Pharmac. Ther. Vol. 52, pp. 211-225, 1991 Printed in Great Britain.All rights reserved

0163-7258/91 $0.00+ 0.50 © 1992PergamonPresspie

Free-Standing Review

OLIGONUCLEOTIDES AS THERAPEUTIC AGENTS JACK S. COHEN Pharmacology Department, Georgetown University Medical School, Washington DC, U.S.A. A~tract~ligodeoxynucleotides can act as antisense complements to target sequences of mRNAs to selectively regulate gene expression. Chemically modified analogs that are nuclease-resistant enable this antisense strategy to be utilized in practice. Studies with oligodeoxynucleotideanalogs in cell free systems, and their cellular uptake will be described. Certain analogs have been found to regulate viral and cellular gene expression. However, some also inhibit in a non-specificmanner, that may be traced to their selective inhibition of viral and cellular polymerases. A chemically modified oligodeoxynucleotideanalog can be regarded as an informational drug.

CONTENTS 1. Introduction 2. Properties of Oligodeoxynucleotides 2.1. Synthesis 2.2. Characterization 2.3. Conjugates 2.4. Cellular uptake 3. Antisense Applications of Oligodeoxynucleotides 3.1. Cell-free and model systems 3.2. Viruses 3.2.1. Antisense inhibition 3.2.2. Sequence non-specific inhibition 3.3. Cellular genes 3.4. General conclusions 4. Other Applications of Oligonucleotides 4.1. Inhibition of polymerases 4.2. Triplex formation 4.3. Ribozymes 4.4. Aptamers 5. Therapeutic Considerations References

1. INTRODUCTION Oligodeoxynucleotides (Fig. 1) are a potential novel form of therapeutic agent (Cohen, 1989; Uhlmann and Peyman, 1990). They are intermediate in size between small drug molecules usually derived from natural products, and large biological macromolecules. Small drug molecules usually function by binding to a protein active site (Fig. 2), and by inhibiting the enzyme action thereby preventing operation of a metabolic pathway. By contrast, oligodeoxynucleotides are small segments of DNA that are synthesized in the laboratory, and that have informational properties through their base sequence (Fig. 2). Their base sequence (the antisense sequence) is made complementary to that present in the mRNA (the sense sequence) of a target gene

211 214 214 215 215 216 218 218 219 219 219 219 220 221 221 221 222 222 222 223

in a cell or virus. By binding specifically to that target sequence, by a process termed hybridization, they can form a hybrid D N A - R N A duplex and prevent the translation of that gene into protein, a process termed translation arrest (Fig. 3). If a cell or virus depends for its survival on that particular gene product then there is the basis for very selective drug action. It should be noted that the production of antisense mRNA as a natural regulatory mechanism has been characterized in prokaryotes (Inouye, 1988), and eukaryotes (Krystal et al., 1990). Since it has taken cons of evolution to produce the genetic code that depends upon Watson--Crick base-pairing, it is an appropriate strategy to exploit that highly specific interaction to produce a new generation of informational drugs (Cohen, 1991a) based upon what we now know of the molecular biology of the cell and the base sequences

211

212

J. S. COHEN

H H _j

X

Protein-drug binding

OPHOSPHATE OR PHOSPHATETRIESTER Me METHYLPHOSPHONATE SPHOSPHOROTHIOATE NR2 PHOSPHORAMIDATE

O%p/O

DNA or RNA duplex formation

i

Drug

Activ~ site

x/ \o-~ C H U B o% /o

Number of H-bonds ~ 4

--I x/P\O--in

Number of H-bonds ~ 60

FIG. 2. Representation of the binding of a small organic drug molecule to a protein active site, and comparison of the binding of an oligonucleotide. Note that in the latter case one of the strands in the duplex could be a cellular nucleic acid and the other an exogeneonsly added oligomer.

HO FIG. 1. The structure of an oligodeoxynucleotide showing some common modifications at the phosphodiester group

Translation Arrest

(B = A, T, C, G).

of many genes. In general the properties desirable in an oligonucleotide for antisense applications are listed in Table 1. Normally, single and double-stranded D N A is degraded by nucleases found in cells and in the body (Fig. 4), so that it is necessary to protect these information-containing drugs from degradation. Note that ribo-oligomers are even more rapidly cleaved by ubiquitous ribonucleases (as well as being more difficult to synthesize), and consequently the deoxyribooligomers are used for these applications. Protection against nucleases is achieved by using chemically modified oligodeoxynucleotide analogs. Several forms of analog have been developed for this purpose; in principle it is possible to modify the base, the deoxyribose moiety, or the phosphodiester backbone (Fig. 5). In general it is preferable not to modify the bases, because the whole strategy of the antisense approach depends upon the intactness of the Watson-Crick base pairing. In some cases modified bases might be indicated for the covalent addition of active groups (Bashkin et al., 1990), although they may also be expected to interfere with the base pairing and stacking in the resultant duplex. Several examples of sugar-modified oligos have been developed that are nuclease-resistant. Of these, the -oligo, in which the base is attached to the glycosidic bond in the non-natural ~-configuration, opposite to the natural #-configuration (Fig. 1) has received the most attention (Morvan et al., 1987). In general, most antisense applications of oligonucleotides

