Antisense oligonucleotides for therapeutic intervention Stephen L. Eck and Gary J. Nabei University of Michigan Medical Center, A n n Arbor, Michigan, USA Advances have been made in defining the best target sequences for use in antisense oligonucleotide technology, and new chemical derivatives of oligonucleotides are being investigated. Although the potential use of antisense oligonucleotide agents in the treatment of neoplastic, viral and parasitic diseases continues to be explored, they are not yet suitable for administration to humans for reasons that are discussed. Current Opinion in Biotechnology 1991, 2:897-904

Introduction Selective inhibition of gene expression by complementary nucleic acid sequences provides a method to define the role of specific genes in disease and during development. Although the concept of 'antisense' RNA targeting has long proven attractive [1,2], its application to biological systems has remained difficult. More recently, the development of synthetic oligonucleotide technology has attracted renewed interest in this subject and its potential therapeutic applications. Antisense oligonucleotides are short synthetic nucleic acids that are complementary to a specific mRNA or DNA sequence. Oligonucleotide binding to a target sequence can potentially interfere with gene expression through a variety of mechanisms. When sequence-specific binding can be achieved within cells, this approach can serve to selectively eliminate gene expression, providing a pharmacologic alternative to other methods of gene targeting, such as homologous recombination, tmnsgenic expression of recombinant DNA, or the use of transdominant inhibitors, which are more difficult to apply to the treatment of human disease. It also complements gene transfer technologies that deliver recombinant genes encoding antisense n m N ~ [3]. Biologic control by naturally occurring antisense RNAs in prokaryotic [4] and eukaryotic [5,6] systems provides a precedent for the use of artificial antisense oligonucleotides and provides clues to potential sites of intervention. In many cases, chemical modification of the phosphodiester backbone has been developed to improve the performance of unmodified oligonucleotides [7]. The methytphosphonate, phosphorothioate, phosphotriester, 2'-O-alkyl and ~x-monomeric oligonucleotide congeners are nuclease resistant and have a prolonged half-life in tissue culture [7,8.]. Intercalating agents have been coupled to oligonucleotides to increase their target aff'anity, and functional derivatives including crosslinldng agents and metal complexes have been incorporated to provide

for chemical modification of the target site [7,8°]. Of these synthetic derivatives, unmodified oligonucleotides, methytphosphonates, and phosphorothioates are the easiest to synthesize and are the most widely used. Despite these advances, several fundamental problems remain to be solved before antisense therapy can become a reality (Table 1). A major issue is the concentration required to achieve therapeutic effects and to avoid potential toxicities. The oligonucleotides currently In use require significant extracellular concentrations to ensure adequate cellular uptake. In many instances, these concentrations cause non-specific binding to cellular proteins or nucleic adds, which will probably contribute to systemic side-effects. In other cases, contaminants and variation in oligonucleotide preparations may cause biologic effects that might then be improperly attributed to the antisense agents being used [9,10]. In every case, these problems must be carefully addressed. Other potential problems are that the immune system may respond to high serum concentrations of extracellular DNA and that antisense agents may prove to have organ toxicity. Nonetheless, many recent reports iUustrate the use of antisense oligonucleotides in the laboratory that have potential application to human diseases (Table 1).

Selection of target sequences Several mechanisms have been postulated to account for oligonucleotide-mediated inhibition of gene expression (Fig. 1). In theory, antisense agents could inhibit transcription, RNA processing, or translation: formation of triple helical DNA could interfere with transcriptional initiation or elongation; binding to RNA transcripts could prevent processing, splicing, or transport from the nucleus; and other sites can affect protein synthesis (for example, blocking the ribosomal binding site can inhibit translational initiation), or might interfere with translational elongation. Other potential mechanisms in-

Abbreviations bFGF~basic fibroblast growth factor; CSF~olony-stimulating factor, HW--human immunodeficiencyvirus; HSV--herpes simplex virus; PK---protein kinase. ~) Current Biology Ud ISSN 0958-1669

