Uses and Applications of Antisense Oligonucleotides Society/Special Colloquium Organized by A. D. B. Malcolm (Charing Cross and Westminster Medical School, London). 643rd Meeting held at the University of Warwick, 22-23 July 1992
Uses of antisense nucleic acids
- an introduction
Alan D. B. Malcolm Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, U.K. The realization that translation of mRNA molecules could be inhibited by suitable complementary nucleic acids dates back to 1977 [l]. Although Miller et al. originally used an antisense oligodeoxynucleotide (ODN), further applications were held up by the difficulty and expense of obtaining the necessary range of ODNs. This was dramatically changed by the development of inexpensive automated ODN synthesizers using a reliable chemistry based on the use of phosphoramidites [Z]. Later, the development of transgenic organisms [3-51 enabled antisense RNA to be produced in vivo, thus eliminating one of the major problems - that of the introduction of large negatively charged molecules into cells. The subsequent papers will address a variety of topics where antisense ODNs and RNAs are finding applications in agriculture, cell biology, developmental genetics and clinical medicine as well as fundamental studies in biochemistry. The technology depends on the fact that while most DNA is double stranded, RNA is predominantly single stranded and is usually only transcribed from one strand (although see Wolfgang Nellen’s paper later in this colloquium). Hence a DNA sequence complementary to the strand which is not transcribed is therefore complementary to this RNA. Such an antisense sequence can therefore bind to this RNA and thereby inhibit translation. With the correct choice of sequence, such inhibition can have a profound effect on the cell, the tissue and the organism. Such projects can therefore easily be stated, but not surprisingly are more difficult to achieve in practice. First of all the delivery of a suitable antisense nucleic acid is likely to be a problem, Abbreviation used: ODN, oligodeoxynucleotide.
although in cell free systems it has been comparatively easy to demonstrate inhibition of translation. Additionally, the efficiency with which any particular antisense nucleic acid inhibits may depend on the secondary structure of the RNA to which it is binding. Although computer modelling of secondary structure can be useful here, in practice the number of potentially stable structures generated often exceeds our ability to test such options experimentally. A certain degree, therefore, of trial and error is still required. Usually the most effective region to target for inhibition is the start point for translation since this can interfere with ribosome binding. However, inhibiting RNA splicing by using antisense nucleic acids complementary to splice junctions has also proved useful. Not only is uptake into cells a problem, but once inside the cells intracellular nucleases may degrade the oligonucleotide and further reduce its effectiveness. A consequence of this is that quantitatively measuring uptake of the antisense molecule is not a trivial problem since the cellular content of 32P,for example, is not necessarily an indication of the concentration of biologically active nucleic acid. Although it is easy to measure the stability, stringency and melting temperature of a particular heteroduplex in vitro, in many cases inkacellular conditions of temperature, pH, ionic strength and counter ions are either unknown or certainly uncontrollable, and may therefore not provide ideal hybridization conditions. As mentioned already, some of the above problems can be overcome by the production of transgenic organisms. Any particular project, therefore, involves the investigation of many variables. First of all the exact gene to be ‘attacked; secondly, the precise sequence within the gene to which the antisense will be com-
I992
Biochemical Society Transactions
746
plementary; and thirdly, how long should the antisense be (the shorter the better from the point of view of uptake, the longer the better from the point of view of specificity and stability)? The problems of length of oligonucleotides may be tackled by the use of two consecutive short oligonucleotides rather than one long one. The use of modified nucleotides to increase resistance to intracellular degradation has many merits although, of course, such modifications are likely to alter the stability of the relevant heteroduplex. In practice it is more common not to use 100% substituted modified nucleotides, but to use a proportion, and therefore the exact proportion and position of the modified nucleotides also needs to be determined. Although, as stated above, intracellular conditions are likely to differ from those of a cell free system, it is still nonetheless important to determine the stability of the heteroduplex in vitro. Several chemically modified bases, including a-anomers, phosphorothioates and methylphosphonates will all be decided in this colloquium. When these parameters have been determined a cell free system can be tested to monitor the efficiency of translation of the RNA, and additionally if it is possible to determine the presence of ribonuclease H in the cells under investigation, this is also useful. (Ribonuclease H degrades the RNA of a heteroduplex.) A variety of methods for introducing antisense oligonucleotides into cells ranging from conjugation to peptides such as polylysine, encapsulation within liposomes and coating tungsten particles with the nucleic acid and using these as a projectile at high velocity, have all been used.
