Tohoku

J. Exp.

Med., 1992,

Selective Principles

168, 351-359

Anti-Gene and

Therapy

for

Cancer

:

Prospects

JACK S. COHEN Cancer Medical

Pharmacology School,

Department,

Rockville

MD,

Georgetown

University

USA

COHEN,J.S. Selective Anti-Gene Therapy for Cancer : Principles and Prospects. Tohoku J. Exp. Med., 1992, 168 (2), 351-359 Oligodeoxynucleotides can act as antisense complements to target sense sequences of natural mRNAs to selectively regulate gene expression by translation arrest. This is a form of interventional gene therapy. Chemically modified analogs that are nucleaseresistant enable this strategy to be utilized in practice. Of the chemically modified backbone analogs of oligodeoxynucleotides we have used the phosphorothioate (PS) analog, in which a non-bridging phosphate oxygen atom is substituted with a sulfur atom. We have shown that these oligodeoxynucleotide analogs inhibit f-globin expression in cell free systems, and that they are taken up by cells. Specific sequences have been shown to selectively regulate viral and cellular gene expression, for example the bcl-2 oncogene that is found in ca. 90% of lymphomas. However, the PS analog has certain disadvantages, notably reduced hybridization and non-selective inhibition of translation. We have therefore synthesized a series of (PS-PO) co-polymers and characterized their properties. Other related approaches include catelytic ribozymes, and formation of triplexes by direct interaction of oligomers in the major groove of DNA. In general, a chemically modified oligodeoxynucleotide analog can be regurded as a novel form of informational drug. oligodeoxynucleotide ; antisense ; DNA ; RNA ; phosphorothioate ; cancer ; bcl-2

The advent of genetic engineering and gene therapy has raised ethical issues regarding the modification of the gene pool in an individual. An alternative approach to gene therapy is the use of shorter sequences of informational molecules that are targeted against a specific cellular nucleic acid sequence, in order to intervene in the flow of genetic information in the cells. Although it is realistically still years away, the basis for such an interventional gene approach can currently be seen in the use of oligodeoxynucleotide analogs as anticancer agents. While there have been several ways of referring to these novel therapeutics, I have proposed the term informational drugs, since these compounds differ from normal types of drugs specifically because they are encoded with an informational content in their base sequence (Cohen 1991). The principle of this method involves an intervention in the normal transfer of genetic information in the cell from DNA to mRNA to protein. Knowing the Addressfor reprints: 4 ResearchCourt,Rockville,Maryland20850USA. 351

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sequence of bases provides the selectivity of a target, since genes differ from each other precisely in their base sequences. The use of such a compound involves a direct interference with either transcrription or translation. In translation arrest the base sequence of the mRNA being targeted is termed the sense sequence and the antisense sequence is the complement. mRNA is a single strand, and contains accessible regions that allow an exogenous complement to form a hybrid duplex. The term antisense was first applied to the complementary mRNA that was expressed endogenously via a cDNA vector (Eguchi et al. 1991) Subsequently it was found that such antisense mRNA's are a natural regulatory mechanism (Inouye 1988; Krystal et al. 1990). What transforms the antisense method into a potential therapeutic approach is the use of synthetic oligodeoxynucleotide analogs (Fig, 1). These "oligos" can form hybrid duplexes with m1~NA and thereby bring about translation arrest. This requires that the oligo have a degree of resistance to naturally occuring nucleases that cleave phosphodiester bonds, and this is conferred by chemical modification of the oligodeoxynucleotide structure. It should be emphasized that there are now many examples of such translation arrest by oligos in the literature (Cohen 1989; Uhlmann and Peyman 1990; Mol and van der Krol 1991). There are many factors that play a role in determining the efficiency of an antisense oligodeoxynucleotide, and some of these are listed in Table 1. Another approach to antisense inhibition in the use of a catalytic RNA molecule known as a ribozyme (Cech 1986). The fact that this is an oligo-RNA means that it is currently more difficult to synthesize than a oligo-DNA, and is also susceptible to degradation due to the presence of ribonucleases. Methods for the inhibition of transcription are less well developed at present. DNA is the target of transcription arrest, and this should be very efficient since these is only one copy of the gene gives rise to many copies of mRNA, each of which in turn gives many copies of protein. Since the DNA duplex is so stable, current attention is focussed

Fig. 1. Structure of an oligodeoxynucleotide ; natural phosphodiester, methylphosphonate, X-Me ; phosphorothioate, X = S-.