Ant isense Oligo~

Fla. 3. A diagrammatic representation of how an antisense oligonucleotide would bind to its complementary sense mRNA target sequence in order to inhibit translation. Reproduced from Cohen, 1990, with permission of the copyright holder, Elsevier, New York.

have focused on chemical modifications of the phosphodiester group. We have chosen to concentrate on the phosphorothioate (or thiophosphate) analog, in which one of the oxygen atoms of the phosphate group of D N A is substituted with a sulfur atom (hence the convenient terminology S-oligo). This confers nuclease-resistance on the molecule, but without changing its properties too much, so that it can still hybridize with its target m R N A sequence (Stein et al., 1988). This is an important consideration since the chemical modification of a complex molecule like an oligodeoxynucleotide can significantly affect its ability to form duplexes. In fact, while the substitution of a sulfur atom for an oxygen is a very conservative substitution, nevertheless it does decrease the ability to hybridize, or anneal, with the target sequence. But,

TABLE 1. Properties of Antisense Oligonucleotides Property Stability Length Composition Sequence Hybridization

Description Nucleases degrade PO bonds 15--30 mers >50% GC content 5' initiation codon target Tm> 37~C

Comment Use resistant analog One target site/avoid mismatches For good hybridization Passive inhibition Measure melting curve

Oligonucleotides as therapeutic agents *~ Exonuclease

213 BASE

DNA

f Endonuclease

FIG. 4. Representation of the nuclease that are present in organisms, cells and media. Exonucleases, which are most abundant, cleave at the ends of the oligomer, while endonucleases cleave in the middle of the chain. Reproduced from Cohen, 1990, with permission of the copyright holder, Elsevier, New York. the decrease is not too large, so that an S-oligo of 15-30 bases in length can still be expected to interact selectively with its target sequence at 37° (Stein et al., 1988). It is fortuitous that the length of an oligonucleotide required to hybridize effectively with its complement is also approximately the same length required to give a unique sequence in the human genome, composed of several billion bases. This ensures the selectivity of the antisense strategy. Many examples of the use of oligodeoxynucleotides and their analogs to bring about translation arrest in viruses and mammalian cells have been reported (Cohen, 1989; Uhlmann and Peyman, 1990). Note that natural phosphodiester oligomers will effect antisense inhibition in vitro provided all nucleases in the culture medium are removed,

OLIGONUCLEOTIDE =1 PRODUCT

DEPROTECTION

N2

X~p/U

PHOSPHATE

FIG. 5. Indication of the three moieties in the nucleotide monomerie unit, base, sugar and phosphate, that could be chemically modified to confer nuclease resistance. Reproduced from Cohen, 1991a, with permission of the copyright holder, Mary Ann Liebert, Inc., New York. either by using serum-free medium, or heat-treating the medium, or adding nuclease inhibitors. Note that these approaches are not available in vivo if one wishes to use oligonucleotides as therapeutic agents. Following consideration of some of the salient features of oligodeoxynucleotides, three examples of the application of oligomers to inhibit gene expression will be described, (a) in a model system, (b) against HIV in T-cells, and (c) in a proto-oncogene system. General considerations in the application of such molecules to regulate gene expression will be discussed subsequent to these examples.

°"",,,I ;'oJ

~ H20/12 .

/

o.r

ro,H ,,

OCH3

N2

N1 r~

X N1

X=O,S STEP 5: OXIDATION

Y/ XZ--~--

~

STEP 1:

/

GETRT~[ATION / S T A R T OF T C ~ / SYNTHESIS

OR SULFURIZATION N1

N1

$8

OCH~ -98-100% STEP 4~,,~

STEP 3:

ADDITION ~

°'2~j1~(Ac)2O o

.: f

CH3~O~ N ~ O ' ~ / V ~ WILL NOT REACT FURTHER

DM.P

:g;vI".,o.

.,

TETRAZOLE + ~MTr~ O ' ~ --N(C3H7)2 OCH3

FIG. 6. Automated step-wise synthesis of an oligodeoxynucleotide from phosphoramidite precursors (step 2). Note that the oxidation step (step 5) from P(III) to P(V) can be replaced with a sulfurization step to make phosphorothioates. Reproduced from Cohen, 1990, with permission of the copyright holder, Elsevier, New York.