897

898 Pharmaceuticalapplications Table 1.1he limitations and potential applications of antisense oligonucleotides for the treatment of human disease. limitations tack of effective delivery to specific cells Poor cellular uptake Non-specific binding to cellular proteins limited half-life within cell Difficulty in the identification of relevant target sequences Unknown mechanisms of action Need for large-scale synthesis Potential toxicity of in vivo treatment Potential applications £x v/vo purging of cancer cel/s from bone marrow Topical treatment of skin disorders Antiviral therapy Suppress chromosomal translocation expression Antiparasitic therapy Inhibitors of inflammation Identification of potential targets of drug design and gene therapy

volve oligonucleotide-promoted degradation of mRNA by RNase H, interference with reverse transcription of viral genomic RNA, or the use of double-stranded oligonucleotides to inhibit DNA-binding proteins. Although a large number of target sequences can be targeted in theory, in practice oligonucleotides often do not act as intended. Limiting factors include the distribution of RNase H activity within the cells [11], interference by RNA secondary structure [11,12-.] and inefficient cellular uptake and transport of oIigonucleotides [11,13,14"]. In one study, the di~culty in predicting effective target sequences was illustrated using oligonucleotides (15 bp each) directed to 10 sequences of c-myc mRNA [12-o]. These agents were designed on the basis of the calculated secondary structure of the mRNA. Despite predictions that the translation initiation (AUG) site would be more accessible than the cap site, the oligonucleotide complementary to the cap sequence was twice as effective in inhibiting cell proliferation. in general, the degree of antisense inhibition does not correlate with predicted awdlability of the target region using currently available algorithms. In the case of c-myc, most consistent success was achieved with the 5'-cap site, followed by the first splice donor, translational initiation site, and other targets in the 5' untranslated leader sequence [12-°]. An increase in the oligonucleotide length beyond that required to hybridize to a unique sequence also increased efficacy. For example, the degree of inhibition at the translational initiation site was found to decrease as the length of the oligonucteotide decreased from 18 to 15 to 12 bases [12--]. These findings parallel results with c-Ha-ras mRNA in NIH 3T3 cells [11].

Anlineoplastic agents Cancer chemotherapy often suffers from a lack of cell specificity, which results in a myriad of dose-limiting side

effects. In many instances, activated oncogenes provide a molecular genetic difference between neoplastic and normal cells. Because the products of these genes contribute to transformation, they represent attractive targets for the design of new therapeutic agents. The c-rnyb proto-oncogene encodes a protein that is an important regulator of lymphocyte proliferation and hematopoiesis. It is also expressed in a variety of malignancies. An anti.sense oligonucleotide complementary to the translation initiation site of c-myb mRNA inhibited cell-cycle S-phase entry and cell proliferation in phytohemagglutinin-stimulated human peripheral blood mononuclear cells [15] and in CCRF-CEM leukemic cell cultures [16-.]. This phenotype was attributed to a decrease in DNA polymerase-ec activity observed after cmj~ antisense treatment [16-.1. To explore the potential application to human leukemias, leukemic cells from 32 patients with T-cell leukemias were incubated with the antisense or sense c-myb oligonucleotides. A decline in DNA synthesis was noted in cells from 20 patients [16 .o] after antisense treatment; c-myb mRNA was not detectable in any of these cultures. Leukemic cell cultures in which DNA synthesis was unaffected by antisense treatment showed no change in DNA polymerase-0c activity. This finding suggests that some ceils might proliferate by mechanisms independent of c-myb [16oo]. Normal and leukemic ceils may also have different sensitivity to antisense c-myb treatment in vitro T leukemia (CEM) cells were specifically growth inhibited by antisense cmyb oligonucleotides under conditions where normal marrow ceils were unaffected [17oo]. Primary leukemic progenitor cell growth was also inhibited in 78% of primary acute myelogenous leukemia cultures and in four of five primary chronic myelogenous leukemia blast phase cell cultures treated with c-rnyb antisense oligonucleotide [17o.], suggesting that tumor cells may be preferentially affected by this treatment. Philadelphia chromosome positive CML cells also express the tumor specific bcr-ablfusion gene, which is a marker of the malignant phenotype. After c-myb antisense treatment, the CML cell cultures showed a significant decline in bcr-abl mRNA expression [17.o]. These and other studies suggest that myb antisense oligonucleotides could be used as an ex vivo bone marrow purging agent prior to autologous bone marrow transplantation. Normal hematopoiesis appears to require c-myb [ 18] and, despite the fact that no toxicity was observed in this short-term cell culture study, the question remains whether this result will apply in viva Chromosomal translocations in many lymphomas result in the formation of tumor-specific chimeric transcripts that could also be the targets of antisense oligonucleotides. A recent study suggests that targeting of these chimeric transcripts may provide therapeutic effects in vitro [19o-]. Another gene that may be an important etiologic factor in B-cell lymphomas is bd-2, which has been implicated in programmed cell death (apoptosis) [20]. Expression of the bd-2 gene may inhibit this process [21] and could contribute to oncogenesis. Suppression of bcl-2 expression in follicular lymphomas could be the basis of a therapeutic intervention. Antisense oligonucleotides complementary to /x:l-2 mRNA have been