X = 0-

;

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oligonucleotides

on the formation of triple helices (Moffat 1991) that result from binding of a third strand of an oligo in the major groove of the DNA. Triplexes are expected to bring about selective transcription arrest (Cooney et al. 1988 ; Povsic and Dervan 1990). An alternative approach involves the sequestration of transcription factors by DNA mini-helices with the appropriate recognition sequence (Bielinska et al. 1990). Antisense inhibition Oligodeoxynucleotide analogs. The methylphosphonate analog (Fig. 1), that was developed as a nuclease-resistant analog for antisense applications, is also uncharged and hence lipophilic (Miller 1989). Phosphorothioates, that were also developed for this purpose, have a sulfur atom substituted at phosphorus (PS) (Fig. 1). One advantage of the latter analogs is that they retain the charge, and hence the water solubility, of the natural phosphodiester (P0) compounds. Synthesis of the PS compounds was based upon earlier work (Eckstein and Gish 1989). Originally it was reported that these chiral analogs, that exist as two stereoisomers at each substituted P atom, were very resistant to nucleases (Stein et al. 1988). However, while it has been confirmed that this is true of exonucleases, endonucleases cleave an all-PS oligomer (anti-/3-globin 17-mer), although not as readily as the all-PO congener (Fig. 2) (Ghosh et al. 1991a). It should also be noted that the melting temperature (reflecting the ability to hybridize) is reduced as expected with the increase in PS content. Thus for improved hybridization it may be preferable to utilize PO/PS copolymers (Ghosh et al. 1991a), or other copolymers in which the overall charge and properties of the oligomer can be optimized.

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Fig.

2. Gel electrophoresis of 32P-end labeled anti-f3-globin 17-mers (P0 and PS) showing cleavage by S1-endonuclease. PO is the more rapidly cleaved.

Note that all mono-substituted analogs (Fig, l) exhibit stereo-isomerism at each P atom. Attempts to overcome this apparent problem have been made, for example using the achiral phosphorodithioate, which contains two sulfur substitutions for non-bridging oxygens on the phosphodiester group (Brill et al. 1989; Dahl et al. 1989). However, it is not clear that the advantage of achirality overcomes other disadvantages of this analog. It should also be noted that molecular simulations indicate that the Rp mono-phosphorothioate isomer (with

Fig

. 3. Variations in total energy during a 10 ps molecular dynamics trajectory of d(CGCGAATTCGCG) as normal phosphodiester (P0), and as both homochiral Rp and Sp diastereomers. The dashed line represents the mean of the Rp and Sp, representing the 50% synthetic mixture, and the dotted line represents the sum of Rp and Sp, that would be the minimum energy expected for the dithio analog.

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sulfur pointing into the minor groove) is destabilised relative to the Sp isomer (Jaroszewski et al. 1992), and since the di-thioate always contains a sulfur in the Rp orientation, it will always be destabilized (Fig. 3). Consistent with this conclusion it has been found that the dithio oligomers exhibit significantly reduced melting temperatures (Ghosh et al. 1991b). Inhibition of expressionin model systems. Oligodeoxynucleotidesand their analogs cause both selective and non-selective inhibition of expression in cell-free systems (Ghosh et al. 1991a,b). Inhibition of /3-globin expression in Xenopus oocytes was chosen to compare the efficacy of different oligo analogs while avoiding the problems of relative cellular uptake, since the globin mRNA and the oligosare injected (Cazenave et al. 1989). The amount of 35S-methionineincorporated into f3-globin expressed in the presence of an antisense oligo was compared with a control, and it was found that a natural (P0) anti-f3-globin-17-merwas quite effective in inhibiting expression, but that the S-oligo was even more effective. The activity of the natural oligos was completely lost when the oligo was injected 6 hr prior to the mRNA, indicating, as expected, that it was premature degradation prior to hybridization that was the main cause of lower activity of the natural oligo. By contrast the S-oligo showed only a small decrease in activity when injected 6 hr prior to the mRNA. Results of Ghosh et al. (1991a,b) on selective vs. nonselective inhibition in reticulocyte compared to wheat germ cell free systems4(Fig.4) confirm that RNase H activity is important (Cazenave et al. 1987). This enzyme cleaves the mRNA in the DNA-RNAhybrid duplex (Dash et al. 1987). This conclusion was supported by the finding that the a-oligo analog, in which the bases are oriented in the direction opposite to the natural configuration at the glycosidic bond (Fig. 1),