In spite of the various problems outlined above, commercially viable examples already exist. The inhibition of polygalacturonase slows the ripening of tomatoes, thereby increasing their shelf life. Antisense RNA against chalcone synthase can change the colour of petunias (although in a nonpredictable manner). A transgenic tobacco plant able to produce an antisense RNA against a viral coat protein for cucumber mosaic mosaic virus offers partial protection to the plant against such infection. In vitro and in cellular systems a variety of antisense molecules against oncogenes and growth factors show potential as antiturnour agents, although the problems of delivery to the affected tissue in patients are a long way from being solved. Similarly, in cell culture it is comparatively easy to demonstrate their use as antiviral agents, for example against human immunodeficiency virus [6], herpes simplex virus, Rous sarcoma virus etc. 1. Ts’o. P. 0. P., Miller, 1’. S. & Gren. J. J. (1983) in Development of Target-Oriented Anticancer Drugs (Cheng, Y.-C. etal., eds.), Kaven Press, New York 2. Alvarado-Urbina, G., Sathe, G. M., Liu?W.-C., Gillen, M. F., Duck, 1’. D.. Bender, R. & Ogilvie. K. K. (1981) Science 214,270-274 3. Hrinster, R. I,. & Palmiter. K. I). (1982) Trends Hiochem. Sci. 3,438-440 4. Stalker, D. M.. McBride. K. E. & Malyj. I,. L). (1988) Science 242,419-422 5. Melton, L). W. (1990) Hiochem. SOC. Trans. 18,
1035-1039 6. Agarwal. S.. Goodchild. J.. Civeira, M. P., Thornton, A. H., Sarin, 1’. S. & Zamecnik, P. C. (1988) I’roc. Natl. Acad. Sci. U S A . 85.7079-7083 Received 24 July 1992
Methylphosphonodiester/phosphodiester chimeric oligodeoxynucleotides David M. Tidd Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K.
The clear demonstration that gene expression may be inhibited by antisense oligonucleotides in cellfree protein synthesizing systems and microinjected oocytes has served to establish that, in principle at least, these species could form the basis of new and highly specific drugs for the treatment of a variety of diseases, including viral infections and cancer [ 13. At the same time, it was evident that the normal phosphodiester structure of DNA alone would be inadequate for drug development as a result of the
Volume 20
poor uptake of oligodeoxynucleotides into intact human cells and their susceptibility to rapid nucleolytic degradation in tissue and biological fluids. Consequently, the accent has been upon the design of structural analogues of the nucleic acids which would circumvent these problems, while retaining the capacity for hybridization with complementary base sequences. However, it would be fair to say that no structure tested so far has proved to be ideal. Indeed, it is sobering to contemplate the list of
Anti-sense Oligonucleotides
Fig. I
General problems in the development of anti-sense oligonucleotide analogues as therapeutic agents 747
SolubiI ity Patient
targeting ( Tissue Pharmacokinetics ‘Biological stability Non-specific toxicity Hybridization efficiency Hybridization stringency Partial complementarity Selective targeting point mutated oncogene mRNA in the face of normal c-ONC expression Cell uptake
Target cell
Activation of RNase H lntracellular participation of RNase H, Optimum target sequence selection
-
Undesired effects on non-targeted mRNA
lntracytoplasmic delivery to target mRNA
* Masking of sequences in native mRNA by secondary and tertiary structure
Fig. 2
Structures of normal phosphodiester and non-ionic methylphosphonate analogue oligodeoxynucleotides Y
Z
A, G,C, or T A, G.C. or T
H H
X Phosphodiester Methylphosphonate
0 CHI
HOCH2
1
Hi)
problems and considerations which must be taken into account in going from the cell-free system to the intact patient (Fig. 1). We originally became interested in the possibility of studying effects of inhibiting oncogene expression on the behaviour of malignant cells in culture, using antisense non-ionic methylphosphonate oligodeoxynucleotide analogues (Fig. 2) developed by Miller and Ts’o [2]. This followed from the authors’ claims that methylphosphonate oligomers were nuclease-resistant, readily entered mammalian cells by passive diffusion across the plasma membrane, and formed stable hybrids with complementary sequences in mRNA, thereby inhibiting gene expression. However, in our hands, methylphosphonate antisense oligodeoxynucleotides simply did not work [ 3 ] . In attempting to account for our failure, it became apparent that the application of antisense oligodeoxynucleotides against intact cells is not as simple or straightforward as at first it might seem, and that problems still remain to be addressed at this level (Fig. 1) if effective drugs are to be developed, and in spite of the current rush into clinical trials with existing structures [4]. Table 1 lists some of the factors affecting biological activity of antisense oligodeoxynucleotides and compares normal phosphodiester and methylphosphonate oligodeoxynucleotide structures with respect to each factor in terms of the extent to which the requirements for antisense efficacy are fulfilled; the more plus signs the greater the poten-
I992
Biochemical Society Transactions
Table I
Factors affecting biological activity of anti-sense oligodeoxynucleotides 748 I. 2. 3. 4. 5. 6.