Antisense oligonucleotides for therapeutic intervention Eck and Nabel

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TITITFFF Fig. 1. Potential antisense targets and mechanisms of inhibition. Inhibition of DNA transcription (a) by blocking of transcription factor binding by double-stranded oligonucleotides, (b) by triplex formation, or (c) by blocking RNA polymerase activity. Inhibition of RNA processing (d) by binding to nascent transcript, (e) by binding to the splicing site, or (13 by preventing transport from the nucleus. Inhibition of translation (g) by binding to the cap site, (b), translation initiation site or by (i) forming a duplex substrate for RNase H mediated cleavage. (j) Oligonucleotide binds protein necessary for nucleic acid processing.

shown to suppress proliferation of lymphocytic leukemia cell lines in vitro [22..,23]. Unmodified and phosphorothioate antisense oligomers decreased bcl-2 protein levels and cell proliferation in a dose- and sequencedependent manner, with the phosphorothioates being five to ten times more potent, but slower in onset of action [22-]. This delayed effect may reflect the previously recognized greater stability but slower uptake of the phosphorothioate oligonudeotides in general. Antisense agents to the bcl-2-immunoglobulin fusion mRNA might also potentially be used to treat lymphomas carrying the t(14 : 18) translocation [22*°]. Another aberrant mRNAinvolves the c-mycgene on chromosome 8. In a high proportion of Burkitt lymphomas, chromosome translocation leads to transcription and initiation from cryptic promoters in the first intron of c-myc [24-.], and this gene has been the target of several antisense oligonucleotide agents. An oligonucleotide complementary to a 21-base sequence within the first intron of c-myc resulted in a specific decrease in c-Myc protein

levels and a 76% to 111% growth inhibition in Burkitt lyrnphoma cell lines, whereas the translation of normal c-myc mRNA was unaffected [24°°] compared with untreated or gene controls. The assay doesn't distinguish between cell dection and growth arrest. Similar investigations have been performed with N-myc [25"], which has a more limited tissue expression (fetal tissue, immature lymphocytes, and certain neuroendocrine tumors) than c-mya Ant/sense oligonudeotides complementary to N-myc mRNA decreased N-Myc protein levels and sequence-specific growth inhibition in a neuroepithelial cell line [25*]. Antisense agents that distinguish between members of the ras oncogene family have also been described [26]. Other successful attempts at blocking the autostimulatory effects of oncogenes have also been described. Colonystimulating factor (CSF-1), a transmembrane glycoprorein that is cleaved to form a soluble growth factor, is necessary for the normal growth and differentiation of monocytes. Antisense oligonucleotides directed at CSF-1