Fig. 4. Inhibition of translation in wheat germ extract with a 17-mer anti-lglobin sequence (2 ,uM ; oligo : mRNA=10) targeted towards the coding region of rabbit /l-globin mRNA (open bars), with a control for nonselective inhibition using brome mosaic virus (dashed bars). The difference (filled bars) corresponds to the selective antisense inhibition.

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showed no translation arrest activity in the wheat germ system (Cazenave et al. 1989). These results are consistent with the view that RNase-H, if present, is a useful activator of antisense inhibition. Cellular uptake. It has been found that DNA enters cells by transport mechanisms (Bennett et al. 1988; Loke et al. 1988), and several groups have found extensive uptake of oligos (Jaroszewski and Cohen 1991). The uptake of acridine-linked oligothymidylates is length dependent and saturable, and they are mostly in the cytoplasm, with very little in the nucleus (Loke at al. 1989). In competition experiments with acridine-linked natural oligos, it was found that S-oligos are very slowly taken up by HL60 cells. One or two oligonucleotide binding proteins have been detected in cells (Loke et al. 1989; Yakubov et al. 1989). Neutral methylphosphonate oligos did not compete for uptake via this mechanism, although charged normal and S-oligos did (Loke et al. 1989). Thus, there appear to be two different mechanisms of cellular uptake : for uncharged oligos by passive diffusion (Miller 1989) and for charged oligos by endocytosis. The latter mechanism may have the disadvantage of long-term sequestration of oligos in endosomes, reducing their intracellular availability and stability. Cancer related inhibition. To demonstrate the antisense approach in a cellular system it is preferable to have an example that has clinical significance. The bcl-2 gene is implicated in most lymphomas and many leukemias. Inhibition of expression of this oncogene has been reported using a 20-mer sequence antisense to the translation initiation region (Reed et al. 1990). This was true for both natural and S-oligos. Although the natural oligodeoxynucleotide was faster acting than the S-oligo of the same sequence, the latter was much more potent

Fig.

5. Concentration dependence of inhibition of 697 leukemia cell production of bcl-2 protein using an antibody assay by bcl-2 antisense 20-mer oligomers (hatched bars), and control sense oligomer (black bars) both all-PO and all-PS.

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(Fig. 5).