Normal phosphodiester
Methylphosphonate analogues
+
++ +++ ++ +
Cell uptake Biological stability Non-toxicity Hybridization efficacy Hybrid arrest RNase-H activation
tial contribution to overall activity from the properties of the structure in that particular aspect. Considering first the question of intracellular transport: it has been demonstrated by fluorescence microscopy and flow cytometry, using oligomers tagged with fluorescent reporter groups, that normal phosphodiester oligodeoxynucleotides are taken up very poorly by mammalian cells, through a saturable process, apparently, of receptor mediated endocytosis [S-71. Intracellular oligodeoxynucleotide is localized in endosomes, still separated by a membrane from the target mRNA, and very little if any enters the cytoplasm. In contrast, methylphosphonate oligodeoxynucleotide analogues are taken up poorly, but at a somewhat faster rate by a non-saturable, concentration dependent process, the kinetics of which appeared initially to be compatible with passive diffusion across the plasma membrane [7]. However, the actual mechanism of intracellular transport is almost certainly not diffusion but fluid phase or adsorptive pinocytosis [81. Again, the intracellular oligomer is bounded by a membrane, although the observation, in addition, of a diffuse cytoplasmic fluorescence would suggest either that some entry into the cytoplasm does occur, or that oligodeoxynucleotide is distributed throughout the cell in a multitude of submicroscopic vesicles [7, 81. Normal phosphodiester oligodeoxynucleotides are rapidly degraded in biological environments, the major nuclease responsible in serum and cells being a 3’-phosphodiesterase. Low endonuclease activity may also be demonstrated in serum by blocking the 3’ end of the molecules against exonuclease attack [9], although the former enzyme apparently derives from cells during clotting and is not present in plasma [ 101. Methylphosphonates, in contrast, are totally resistant to nucleases. The nuclease susceptibility of phospho-
Volume 20
-
f
+++ +? +++
_+? -
diester oligodeoxynucleotides may be responsible, at least in part, for non-specific toxic or growth inhibitory effects observed in cell culture and microinjected oocytes, since high concentrations of deoxynucleosides released thereby may severely affect cell proliferation, as exemplified by the dire consequences of congenital adenosine deaminase deficiency, and the technique of synchronizing cultured cell populations at the start of S-phase of the cell cycle with thymidine. On the other hand, the nuclease resistance of methylphosphonates would account for their comparative innocuity [ 31. Unfortunately, and despite earlier claims to the contrary, it turns out that, in fact, methylphosphonates hybridize poorly to complementary base sequences in RNA in comparison with antisense oligodeoxynucleotides with the normal phosphodiester structure [ 1, 111, and since, unlike the latter, the analogue is unable to direct destruction of the RNA component of the heteroduplex by ribonuclease H (RNase H) [ l l ] , antisense activity of methylphosphonates may only result from physical blockade of ribosome binding or splicing reactions. Indeed, the overwhelming evidence that fully loaded ribosomes are readily able to disrupt secondary structure in their path has cast doubt upon the original concept of hybrid arrest, in which antisense oligonucleotides targeted at translated sites in mRNA were viewed as presenting a ‘roadblock‘ to ribosome progression [ 11. However, notwithstanding these considerations, differential inhibition of point mutant Ha-ras $1 translation in the face of wild type synthesis and vice versa has been reported, at a temperature above the heteroduplex melting temperature, with methylphosphonate 11mers targeted at the codon 61 region of the gene
[121. A further potential limitation on the development of non-ionic methylphosphonate oligonucleo-
Anti-sense Oligonucleotides
