is the basis for an observed experi¬ mental effect. Thus, we are establishing the following guide¬ lines for manuscripts submitted to Antisense Research and Development, when the target is mRNA. 1. It is virtually imperative that the experimenter demon¬ a decrease in the target protein if an antisense mechanism is proposed. In some systems this may not be possible, but a strong case must be made for omitting this critical measure¬ ment. For example, in primary cells, oligo uptake may be het¬ erogeneous and quite poor (Krieg et al., 1991); target protein levels may change in a subset of cells, but overall target protein levels may not change significantly. However, if measurement of target protein levels is omitted, additional controls should be included that demonstrate a lack of effect on cell lines that do not have the target sequence. Furthermore, it is not reasonable to make an argument for specificity if the target protein has, for example, a short half-life while the control protein has a long half-life. Decreased levels of the target mRNA, as demon¬ strated by reverse transcriptase-polymerase chain reaction (RTPCR), have frequently been demonstrated, but this measure¬ ment alone is not proof of mechanism. At this time, we will not require demonstration of decrease levels of target mRNA, be¬ cause that implies that blockade of ribosomal readthrough is ir¬ relevant, thus implicitly endorsing the RNase H mechanism. The latter may indeed be the major contributor to an antisense effect, but rigorous proof for this in mammalian cells is at this time lacking. Measurement of other biological end points, for example, cellular proliferation and ability to clone in soft agar, also is not acceptable as sole proof of mechanism. 2. An acceptable number of appropriate controls must be performed. There are four types of control oligos that should be considered: a. Sense control: This type of control maintains structural features (e.g., palindromes, stem loops, class), but does not maintain composition. b. Scrambled control: This type of control does not maintain strate
EDITORIAL structural features, but does maintain composition. This type of control does not detect the presence of a G-quartet. c. Mismatched control (i.e., with one or two mismatches in the central section of the oligo): this type of control demon¬ strates target hybridization selectivity, and can maintain com¬ position if two mismatches are made, but may not be able to maintain structural features, depending on where the mis¬ matches are made. d. Mismatched target control (i.e., using cells with a mutant gene, deleted gene, etc.): This control may demonstrate a lack of nonsequence specificity, but the control cells may thus be significantly different from the target cells with regard to other, critical parameters, such as oligo internalization and compartmentalization. At this time, there is no scientifically validated "correct" number of control oligos. However, because of the potential difficulties discussed above, it is not acceptable to proffer mechanistic claims after use of only an antisense oligo and a single sense control. The more control oligos that are employed in any experiment, the more likely it is that the observed end point (if the controls are negative, of course) has resulted from a true antisense mechanism. With this in mind, we have arbi¬ trarily chosen three oligos (antisense plus two controls) as the absolute minimum number ofoligos that can justify any mecha¬ nistic claims. Furthermore, a justification of the selection of each control should be given. Manuscripts that claim antisense effects, and that do not use at least two controls, will be re¬ turned without review. Authors must be careful to choose con¬ trol oligos that are appropriate for their own antisense construct. If the appropriate control oligos are not used, mechanistic claims may not necessarily be valid, and the scientific literature will become increasingly confused. This editorial has not sought to comment on the validity of results, but merely on their interpretation. Furthermore, this policy may change in the future, as additional data are accumu¬ lated. This change in editorial policy is being driven by the fact that synthetic oligodeoxynucleotides appear to interact with bi¬ ological systems in many ways, and can specifically associate with mRNA, proteins, and duplex DNA. It is impossible at pre¬ sent to "target" an oligo to only one class of biologically impor¬ tant substance. Nevertheless, the effects that are seen are wor¬ thy of intense scrutiny, and may themselves have therapeutic utility. It is in the interest of everyone involved in this field to promote a clear understanding of these mechanistic distinc¬ tions.
ACKNOWLEDGMENTS We wish to thank members of the Antisense Research and
Development Editorial Board, particularly A. Gewirtz, E. Wickstrom, R. Narayanan, D. Tidd, M. Caruthers, H. Soreg, and J. Reed, for helpful comments. We also wish to thank M. Gait for his particularly insightful comments on proper con¬ trols, and for his division of the controls into four general cate¬ gories. C.A.S. is the Irving Assistant Professor of Medicine and Pharmacology, recipient of an American Cancer Society Clinical Career Development Award, and is partially supported
by NCI R29-60639 and the Mathieson Foundation. A.M.K. is a
69
EDITORIAL Pfizer Scholar, and is supported by NIH R29-AR42556 and by grants from the Veterans Administration of the Arthritis Foundation.
Phosphorothioate oligodeoxynucleotides—antisense gene expression? Pharmacol. Therapeut. 52, 365-384.
inhibitors of
STEIN, CA., CLEARY, ., YAKUBOV, L., and LEDERMAN, S.
(1993). Phosphorothioate oligodeoxynucleotides bind to the third variable loop domain (V3) of HIV-1 gpl20. Antisense Res. Dev. 3,
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