Perspectives Recently published results of selective inhibition of bcr-abl sequences by natural antisense oligos in cells of patients with chronic myelogeneous leukemia (Szczylik et al. 1991) indicate the clinical potential of this approach. Similarly indicative is a report of effective targeting a splice site in the c-myc expression in lymphoma (McManaway et al. 1990). Two recent publications also utilized phosphorothioate oligomers successfully in inhibiting growth in oocytes (Vgl ) (Woolf et al. 1990) and c-myc in breast cancer cells (Watson et al. 1991). The cost to produce oligodeoxynucleotides are currently very high (Geiser 1990), but are rapidly being decreased. Preliminary results of pharmacokinetics of natural and phosphorothioate analogs in mice and rats have recently been described. The results of single injections in mice showed a significant difference between natural phosphodiester and phosphorothioate analogs ; the latter had a longer retention time that make them potential therapeutic agents (Iversen et al. 1991). No significant toxicity was observed in an extensive study of phosphorothioate oligos in mice and rats (Agrawal et al. 1991). Problems of efficient production, effective delivery, and higher potency, that are the basis of any new drug development, will have to be taken particularly seriously for such costly substances as oligodeoxynucleotides. Likely initial applications of oligonucleotides are for topical rather than systemic applications, and/or as adjuvants to established therapies. References 1) Agrawal, S., Temsamani, J. & Tang, J.Y. (1991) Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc. Natl. Acad. Sci. USA, 88, 7595-7599. 2) Bennett, R.M., Hefeneider, S.H., Bakke, A., Merritt, M.J., Smith, D., Mourich, D. & Heinrich, M.L. (1988) The production and characterization of murine monoclonal antibodies to a DNA receptor on human leukocytes. J. Immunol., 140, 2937-2972. 3) Bielinska, A., Shivdasani, R.A., Zhang, L. & Nabel, G.J. (1990) Regulation of gene expression with double-stranded phosphorophioate oligonucleotides. Science, 250, 997-1000. 4) Brill, W.K.-D., Nielsen, J. & Caruthers, M.H. (1989) Synthesis of dinucleoside phosphorodithioates via thioamidites. Tetrahedron Lett., 30, 5517-5520. 5) Cazenave, C., Loreau, N., Thuong, NT., Toulme, J.J. & Helene, C. (1987) Enzymatic amplification of translation inhibition of rabbit /3-globin mRNA mediated by anti-message oligodeoxynucleotides covalently linked to intercalating agents. Nucleic Acids Res., 15, 4717-4736. 6) Cazenave, C., Stein, C.A., Loreau, N., Thuong, N.T., Neckers, L.M., Subasinghe, H., Helene, C., Cohen, J.S. & Toulme, J.J. (1989) Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodexynucleotides. Nucleic Acids Res., 17, 4255-4273. 7) Cech, T.R. (1986) The generality of self-splicing RNA : Relationship to nuclear mRNA splicing. Cell, 44, 207-210.

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8) Cohen, J.S., ed. (1989) Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, Fl. 9) Cohen, J.S. (1991) Informational drugs : A new concept in pharmacology. Antisense Res. Dev., 1, 191-193. 10) Cooney, M., Czernuszewicz, G., Postel, E.H., Flint, S.J. & Hogan, ME. (1988) Site specific oligonucleotide binding represses transcription of the human c-myc gene in vitro. Science, 241, 456-459. 11) Dahl, B.H., Bjergarde, K., Sommer, V.B. & Dahl, 0. (1989) Deoxyribonucleoside phosphorothioates. Preparation of dinucleoside phosphorodithioates from nucleoside thiophosphoramidites. Acta Chem. Scand., 43, 896-901. 12) Dash, P., Lotan, I., Knapp, M., Kandel, ER. & Goelet, P. (1987) Selective elimination of RNAs in vivo : Complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity. Proc. Natl. Acad. Sci. USA, 84, 7896-7900. 13) Eckstein, F. & Gish, G. (1989) Phosphorothioates in molecular biology. TIBS, 14, 97-100. 14) Eguchi, Y., Itoh, T. & Tomizawa, J. (1991) Antisense RNA. Annu. Rev. Biochem., 60, 631-652. 15) Geiser, T. (1990) Large-scale economic synthesis of antisense phosphorothioate analogues of DNA for preclinical investigation. Ann. N. Y. Acad. Sci., 616, 173-183. 16) Ghosh, M., Ghosh, K. & Cohen, J.S. (1991a) Oligonucleotide phosphorothioatephosphodiester co-polymers : Assessment for antisense applications. Anti-Cancer Drug Design. in press 17) Ghosh, M., Ghosh, K., Dahl, 0. & Cohen, J.S. (1991b) Evaluation of phosphorodithioate oligodeoxyribonucleotides for antisense applications. in preparation 18) Inouye, M. (1988) Antisense RNA : Its functions and applications in gene regulation - A review . Gene,72, 25-34. 19) Iversen, P., Mata, J. & Zon, G. (1991) The single-injection pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev (art/ trs) from the human immunodeficiency vius (HIV) in the adult male rat. J. Pharmacol. Exp. Ther. in press 20) Jaroszewski, J.W. & Cohen, J.S. (1991) Cellular uptake of antisense oligodeoxynucleotides. In: Advanced Drug Delivery Reviews, edited by R.L. Juliano, Elsevier, Amsterdam, pp. 235-250. 21) Jaroszewski, J.W., Syi, J.-L., Maizel, J. & Cohen, J.S. (1992) Towards rational design of antisense DNA : Molecular modelling of phosphorothioate analogues. Anti-Cancer Drug Des., 1, 253-262. 22) Krystal, G.W., Armstrong, B.C. & Battey, J.F. (1990) N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts. Mol. Cell. Biol., 10, 41804191. 23) Loke, S.L., Zhang, X.H., Stein, C.A., Avigan, M., Cohen, J.S. & Neckers, L.M. (1988) Delivery of c-myc antisense phosphorotioate oligodeoxynucleotides to hematapoietic cells in culture by liposome fusion. Curr. Top. Microbiol. Immunol., 141, 282-289. 24) Loke, S.L., Stein, C.A., Zhang, X.H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J.S. & Neckers, L.M. (1989) Characterization of oligonucleotide transport into living cells. Proc. Natl. Acad. Sci. USA, 86, 3474-3478. 25) McManaway, ME., Neckers, L.M., Loke, S.L., Al-Nasser, A.A., Redner, R.L., Shiramizu, B.T., Goldschmidts, W.L., Huber, BE., Bhatia, Kishor & Magrath, I.T. (1990) Tumor-specific inhibition of lymphoma growth by an antisense oligodeoxynucleotide. Lancet, 335, 1, 808-811. 26) Miller, P.S. (1989) Non-ionic antisense oligonucleotides. In : Oligodeoxynucleotides. edited by J.S. Cohen, CRC Press, Boca Raton, FL. 27) Moffat, AS. (1991) Triplex DNA finally comes of age. Science, 252, 1374-1375. 28) Mol, J.N.M. & van der Krol, A.R., eds. (1991) Antisense Nucleic Acids and Proteins.