tide analogues as drugs is their poor aqueous solubility in contrast to the ready solubility of polyanionic phosphodiester species. This final point underscores the general impression gained from consideration of Table 1, in that each structure is apparently good at the things in which the other is lacking. Therefore, we decided that it would be sufficiently interesting to combine the structures and produce chimeric oligodeoxynucleotides in the hope that the desirable properties of each would contribute to improvement in antisense efficacy [3, 91. As expected, chimeric oligodeoxynucleotides with methylphosphonate sections at each end and a central phosphodiester region proved to be readily soluble and exhibited significantly enhanced biological stability relative to all phosphodiester molecules [7, 91, while retaining the capacity to direct RNase H degradation of target RNA [ 1, 111. In fact, progressive replacement of terminal phosphodiester linkages with methylphosphonates resulted in enhanced RNase H activity, a counterintuitive result in view of the concomitant reduction in RNA:oligonucleotide heteroduplex stability [ 113. Reducing the internal phosphodiester section to four linkages abolished the non-specific cell growth inhibitory effects of the oligodeoxynucleotides [ 131, and, at the same time, reduced to minimal levels RNase H attack at non-targeted sites of partial complementarity in RNAs transcribed in vitro [ 141. The latter result demonstrated the potential for controlling the stringency of hybridization and RNase H cleavage specificity within intact cells by judicious incorporation of helix destabilizing backbone modifications on the antisense oligodeoxynucleotides. Also, it would be predicted that differential targeting of point mutations might be achieved with chimeric oligodeoxynucleotides by centring a short phosphodiester section on the site of mutation. Methylphosphonodiester/phosphodiester chimeric 15-mers with just four phosphodiester linkages were still only taken up poorly by MOLT-4 cells via the putative receptor mediated endocytotic route of all phosphodiester molecules, and were likewise restricted to the intracellular endosomal compartment [7]. In cont ast, 15-mers with two phosphodiester linkages entered cells more efficiently, apparently by the same mechanism
as all-methylphosphonate molecules, and also directed specific cleavage of in vitro transcribed and cell-extracted RNA by RNase H. However, whether or not these chimeric oligodeoxynucleotides truly gain access to the target mRNA in the cytoplasm/ nucleus, and the potential involvement of RNase H in antisense effects in intact human cells are questions that remain to be answered.
I should like to thank Richard Giles, David Spiller and Jean Wood for their expertise and commitment to this project. The support of the Cancer Research Campaign and the Cancer and Polio Research Fund is gratefully acknowledged. 1. Tidd, D. M. (1 990) Anticancer Kes. 10,1169- 1 182 2. Miller. P. S. & Ts’o, P. 0.P. (1987) Anti-Cancer Drug Des. 2, 1 17- 128 3. Tidd, D. M., Hawley, P., Warenius, H. M. & Gibson, 1. (1988) Anti-Cancer Drug Des. 3, 117-127 4. McKie, R. (1992) The Observer, 23 February, p. 3 5. Loke. S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J. S. & Neckers. I,. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86,3474-3478 6. Yakubov, I,. A,, Deeva, E. A., Zarytova, V. F., Ivanova. E. M., Ryte. A. S., Yurchenko, I,. V. & Vlassov, V. V. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6454-6458 7. Spiller, D. G. & Tidd. D. M. (1992) Anti-Cancer Drug Des. 7, 1 15- 129 8. Shoji, Y., Akhtar, S., Periasamy, A., Herman, B. & Juliano, R. I,. (1991) Nucl. Acids Res. 19,5543-5550 9. Tidd, D. M. & Warenius, H. M. (1989) Br. J. Cancer 60,343-350 10. Eder, P. S.,DeVine, R. J., Dagle, J. M. & Walder, J. A. (1991) Antisense Res. Dev. 1, 141-151 11. Giles, R. V. & Tidd, D. M. (1992) Anti-Cancer Drug Des. 7,37-48 12. Chang, E. H., Miller, P. S., Cushman, C.. Devadas, K., Pirollo, K. F.. Ts’o, P. 0. P. & Yu, Z. P. (1991) Biochemistry 30,8283-8286 13. Tidd, D. M. (1992) in Molecular Aspects of Anticancer Drug-DNA Interactions (Neidle, S. & Waring, M., eds.), Macmillan, Basingstoke, in the press 14. Giles, R. V. & Tidd, D. M. (1992) Nucl. Acids. Res. 20,703-770 Keceived 22 July 1992.