899

900 Pharmaceuticalapplications mRNA in a growth-factor-independent cell line (which constitutively expresses CSF-1) demonstrated a 60-75% growth inhibition [27]. Simultaneous administration of a monoclonal antibody recognizing CSF-1 resulted in a 95% inhibition of cell growth. Removal of the antibody and antisense agent resulted in reversal of the inhibition [27], suggesting that CSF-l-dependent cell growth can be inhibited by a combination of antisense and antibody agents. Similarly, suppression of basic fibroblast growth factor (bFGF) by antisense oligonucleotides [28] resulted in growth inhibition of transformed but not non-transformed glial cells. Removal of the ollgonucleotide or addition of exogenous bFGF reversed the growth inhibitory effects [28]. Myeloma cell division has been inhibited by antisense oligonucleotides which block prothymosin-ct synthesis [29]. Although prothyrnosin-cx appears to have an important rote in cell division, its inhibition will be of limited value as an antineoplastic measure because it is present in most mammalian tissues. Proteins involved in signal transduction may function abnormally in cancer ceils and have also been investigated as potential sites of antisense intervention. For example, isoforms of cAMP-dependent protein kinase (A) regulatory subunits are differentially expressed during ontogeny and cell differentiation [30] and its activated form promotes cell growth inhibition, differentiation, and reversal of the transformed phenotype of several cancer cell lines. PKA is a tetramer (RzC2) of two regulatory subunits (R), which bind cAMP, and two catalytic subunits (C). Four isoforms of the regulatory subunit (RIa, RII~, Rlla, RIII3) have been identified, and antisense oligonucleotides have been used to suppress transcription of Riot and RUl3mRNA [30~,1,32"]. In HL60 promyelocytic leukemia ceils, a RIII~ antisense oligonucleotide, unlike RIII~ sense, R/I~ anti.sense, RIIct antisense, or irrelevant oligonucleotides, decreased RIII3protein levels and prevented cAMP analog-induced growth inhibition and differentiation [31]. Phorboli myristale acetate-induced differentiation was unaffected. In contrast, RIa antisense DNA produced a decline in RIa mRNA and protein levels and an increase in RIII3 protein levels, as well as promoting cell growth inhibition and monocytic differentiation. This process was not augmented by addition of phorbol myristate acetate or a cAMP analog [32"]. Interestingly, the effect of RIa antisense DNA could be abolished by the addition of RIII3 antisense DNA, consistent with the hypothesis that RIet is an oncogenic protein, whose constitutive expression can lead to neoplastic growth. Blockhag RIctprotein synthesis therefore remains an oncogenic stimulus and promotes differentation. Growth inhibition in these experiments was not associated with a decline in cell viability; however, RIccantisense-induced growth inhibition was associated with morphological changes indicative of differentiation. RIa antisense ollgonucleotide increased the RII3:RIa ratio 25-fold, much like treatment with cAMP analysis. The RL, antisense DNA has also been tested in human colon, gastric, and breast carcinoma cell lines with similar results33"]. Taken together, these studies suggest that an alteration in the balance of these subunits may result in malignant transformation, a process which can be potentially reversed by RI~ antisense

intervention [32",33"', 34 ]. Antisense inhibition of the catalytic subunit of PKA in normal mouse mammary cells has also been described [35]. Other processes involved in tumor progression can conceivably be inhibited for therapeutic benefit. Suppression of urokinase expression by a tmnsfected gene encoding an antisense mRNA correlated with loss of metastatic potential in a melanoma cell line [36]. Other potential areas of antisense application will become apparent as our knowledge of tumor biology increases.

Antiviral therapy Many viral pathogens, most notably human immunodeficiency virus (HN), cannot be successfully treated yet with conventional therapeutics or immunizations. Because viral-specific sequences can serve as unique targets which will not interfere with normal cellular function, antisense or ribozyme agents could provide an attractive treatment for these diseases. Transfected genes encoding antisense RNA have been used to inhibit viral replication in ceils harboring human T-cell leukemia virus-1 [36] and H1V [37]. Recently, analogs of the H1V tat-I target RNA structure have been used successfully to prevent HIV infection in vitro [38]. In addition, ribozymes have shown promise against HIV mRNA in vitro and in cells [39]. Expression of ribozymen are entended to degrade H N to mRNA selectively. These approaches have considerable promise as potential gene therapies for HIV infection. To improve the efficiency of antisense oligonucleotides as an acquired immune deficiency syndrome treatment, Renneisen et al. [40] have used an antibody (anti-CD3) coated liposome to target antisense RNA into H1V-infected cells. The in vitro synthesized mRNA, complementary to exon II of the HIV tatgene, specifically and almost completely inhibited HIV production. Several studies have also analyzed the sensitivity of H1V to antisense oligonucleotides. For example, laurence et at [41] prepared antisense methylphosphonate oligonucleotides to several regions within genomic RNA and mRNA transcripts of HIV-1, including initiation sites, splice sites, the tar region, the negative regulatory element and the ×B enhancer element. Of all these only the last was effective, but these findings are difficult to interpret because the methylphosphonates were short (8-12reefs) and do not provide substrates for RNase H mediated cleavage on binding to the target RNA site. Both of these factors have been shown to be important in other studies

[8.1. Methylphosphonate oligonucleotides complementary to the c~acting ×B enhancer dement within the H1V long terminal repeat (LTR) blocked PMA-mediated upregulation of the virus in chronically infected cells [41], although the mechanism of this effect is unclear. In the case of viral replication, modified oligonucleotides have several advantages over transfected antisense RNA, but their mechanism of action is unknown. For example, phosphorothioate oligonucleotides are nuclease-re-