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Marcel Dekker Inc., New York. Povsic, T.J. & Dervan, P.B. (1990) Sequence-specific alkylation of double-helical DNA by oligonucleotide-directed triple-helix formation. J. Am. Chem. Soc., 112, 9428-9430. Reed, J.C., Stein, C.A., Subasinghe, C.A., Haldar, S., Croce, CM., Yum, S. & Cohen, J.S. (1990) Antisense-mediated inhibition of BCL2 protooncogene expression and leukemic cell growth ; comparisons of phosphorothioate oligonucleotides. Cancer Res., 50, 6565-6570. Stein, C.A., Shinozuka, K., Subasinghe, C. & Cohen, J.S. (1988) Physiochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res., 16, 32093221. Szczylik, C., Skorski, T., Nicolaides, NC., Manzella, L., Malagunera, L., Venturelli, D., Gerwitz, A.M. & Calabretta, B. (1991) Selective inhibition of luekemia cell proliferation by BCR-ABL antisense oligonucleotides. Science, 253, 562-565. Uhlmann, E. & Peyman, A. (1990) Antisense oligonucleotides : A new therapeutic principle. Chem. Rev., 90, 543-584. Watson, P.H., Pon, R.T. & Shiu, R.P.C. (1991) Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role in the growth of human breast cancer. Cancer Res., 51, 3996-4000. Woolf, TM., Jennings, C.B.G., Rebagliati, M. & Melton, D.A. (1990) The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic Acids Res., 18, 1763-1769. Yakubov, L.A., Deeva, E.A., Zarytova, V.F., Ivanova, EM., Ryte, AS., Yurchenko, L.V. & Vlassov, V.V. (1989) Mechanism of oligonucleotide uptake by cells : Involvement of specific receptors? Proc. Natl. Acad. Sci. USA, 86, 6454-6458.

Selective anti-gene therapy for cancer: principles and prospects.

Oligodeoxynucleotides can act as antisense complements to target sense sequences of natural mRNAs to selectively regulate gene expression by translati...
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