I992
749
Uses of antisense nucleic acids
- an introduction
Alan D. B. Malcolm Department of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London W6 8RF, U.K. The realization that translation of mRNA molecules could be inhibited by suitable complementary nucleic acids dates back to 1977 [l]. Although Miller et al. originally used an antisense oligodeoxynucleotide (ODN), further applications were held up by the difficulty and expense of obtaining the necessary range of ODNs. This was dramatically changed by the development of inexpensive automated ODN synthesizers using a reliable chemistry based on the use of phosphoramidites [Z]. Later, the development of transgenic organisms [3-51 enabled antisense RNA to be produced in vivo, thus eliminating one of the major problems - that of the introduction of large negatively charged molecules into cells. The subsequent papers will address a variety of topics where antisense ODNs and RNAs are finding applications in agriculture, cell biology, developmental genetics and clinical medicine as well as fundamental studies in biochemistry. The technology depends on the fact that while most DNA is double stranded, RNA is predominantly single stranded and is usually only transcribed from one strand (although see Wolfgang Nellen’s paper later in this colloquium). Hence a DNA sequence complementary to the strand which is not transcribed is therefore complementary to this RNA. Such an antisense sequence can therefore bind to this RNA and thereby inhibit translation. With the correct choice of sequence, such inhibition can have a profound effect on the cell, the tissue and the organism. Such projects can therefore easily be stated, but not surprisingly are more difficult to achieve in practice. First of all the delivery of a suitable antisense nucleic acid is likely to be a problem, Abbreviation used: ODN, oligodeoxynucleotide.
although in cell free systems it has been comparatively easy to demonstrate inhibition of translation. Additionally, the efficiency with which any particular antisense nucleic acid inhibits may depend on the secondary structure of the RNA to which it is binding. Although computer modelling of secondary structure can be useful here, in practice the number of potentially stable structures generated often exceeds our ability to test such options experimentally. A certain degree, therefore, of trial and error is still required. Usually the most effective region to target for inhibition is the start point for translation since this can interfere with ribosome binding. However, inhibiting RNA splicing by using antisense nucleic acids complementary to splice junctions has also proved useful. Not only is uptake into cells a problem, but once inside the cells intracellular nucleases may degrade the oligonucleotide and further reduce its effectiveness. A consequence of this is that quantitatively measuring uptake of the antisense molecule is not a trivial problem since the cellular content of 32P,for example, is not necessarily an indication of the concentration of biologically active nucleic acid. Although it is easy to measure the stability, stringency and melting temperature of a particular heteroduplex in vitro, in many cases inkacellular conditions of temperature, pH, ionic strength and counter ions are either unknown or certainly uncontrollable, and may therefore not provide ideal hybridization conditions. As mentioned already, some of the above problems can be overcome by the production of transgenic organisms. Any particular project, therefore, involves the investigation of many variables. First of all the exact gene to be ‘attacked; secondly, the precise sequence within the gene to which the antisense will be com-
I992
Biochemical Society Transactions
746
plementary; and thirdly, how long should the antisense be (the shorter the better from the point of view of uptake, the longer the better from the point of view of specificity and stability)? The problems of length of oligonucleotides may be tackled by the use of two consecutive short oligonucleotides rather than one long one. The use of modified nucleotides to increase resistance to intracellular degradation has many merits although, of course, such modifications are likely to alter the stability of the relevant heteroduplex. In practice it is more common not to use 100% substituted modified nucleotides, but to use a proportion, and therefore the exact proportion and position of the modified nucleotides also needs to be determined. Although, as stated above, intracellular conditions are likely to differ from those of a cell free system, it is still nonetheless important to determine the stability of the heteroduplex in vitro. Several chemically modified bases, including a-anomers, phosphorothioates and methylphosphonates will all be decided in this colloquium. When these parameters have been determined a cell free system can be tested to monitor the efficiency of translation of the RNA, and additionally if it is possible to determine the presence of ribonuclease H in the cells under investigation, this is also useful. (Ribonuclease H degrades the RNA of a heteroduplex.) A variety of methods for introducing antisense oligonucleotides into cells ranging from conjugation to peptides such as polylysine, encapsulation within liposomes and coating tungsten particles with the nucleic acid and using these as a projectile at high velocity, have all been used.