Antisense oligonucleotidesfor therapeutic intervention Eck and Nabel 901 sistant, lack appreciable secondary structure, may have higher binding constants, and are more sensitive to small sequence differences between host cell and viral mRNA; but several non-sequence-specific effects have been observed. The phosphorothioate analog of oligodeoxycytidine (dC28) binds reverse transcriptase and inhibits HIV replication in a non-sequence-specific fashion [42,43]. It also inhibits herpes simplex virus (HSV)-2 DNA polymerase more than human DNA polymerases [44] and shows a concentration-dependent reduction of cellular uptake of HSV-2 [45"]. This may be due to phosphorothioate interference with HSV binding to heparin sulfate on the cell surface. Interestingly, it was also observed that HSV-2 (but not HSV-1) infection enhanced cellular phosphorothioate uptake and that HSV-2 growth was inhibited to a larger extent than HSV-1 [45-]. Preferential binding of the oligonucleotide to HSV-2 may reflect differences in post-translational modifications of the viral envelope proteins between these HSV types [45-]. Sequence-specific inhibition of reverse transcfiptase has recently been demonstrated. Loreau et aL [46*] prepared unmodified and acridine-linked oligonucleotides that blocked primer extension by avian myelobustosis virus reverse transcriptase in a cell free system. The 'stopper oligonucleotide' led to template RNA degradation by RNase H mediated cleavage [46.]. ev-Anomeric antisense oligonucleotides homologous to RNA primers, which cannot be elongated by reverse transcriptase, have been shown to block primer extension by competing with the endogenous RNA primer [47]. Phosphorothioate oligonucleotides have also been used in a novel fashion to alter viral transcription. Doublestranded phosphorothioates were prepared which contamed the xB or other enhancer elements [48°*]. This oligonucleotide was taken up by cells, bound the transcription factor NF-xB, and specifically blocked PMAinduced (NF-xB-mediated) transcription from an HIV promoter-driven reporter gene [48..]. Targeting specific promoter elements in this fashion may provide another strategy for inhibiting vim/induction in latently infected cells. DNA viruses have also been investigated as potential targets of antisense technology. Goodarzi et at [49] used cap site and translation initiation site directed antisense oligonucleotides to inhibit human hepatitis B surface antigen expression in hepatocellular carcinoma cells that contained hepatitis B virus genomes. Phosphorothioate oligonucleotides produced a greater degree of translational arrest than unmodified oligonucleotides [49]. Such a scheme could potentially be used to ameliorate the deleterious effects of persistent viral antigenemia which, by interaction with the host immune system, may promote liver damage and hepatocel/ular carcinoma [49].

Antiparasitic agents Mature mRNA of trypanosome and leishmania parasites contain a species-specific, conserved nucleotide

sequence (the splice-leader sequence) at the 5'-end. Antisense oligonucleotides complementary to this sequence have been shown to inhibit translation in a cell-free system [50], although modification of the ribonucleotides in this region diminished antisense bindhag. Antisense oligonucleotides have been shown to kill the Trypanosoma bruceiparasite in culture [51,52]. P/asmodium falc.~parum, a protozoal agent causing malaria, synthesizes a bifunctional polypeptide with dihydroreductase and thymidylate synthase activities that is solely responsible for the biosynthesis of thyrnidine-5'-monophosphate [53]. Antisense oligonucleotides have been used to block the synthesis of this key peptide in a cellfree model system. Unfortunately, long oligonucleotides (up to 49mer) were required to achieve significant inhibition of mRNA synthesis. This was due to the low G and C base frequency in P. falcg~arum RNA [54].

New antisense agents The therapeutic potential of oligonucleotides, in general is limited by poor cellular uptake. Shea et aL [54.] demonstrated that phospholipid-antisense oligonucleotide conjugates had a greater cellular uptake and in. hibited protein synthesis more than normal or phosphorothioate oligonucleotides in vesicular stomatitis virus infected cells. Kabanov et aL [55] found that 5'-undecyi antisense oligonucleotides had superior cellular uptake, to antisense and sense oligonucleotides, and that it inhibited influenza A virus, whereas the oligonucleotides had no effect. Degols et al. [56] were able to improve the stability and decrease the dose of an unmodified cmyc antisense oligonucleotide by covalently linking it to polytysine and administering it as a complex with he W arin. This complex may protect the oligonucleotide for nuclease digastion and facilitate entry into cells.

