This document summarizes a speech given by Michael L. Riordan about the development of oligonucleotide therapeutics. Riordan discusses two main avenues for increasing the potency of oligonucleotides: 1) enhancing cellular permeation through modifications that allow passive diffusion, and 2) targeting DNA instead of RNA to reduce the copy number of the target per cell. He also describes a new technique called aptamers, where oligonucleotides are selected and amplified based on their ability to bind target molecules, allowing novel therapeutics with high affinity and specificity to be developed.

1. OUGONUCLEOTIDE1HERAPEUTICS:11fE PLOTnDCKENS
Followingis a transcript of a speech deliveredon January 16, 1991, by Michael L~·Riordan,M.D.,
President and Chief Executive Officer of Gilead Sciences,Inc., at the International Conferenceon
Nucleic Acid Therapeutics sponsored by the International Union of Biochemistry and the U.S.
National CancerInstitute, held in ClearwaterBeach,Florida.
I would like to begin with these insightful words of Claude Bernard, the gifted 19th century French
physiologist: "Art is I; Scienceis we." Certainly his words are true for the sciences represented here:
the field of oligonucleotide chemistry and the emerging biology of sequence-specific gene inhibition.
Our company, Gilead Sciences, is a direct beneficiary of the scientific infrastructure in nucleic acids and
molecular biology that has been built over the past 15 to 20 years, constructed in part by many of the
participants at this conference. Our hats are off to you. Scienceis "we".
We are fortunate among pharmaceutical research organizations to know, in advance, approximately
what our final products will look like. This is .not true of ma~y other pharmaceutical discovery
programs, wherein lead structures are usually identified only after considerable investment has been
made in biological assays,protein crystal structures, inassive screening programs or basic research in
disease etiology.
The general specifications of our would-be products are outlined in Figure 1: a 15-mer oligonucleotide
having a molecular weight of approximately 5,000, containing backbone alterations to enhance
permeation and stabilizing moieties to inhibit nuclease degradation. Depending upon the nature of the
molecular target, the compound will also contain modified bases to augment recognition, enhance
~ffinity, or position a reactive group, such as the aziridinyl moiety Mark Matteucci of Gilead described
yesterday. As you are aware, other modifications can also be made to the intrinsic oligonucleotide
structure.

2. Figure 1
General specifications do not a drug make,
however. At some point along the ongoing
continuum of modifications that confer favor-
able pharmaceutical properties, one must stop
and select individual chemical entities for
costly preclinical and clinical studies. Making
the wrong choice, especially that of choosing a
specificcompound too early in the continuum of
improvements, can be disastrous for two reasons:
(a) Toxicity or poor efficacy may result in large
sums of money being poured down the drain for
fruitless preclinical and clinical evaluation;
and (b) presenting to the FDA a premature,
suboptimal compound with suboptimal support-
ing studies, either at the IND or NOA stages,
may result in a muddying of the waters for this
broad, new class of oligonucleotide therapeutics, and delay introduction of subsequent successful
therapies for desperate patients.
What are the chief objectives in further refining the compound specifications? In our opinion, the chief
objective _is to increase potency. By increasing the potency (i.e. reducing the required dose) of
oligonucleotides, we are assured of reducing toxicities, easing pharmacokinetic constraints, and reducing
the costs of treatment. This evening I would like to address what we believe are the two central
avenues toward radically increasing the potency of oligonucleotides. Then, at the end of my remarks, I
would like to describe a new, promising extension of oligonucleotide technology into the realm of
therapeutics. The two central avenues I refer to for radically increasing potency are, first, marked
enhancement of cellular permeation and, second, attacking a low-copy target, namely DNA.
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3. The first objective, markedly enhanced cytosolic penetration, is best accomplished, we believe, by
altering the oligonucleotide structure so as to enable passive diffusion through the plasma membrane.
It is our opinion that oligonucleotide uptake via a putative membrane protein porting mechanism is
unlikely to be adequate for broad pharmaceutical purposes, because such porting mechanisms are
unlikely to accommodate the various structural modifications of the oligonucleotide that are necessary
to confer various pharmaceutical properties. Passive diffusion through a lipid membrane is known to be
roughly inversely related to size and polar character of the penetrating molecule, but the details of
this relationship between polarity, size and permeability are entirely unknown for molecules in the
molecular weight range of the oligonucleotides we are developing.
There are very few even theoretical touchstones for analyzing this problem or for predicting efficacious
structural alterations of molecules in the molecular weight range of relevance to us, namely the 3,000to
6,000 mw range. As sh own in Figure 2, oligonucleotides in the 5,000 mw range fa 11 i nt o large Iy
MW Spectrum for Human Therapeutics
25K
f f
Protein•
SOK
Penicillin Bleom7cin
(MW ...371) (MW ...1,5H)
75K 100K
f
15-mer
(MW ...5,ttt)
Figure2
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125K 150K
Antibodies
175K
f
Monoclonals
(MW2!180,000)
lOK