In spite of the various problems outlined above, commercially viable examples already exist. The inhibition of polygalacturonase slows the ripening of tomatoes, thereby increasing their shelf life. Antisense RNA against chalcone synthase can change the colour of petunias (although in a nonpredictable manner). A transgenic tobacco plant able to produce an antisense RNA against a viral coat protein for cucumber mosaic mosaic virus offers partial protection to the plant against such infection. In vitro and in cellular systems a variety of antisense molecules against oncogenes and growth factors show potential as antiturnour agents, although the problems of delivery to the affected tissue in patients are a long way from being solved. Similarly, in cell culture it is comparatively easy to demonstrate their use as antiviral agents, for example against human immunodeficiency virus [6], herpes simplex virus, Rous sarcoma virus etc. 1. Ts’o. P. 0. P., Miller, 1’. S. & Gren. J. J. (1983) in Development of Target-Oriented Anticancer Drugs (Cheng, Y.-C. etal., eds.), Kaven Press, New York 2. Alvarado-Urbina, G., Sathe, G. M., Liu?W.-C., Gillen, M. F., Duck, 1’. D.. Bender, R. & Ogilvie. K. K. (1981) Science 214,270-274 3. Hrinster, R. I,. & Palmiter. K. I). (1982) Trends Hiochem. Sci. 3,438-440 4. Stalker, D. M.. McBride. K. E. & Malyj. I,. L). (1988) Science 242,419-422 5. Melton, L). W. (1990) Hiochem. SOC. Trans. 18,
1035-1039 6. Agarwal. S.. Goodchild. J.. Civeira, M. P., Thornton, A. H., Sarin, 1’. S. & Zamecnik, P. C. (1988) I’roc. Natl. Acad. Sci. U S A . 85.7079-7083 Received 24 July 1992
Methylphosphonodiester/phosphodiester chimeric oligodeoxynucleotides David M. Tidd Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K.
The clear demonstration that gene expression may be inhibited by antisense oligonucleotides in cellfree protein synthesizing systems and microinjected oocytes has served to establish that, in principle at least, these species could form the basis of new and highly specific drugs for the treatment of a variety of diseases, including viral infections and cancer [ 13. At the same time, it was evident that the normal phosphodiester structure of DNA alone would be inadequate for drug development as a result of the
Volume 20
poor uptake of oligodeoxynucleotides into intact human cells and their susceptibility to rapid nucleolytic degradation in tissue and biological fluids. Consequently, the accent has been upon the design of structural analogues of the nucleic acids which would circumvent these problems, while retaining the capacity for hybridization with complementary base sequences. However, it would be fair to say that no structure tested so far has proved to be ideal. Indeed, it is sobering to contemplate the list of
Anti-sense Oligonucleotides
Fig. I
General problems in the development of anti-sense oligonucleotide analogues as therapeutic agents 747
SolubiI ity Patient
targeting ( Tissue Pharmacokinetics ‘Biological stability Non-specific toxicity Hybridization efficiency Hybridization stringency Partial complementarity Selective targeting point mutated oncogene mRNA in the face of normal c-ONC expression Cell uptake
Target cell
Activation of RNase H lntracellular participation of RNase H, Optimum target sequence selection
-
Undesired effects on non-targeted mRNA
lntracytoplasmic delivery to target mRNA
* Masking of sequences in native mRNA by secondary and tertiary structure
Fig. 2
Structures of normal phosphodiester and non-ionic methylphosphonate analogue oligodeoxynucleotides Y
Z
A, G,C, or T A, G.C. or T
H H
X Phosphodiester Methylphosphonate
0 CHI
HOCH2
1
Hi)
problems and considerations which must be taken into account in going from the cell-free system to the intact patient (Fig. 1). We originally became interested in the possibility of studying effects of inhibiting oncogene expression on the behaviour of malignant cells in culture, using antisense non-ionic methylphosphonate oligodeoxynucleotide analogues (Fig. 2) developed by Miller and Ts’o [2]. This followed from the authors’ claims that methylphosphonate oligomers were nuclease-resistant, readily entered mammalian cells by passive diffusion across the plasma membrane, and formed stable hybrids with complementary sequences in mRNA, thereby inhibiting gene expression. However, in our hands, methylphosphonate antisense oligodeoxynucleotides simply did not work [ 3 ] . In attempting to account for our failure, it became apparent that the application of antisense oligodeoxynucleotides against intact cells is not as simple or straightforward as at first it might seem, and that problems still remain to be addressed at this level (Fig. 1) if effective drugs are to be developed, and in spite of the current rush into clinical trials with existing structures [4]. Table 1 lists some of the factors affecting biological activity of antisense oligodeoxynucleotides and compares normal phosphodiester and methylphosphonate oligodeoxynucleotide structures with respect to each factor in terms of the extent to which the requirements for antisense efficacy are fulfilled; the more plus signs the greater the poten-
I992
Biochemical Society Transactions
Table I
Factors affecting biological activity of anti-sense oligodeoxynucleotides 748 I. 2. 3. 4. 5. 6.