Conclusion Antisense oligonucleotides have many potential therapeutic applications; however, several barriers must be overcome before they can be widely used. The large scale synthesis of these compounds has not been accomplished and their in vivo toxicity is yet to be fully determined. New oligonucleotide analogs, such as the phosphorodiothioate derivatives, may have advantageous biochemical properties, yet effective means of delivery, including targeting specific cells and enhancing cellular uptake, need further development. In the interim, antisense compounds have proved to be of considerable use in the laboratory in the definition of the molecular basis of human disease. Their use in e x vivosettings, for example in bone marrow transplantation or to define chemical structures for rational drug design or for gene therapy, is likely to bring the benefits of this technology to the treatment of human disease.

902 Pharmaceuticalapplications References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •. of outstanding interest 1.

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3.

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VOLK R, KOSTER M, POTING A, HA'I'M&'~ L, KNOCHEL W: An Antisense Transcript from the Xenopus laevts bFGF Gene Coding for an Evolutionarily Conserved 24 kd Protein. EMBO J 1989, 8:2983-2988.

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HELENEC, TOm.ME J-J: Specific Regulation of C_)ene Expression by Antisense, Sense and Antigene Nucleic Acids. B/oc/=n'm B/q0bys Ac.ta 1990, 1049:99-125. The chemical basis of antisense oligonucleotide inhibition is illustrated, along with examples of antisense RNA inhibitors. 9.

BUCK HM, KOOLE LH, VAN GENDEREN MH, SM1Tl.) GEELENJL, J ~ S, GOUDSMrrJ: Phosphate-Methylated DNA Aimed at HIV-I RNA Loops and Integrated DNA Inhibits Viral Infectivity. Sc~,'~ce 1990, 248:208-212.

10.

MOODYHM, QUAEDFtmG PJ, KOOLE Hi, VAN GENDEREN MH, BUCK HM, SMIT L, JURRIAANSS, GEELENJL, GOUDSMITJ: Inhibition of HIV-I Infectivity by Phosphate-methytated DNA: Retraction (of [9]. in Science 1990, 248:208-212). Science 1990, 250:125-126.

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DAAKAY, WICKS'fROM E: Target Dependence of Antisen.~ Oligodeoxynudeotide Inhibition of c-Ha.ra, p21 Expression and Focus Formation in T24-transformed NIH3T3 Cells. Oncogene Re• 1990, 5:267-275.

12. •s

BACON TA, WICKSTROM E: Walking Along Human c-myc mRNA with Antisense Oiigodeoxynucleotides: Matlmtlm Efficiency at the 5' Cap Region. Oncogene Re• 1991, 5:13-19. The calculated secondary structure of omyc mRNA is used to illustrate the dimcuhies encountered in attempting to predict the most efficacious sites for antisense inhibition of mRNA translation. 13.

Ao A, ERICKSON RP, BEVILACQUAA, KAROLYIJ: Anti.sense Inhibition of ~-Glucuronidase Expression in Preimplantation Mouse Embryos: a Comparison of Transgenes and Oligonucleotides. Antisense Research Develop 1991, 1:1-10.

16. **

VENTURELUD, MAmANO MT, SzczYt.m C, VALTmH M, LANGE B, CP.IST W, LINK M, CA1ABRE'I'rAB: Down.regulated c-myb Expression Inhibits DNA Synthesis of T.Leukemia Cells in Most Patients. Cancer Res 1990, 50:7371-7375. Proliferation of some leukemia• is shown to require c-m.)O expression and an antisense agent against c-rr~'b mRNA can inhibit DNA synthesis in these leukemia•. This illustrates the heterogeneity of the neoplastic process, and the need to find targets more essential to neoplastic growth. 17. ..

CALABRETTAB, SIMS RIB, VAL'rmRIM, CARACaOLO D) S/L:ZYIIK C, VENHJRELLID, RATAJCZAKM, BERANM, G~wm~ AM: Norreal and Leukemic Hematopoietic Cells Manifest Differential Sensitivity to Inhibitory Effects of c.myb Andsense Oligodeoxynucleotides: an In Vitro Study" Relevant to Bone Marrow Purging. Proc Natl Acad Sci USA 1991, 88:2351-2355. This paper illustrates selective interference with neoplastic cell metabolism while sparing normal cell function. The potential application to the treatment of leukemias by autologous bone marrow transplamadon is discussed. 18.

MUCENSK1ML, McLAIN K, KmR AB, SWERDLOWSH, SCHREINER CM, MM.ER TA, PmJ~cY'GADW, ScoTr WJ, POTTER SS: A Functional c-myb Gene is Required for Normal Murine Fetal Hepatic Hematopoiesis. Cell 1991, 65.677-689.