214

J.S. COHEN 2. PROPERTIES OF OLIGODEOXYNUCLEOTIDES

2.1. SYNTHESIS Methylphosphonamidite

It is instructive to consider the most common synthesis method used in the automated machines (Caruthers, 1985). This is a cyclic process whereby nucleosides are added sequentially as their phosphoramidites to the growing oligonucleotide chain (Fig. 6). The nature of the chemical steps and the synthetic precursors, or synthons, can be altered to produce chemically modified analogs of oligos. For example, if a methylphosphonate analog is desired then a methylphosphonamidite precursor containing a P-C bond would be required (Fig. 7). For the synthesis of phosphorothioate analogs, it is necessary to substitute a sulfurization for the oxidation step from the P(III) level of oxidation to the P(V) level (Fig. 6). This can be accomplished using a variety of sulfurization agents (Stein el al., 1988; Iyer et al., 1990; Applied Biosystems Inc., 1991). An alternative method that has been used to synthesize oligodeoxynucleotides is the hydrogen-phosphonate

OR CH3

P/

\.-ip~,

FIG. 7. Structure of a methylphosphonamidite required to synthesize methylphosphonates (see Fig. I) via the stepwise automated procedure shown in Fig. 6. method. This involves the use of hydrogen-phosphonate precursors, and the preparation of a hydrogen-phosphonate oligomer, which is then oxidized in one step (Froehler et al., 1986; Andrus et al., 1988). Alternatively, one-step sulfurization of all positions PS

PO

PO-PO-PO-PS

PO-PO-PS

PO-PS

PO-PS-PS

AII-PS

AII-PO

Hl~'~q~'~rN HINHIfNq'~*JI''HI'''*I''''I*'''IIINI''HI'H'I'''~I'*'' 60 50 40 30 20 10 0 --10 PPld

FIG. 8. 31p NMR spectra at 160 MHz of a series of copolymer oligodeoxy-nucleotides with the sequence of natural phosphodiester (PO) and phosphorothioate bonds (PS) shown.

215

Oligonucleotides as therapeutic agents

of 31P NMR. This method allows the nature of the substituent at phosphorus to be identified, because of the effect on the frequency (chemical shift) of the 31P resonance. For example, there is a very large (56 ppm) difference between PO and PS signals (Fig. 8). This allows the detection of impurity levels of PO ( > 3 % ) in PS analogs, and the quantitation of the relative proportions of PO and PS in co-polymers (Fig. 8). FIG. 9. Fluorescence micrograph of HL60 cells containing fluorescently linked oligodeoxynucleotides. Cells were incubated with Acr-dT7 for 16 hr. Reproduced from Loke et al., 1989.

2.3. CONJUGATES Chemical groups of various types can be attached to oligonucleotides, either at the 5' or the 3' end, or in the middle, and these conjugates can confer different properties on the molecule (Goodchild, 1990). For example, the addition of a planar aromatic group improves the binding of a short oligomer due to intercalation of the ring between base pairs (Helene and Toulme, 1989). These molecules are also fluorescent, and this can enable the distribution of the conjugate to be determined by fluorescence microscopy and fluorescent activated cell sorting (FACS). Such groups can also increase the activity of the antisense oligo by adding a reactive component, either as an alkylating group (Knorre et al., 1989), or as a radical producing group. The addition of a peptide (Haralambidis et al., 1987; Bashkin et al., 1990), a polypeptide (Lemaitre et al., 1987), or a cholesteryl group (Letsinger et al., 1989) could aid in cellular uptake. It is, of course, possible to attach two (or more) groups to elicit different properties (Agrawal and Zamecnik, 1990).

can be accomplished (Matsukura et al., 1988), and this approach is particularly useful for the preparation of 35S-labelled oligomers (Stein et al., 1990) for use in cellular uptake and in vivo experiments. 2.2. CHARACTERIZATION The oligomers are purified by reverse phase high performance liquid chromatography (hplc) using acetonitrile solvents, and the phosphorothioates have longer retention times than the natural PO oligomers. The methyiphosphonate oligomers have even longer retention times because they are so much more hydrophobic. Oligomers of lengths greater than ca. 8 are then precipitated by the addition of ethanol. Another more direct means of characterization of phosphodiester backbone modified analogs is the use

Isolation of Oligodeoxynucleotide

HL60 cells

Binding Protein

Sutface prol~lns

todinated

•

eo ~eoe

--...

/\ Concanavallnbeads ~ ~rash& elu~,

~e Oligobindingprob~ln FIG. 10. Schematic representation of the isolation of an oligo-binding protein by affinity chromatography from the radioactively labelled membrane surface proteins of HL60 cell. Reproduced from Jaroszewski and Cohen, 1991, with permission of the copyright holder, Academic Press, New York.

216

J, S. COHEN

Competition with Acr-dTn in HL60 Cells 160

140 ag

120

Z ul

Lu E

~o.

100

a.