Normal phosphodiester
Methylphosphonate analogues
+
++ +++ ++ +
Cell uptake Biological stability Non-toxicity Hybridization efficacy Hybrid arrest RNase-H activation
tial contribution to overall activity from the properties of the structure in that particular aspect. Considering first the question of intracellular transport: it has been demonstrated by fluorescence microscopy and flow cytometry, using oligomers tagged with fluorescent reporter groups, that normal phosphodiester oligodeoxynucleotides are taken up very poorly by mammalian cells, through a saturable process, apparently, of receptor mediated endocytosis [S-71. Intracellular oligodeoxynucleotide is localized in endosomes, still separated by a membrane from the target mRNA, and very little if any enters the cytoplasm. In contrast, methylphosphonate oligodeoxynucleotide analogues are taken up poorly, but at a somewhat faster rate by a non-saturable, concentration dependent process, the kinetics of which appeared initially to be compatible with passive diffusion across the plasma membrane [7]. However, the actual mechanism of intracellular transport is almost certainly not diffusion but fluid phase or adsorptive pinocytosis [81. Again, the intracellular oligomer is bounded by a membrane, although the observation, in addition, of a diffuse cytoplasmic fluorescence would suggest either that some entry into the cytoplasm does occur, or that oligodeoxynucleotide is distributed throughout the cell in a multitude of submicroscopic vesicles [7, 81. Normal phosphodiester oligodeoxynucleotides are rapidly degraded in biological environments, the major nuclease responsible in serum and cells being a 3’-phosphodiesterase. Low endonuclease activity may also be demonstrated in serum by blocking the 3’ end of the molecules against exonuclease attack [9], although the former enzyme apparently derives from cells during clotting and is not present in plasma [ 101. Methylphosphonates, in contrast, are totally resistant to nucleases. The nuclease susceptibility of phospho-
Volume 20
-
f
+++ +? +++
_+? -
diester oligodeoxynucleotides may be responsible, at least in part, for non-specific toxic or growth inhibitory effects observed in cell culture and microinjected oocytes, since high concentrations of deoxynucleosides released thereby may severely affect cell proliferation, as exemplified by the dire consequences of congenital adenosine deaminase deficiency, and the technique of synchronizing cultured cell populations at the start of S-phase of the cell cycle with thymidine. On the other hand, the nuclease resistance of methylphosphonates would account for their comparative innocuity [ 31. Unfortunately, and despite earlier claims to the contrary, it turns out that, in fact, methylphosphonates hybridize poorly to complementary base sequences in RNA in comparison with antisense oligodeoxynucleotides with the normal phosphodiester structure [ 1, 111, and since, unlike the latter, the analogue is unable to direct destruction of the RNA component of the heteroduplex by ribonuclease H (RNase H) [ l l ] , antisense activity of methylphosphonates may only result from physical blockade of ribosome binding or splicing reactions. Indeed, the overwhelming evidence that fully loaded ribosomes are readily able to disrupt secondary structure in their path has cast doubt upon the original concept of hybrid arrest, in which antisense oligonucleotides targeted at translated sites in mRNA were viewed as presenting a ‘roadblock‘ to ribosome progression [ 11. However, notwithstanding these considerations, differential inhibition of point mutant Ha-ras $1 translation in the face of wild type synthesis and vice versa has been reported, at a temperature above the heteroduplex melting temperature, with methylphosphonate 11mers targeted at the codon 61 region of the gene
[121. A further potential limitation on the development of non-ionic methylphosphonate oligonucleo-
Anti-sense Oligonucleotides