19.

SzCZYlm C, SKORSKI T, N ~ C O ~ NC, ~ L, MALAGUARNERA L, VENTUREILI D, GEWEqZ AM, CAI~RETrA B: Selective Inhibition of Leukemia Ceil Proliferation by Bcrabl Antisense Oligonudeotides. Sc/en~ 1991, 253:562-565. Anti.sense oligonucleotides to BCR-ABLselectively suppress leukemia tumor cell growth in v/tra The potential of this approach for treatment of Philadelphia chromosome positive CML is indicated. oo

20. 21.

WIIIJAMSGT: Programmed CeU Death: Apoptosis and Oncogenesis. Ce//1991, 65:1097-1098. HENDERSONS, ROWE M, GREGORYC, CROOM-C_.AKIXRD, WANG F, LONGNECKER R, KmFF E, RICKINSON A: Induction of beg-2

Expression by Epstein--Barr Virus Latent Membrane Protein 1 Protects Infected Cells firom Programmed Cell Death. Cell 1991, 65:1107-1115. 22. **

REEDJC, STEIN C, SUBASLNGHEC, HALOARS, CROCE CM, YUM S, COHEN J: Antisense-mediated Inh~ition of BCL2 Protooncogene Expression and Leukemic Cell Growth and Survival: Comparison of Phosphodiester and Phosphorothioate Oiigodeoxynucleotides. Cancer Re• 1990, 50:6565-6570. The kinetics of phosphodiester and phosphorothioate antisense oligonucleotides are described with respect to their ability to suppress target protein synthesis. Chromosomal translocations provide targets unique to neoplastic cells which may be exploited to therapeutic ad+antage. 23.

REEDJC, CUDDY M, HAtDAR S, CROCE C, NOWELLP, MAKOVER D, BRADLEY K: BCL2-mediated Tumorigenicity of a Human T-Lymphoid Ceil Line: Synergy with myc and Inhibition by bcl-2 Antisense. Proc Naa Acad Sci ~ 1990, 87:3660-3664.

24. **

MC~NAWAYME, NECKER5 LM, LOKE SI., AL-NASSERAA, REDNER RI~ S ~ U BT, GOt.DSCHMm~ WL, HOBER BE, BHATIA K, MCGRAW! IT: Tumor-specific Inhibition of Lymphocyte Growth by an Antisense Oligodeoxynucleotide. Lancet 1990, 335:808--811. An abnormally spliced mRNA provides a tumor specific target which is exploited to inhibit Burkitt bTnphoma cell growth.

CHIN DJ, GREEN GA, ZON G, SZOKA FC, S'I'RAUBINGERRM: Rapid Nuclear Accumulation of Injected Oligodeoxyribonucleotides. New B&~/1990, 2:1091-1100. The mechanism of intracelIular oligonucleotide transport and nuclear incorporation is discussed highlighting sariations within these changes in chemical structure.

ROSEOLENA, WHITESELL L, IKEGAKI N, KENNET1" RH, NECKERS LM: Antisense Inhibition of Single Copy N-myc Expression Results in Decreased Cell Growth Without Reduction of c-myc Protein in a Neuroepithelioma Ceil Line. Cancer Re• 1990, 50:6316-6322. This relx)rt illustrates the selective inhibition of an o~erexpressed oncogene in the presence of other members of the same oncogene family. This may be important for selectively inhibiting cancer cell growth.

I5.

26.

14. •

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25. •

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BmCHENAU.-ROBERTSMC, FEMURC, FEmUS D, FALKLA, KASPER J, WHIIX G, RUSCETn FW: Inhibition of Murine Monocyte Proliferation by a Colony-stimulating Factor-I Antiserise Oligodeoxynucleoflde. Evidence for Autoregulation. J Imm u n 1990, 145:3290--3296.

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SAgromus C, FP~Wa.iN RM: Hybridization Arrest of the C_.eli-free Translation of the Malarial DihydrofoIate Reductase/Thymidylate Synthase mRNA by Andsense Oligodeoxyribonucleotides. Nucleic Acid Res 1991, 19:1613-1618.

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S.L Eck and GJ. Nabel, Howard Hughes Medical Institute, University of Michigan Medical Center, Departments of Internal Medicine and Biological Chemistry, Ann Arbor, Michigan 48109-0650, USA.