0C

80

0

Squaresn=3

\\

Z

.j J

O ~

60

al o

40

E

I,Z --

20

~PhosphorothiS-dT7 oate 1

1

v

v

l

1:0

1:1

1:2

1:4

1:8

DOSAGE

(ACR-OLIGO

: COMPETITOR)

FIG. 11. Competitive inhibition into HL60 cells of Acr-dT3 (squares) and Acr-dT8 (triangles) uptake by phosphorothioate dT7 (open symbols), but not methylphosphonate dT7 (filled symbols). Redrawn from Loke et al., 1989.

2.4.

CELLULAR UPTAKE

It has been the conventional wisdom that charged polyelectrolytes, such as DNA, cannot generally cross the cellular membrane, that is designed to protect the cell against such substances. However, it has been found that quite large molecules, including DNA, can in fact enter cells by definite transport mechanisms (Bennett et al., 1987). The fact that oligos have been found to exert specific antisense effects upon gene expression, that is not found for example by control sense or random constructs, has long been held as indicative of the fact that some intact oligo must be

FIG. 12. Diagrammatic representation of the most likely mechanism of cellular uptake of oligonucleotides, namely endocytosis. Gradual loss of the oligomer into the cytoplasm may also result in nuclear uptake.

entering the cell and reaching the intracellular target (Zamecnik and Stephenson, 1978). Several groups have investigated oligo uptake by cells. All have found extensive uptake, but that is where the similarity in the results ends. 32p-labelled oligos and microradiography have been used to show the intracellular distribution of oligos (Goodchild et al., 1988). Assuming that the oligos remained intact over the time period of the experiments, it was found that the oligos were distributed in both the cytoplasm and the nucleus, but were clustered around the nuclear membrane and the nucleolus. Acridine-linked fluorescent oligos are readily taken up by cells, and by fluorescence measurements are found distributed throughout the cells; for example, Toulme and coworkers in their work on trypanosomes showed that only the nucleolus was not fluorescent (Verspieren et al., 1987). Loke et al. (1989) showed that the uptake of acridine-linked oligothymidylates is length dependent and saturable, and that they are mostly in the cytoplasm, with very little in the nucleus (Fig. 9). They also showed with acridine linked S-oligos, and by competition experiments with acridine linked normal oligo, that S-oligos are very slowly taken up by HL60 cells. By contrast, Egan and co-workers (Marti et al., 1991) found that a fluorescein-linked anti-rev S-oligo was taken up into lymphocytes at approximately the same rate as the neutral congener, and was found predominantly in the nucleus and nucleolus. Thus, it appears that the reporter group could be having a profound influence on the intracellular distribution as well as the mechanism of uptake. Another report of

Oligonucleotides as therapeutic agents

RNase H Function

a) 17PO-p

L

= 1 "=

217

RNase H

a)

RNA f

0.8

: 0.6,

Oligo b)

-~ o.4,

I m

==

c) ['oligonucleot ide)

~M

r

I

b) 17-PS

I

I

FIG. 14. Function of ribonuclease-H. The RNA molecule is cleaved (arrow) where a DNA-RNA hybrid duplex is formed. Reproduced from Cohen, 1990, with permission of the copyright holder, Elsevier, New York.

~0.8

0.1

el .= O.

0.=

0.4

.Q

' i

o~I

4

i

1"15(nM 1

z

[oligonucleot ide]

pM

FIG. 13. Effect of a 17-mer (5'-d-CACCAACTTCTTC-

CACA) antisense to fl-globin mRNA (a) natural (PO) phosphodiester, and (b) its phosphorothioate (PS) analog. Globin synthesiswas determined from densitometer tracings of the autoradiographs, relative to expression determined in the absence of added oligodeoxynucleotide. Circles, oligos coinjected with mRNA; triangles, oligos injected 6 hr prior to mRNA; squares, oligos injected 6 hr after mRNA. The inset in panel (b) is an enlargement of the lowest curve in (b). Reproduced from Cazenave et al., 1989,with permission of the copyright holder, IRL Press, Ltd., Oxford.

15 I

microinjection of several types of oligonucleotides into cells concluded that there is rapid nuclear accumulation (Chin et al., 1990). Loke et al. (1989) have also reported the observation of a single band of ca. 80 kDa from the labelled surface protein of several cell lines that had been selectively bound to oligo-dT beads on a column (Fig. 10). This indicates a receptor protein for specific transport of charged oligos. Similar results have been reported by Vlassov and co-workers (Yakubov et al., 1989). Interestingly the natural methylphosphonate oligos did not compete for uptake via this mechanism, although charged normal and S-oligos did (Fig. 11). Thus, one can envisage completely different mechanisms of cellular uptake for uncharged oligos by passive diffusion (Miller, 1989) or by endocytosis (Fig. 12). However, while the latter mechanism may

3001"

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