tide analogues as drugs is their poor aqueous solubility in contrast to the ready solubility of polyanionic phosphodiester species. This final point underscores the general impression gained from consideration of Table 1, in that each structure is apparently good at the things in which the other is lacking. Therefore, we decided that it would be sufficiently interesting to combine the structures and produce chimeric oligodeoxynucleotides in the hope that the desirable properties of each would contribute to improvement in antisense efficacy [3, 91. As expected, chimeric oligodeoxynucleotides with methylphosphonate sections at each end and a central phosphodiester region proved to be readily soluble and exhibited significantly enhanced biological stability relative to all phosphodiester molecules [7, 91, while retaining the capacity to direct RNase H degradation of target RNA [ 1, 111. In fact, progressive replacement of terminal phosphodiester linkages with methylphosphonates resulted in enhanced RNase H activity, a counterintuitive result in view of the concomitant reduction in RNA:oligonucleotide heteroduplex stability [ 113. Reducing the internal phosphodiester section to four linkages abolished the non-specific cell growth inhibitory effects of the oligodeoxynucleotides [ 131, and, at the same time, reduced to minimal levels RNase H attack at non-targeted sites of partial complementarity in RNAs transcribed in vitro [ 141. The latter result demonstrated the potential for controlling the stringency of hybridization and RNase H cleavage specificity within intact cells by judicious incorporation of helix destabilizing backbone modifications on the antisense oligodeoxynucleotides. Also, it would be predicted that differential targeting of point mutations might be achieved with chimeric oligodeoxynucleotides by centring a short phosphodiester section on the site of mutation. Methylphosphonodiester/phosphodiester chimeric 15-mers with just four phosphodiester linkages were still only taken up poorly by MOLT-4 cells via the putative receptor mediated endocytotic route of all phosphodiester molecules, and were likewise restricted to the intracellular endosomal compartment [7]. In cont ast, 15-mers with two phosphodiester linkages entered cells more efficiently, apparently by the same mechanism
as all-methylphosphonate molecules, and also directed specific cleavage of in vitro transcribed and cell-extracted RNA by RNase H. However, whether or not these chimeric oligodeoxynucleotides truly gain access to the target mRNA in the cytoplasm/ nucleus, and the potential involvement of RNase H in antisense effects in intact human cells are questions that remain to be answered.
I should like to thank Richard Giles, David Spiller and Jean Wood for their expertise and commitment to this project. The support of the Cancer Research Campaign and the Cancer and Polio Research Fund is gratefully acknowledged. 1. Tidd, D. M. (1 990) Anticancer Kes. 10,1169- 1 182 2. Miller. P. S. & Ts’o, P. 0.P. (1987) Anti-Cancer Drug Des. 2, 1 17- 128 3. Tidd, D. M., Hawley, P., Warenius, H. M. & Gibson, 1. (1988) Anti-Cancer Drug Des. 3, 117-127 4. McKie, R. (1992) The Observer, 23 February, p. 3 5. Loke. S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J. S. & Neckers. I,. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86,3474-3478 6. Yakubov, I,. A,, Deeva, E. A., Zarytova, V. F., Ivanova. E. M., Ryte. A. S., Yurchenko, I,. V. & Vlassov, V. V. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6454-6458 7. Spiller, D. G. & Tidd. D. M. (1992) Anti-Cancer Drug Des. 7, 1 15- 129 8. Shoji, Y., Akhtar, S., Periasamy, A., Herman, B. & Juliano, R. I,. (1991) Nucl. Acids Res. 19,5543-5550 9. Tidd, D. M. & Warenius, H. M. (1989) Br. J. Cancer 60,343-350 10. Eder, P. S.,DeVine, R. J., Dagle, J. M. & Walder, J. A. (1991) Antisense Res. Dev. 1, 141-151 11. Giles, R. V. & Tidd, D. M. (1992) Anti-Cancer Drug Des. 7,37-48 12. Chang, E. H., Miller, P. S., Cushman, C.. Devadas, K., Pirollo, K. F.. Ts’o, P. 0. P. & Yu, Z. P. (1991) Biochemistry 30,8283-8286 13. Tidd, D. M. (1992) in Molecular Aspects of Anticancer Drug-DNA Interactions (Neidle, S. & Waring, M., eds.), Macmillan, Basingstoke, in the press 14. Giles, R. V. & Tidd, D. M. (1992) Nucl. Acids. Res. 20,703-770 Keceived 22 July 1992.
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