4. unexplored territory for human therapeutics. At the top of Figure 2 is depicted the entire molecular
weight spectrum of therapeutics, including proteins like human growth hormone at 22 kilodaltons, TPA
at 59 kilodaltons, and antibodies at 180,000and above. The zero to 10,000molecular weight range is
expanded at the lower part of the figure, showing conventional small drugs like penicillin in the 300 to
500 range, and other larger, natural product-derived compounds like bleomycin in the 1500molecular
weight range. Notably, I should add, bleomycin and several other relatively large anti-tumor agents
have the nucleus as their site of action. In the 3,000 to 6,000 molecular weight range one can find very
few examples in today's pharmacopoeia, and even less information regarding membrane permeability
parameters in this size bracket.
The challenge is illustrated crudely in the following computer modeling rendition (Figure 3) of an
oligonucleotide 15-mer bearing 3' methoxyethylamine "endcaps" juxtaposed with a cross-sectional
segment of lipid bilayer membrane.
Figure3
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5. By delineating the structural requirements for membrane permeation of oligonucleotides, I believe
entirely new horizons for medicinal chemistry will be opened up with regard to other classes of
molecules that lie in this unexplored territory - peptides, for example - and the insights garnered
will potentially be used to improve the intracellular disposition of smaller molecules as well.
Performing this rigorous structure-function evaluation of permeability requires cell biology methods
that are far more sophisticated than the techniques that historically have been part of standard
pharmaceutical sciences. High-resolution fluorescence microscopy is but one example of the
sophisticated cell biology tools that have been presented at this conference and are at work in our
laboratories.
At this meeting we have seen hints of the highly refined cellular biology that must be brought to bear
in a field that historically has been driven primarily by chemistry, not biology. The relative paucity
of biological muscle exerted heretofore in the antisense oligonucleotide arena has been unfortunate, and
has resulted in many studies which underestimate the complexity of the cellular response to exogenous
oligonucleotides.
The second major avenue toward markedly increasing potency is to use triple helix oligonucleotides to
inhibit gene expression at an earlier stage - at the level of transcription. Our own program in triple
helix gene inhibition, which began at Gilead's inception, has dramatically expanded in scope, driven
by rapid technical advances and the quest for high potency. An additional reason for our triple helix
emphasis is its attractiveness for attacking viruses whose life cycle includes a double stranded DNA
stage, such as in viral latency. The prospects for a very low dose agent, although speculative until
confirmed in actual pharmacokinetic studies, are outlined below in Figure 4.
On the right in Figure 4 are listed three classes of pharmaceutical candidates: triple helix agents,
antisense oligonucleotides, and conventional small molecules. Most conventional small molecules act
upon a protein, whose copy number per cell is generally in the range of 10,000to 1 million. Antisense
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6. Protein
Triple Helix Drugs: Potentially Very Low Dose
Figure4
# Copies of
Target per Cell
10,000- 1,000,000
Type of
Drug
TripleHelix
Antisense
Conventional
oligonucleotides of course act upon RNA species which typically exist at a copy number of 100to 100,000
per cell, and are being regenerated over time, if the encoding gene is transcriptionally active.
At the DNA level, the copy number of the corresponding sequence is in the range of 1,2, or only a few per
cell, depending on the allelic status of the gene. Unlike RNA, DNA is not being regenerated, except of
course at the usually modest rate of cell division. These considerations, although they are paper
arguments, suggest that DNA-directed agents potentially could be effective at dose levels orders of
magnitudes lower than those of the corresponding antisense agent. Concerns that chromatin structure or
superhelicity might be formidable obstacles to effective triple helix formation have not held up. In
retrospect, one need only look at endogenous transcription factors, whose activity we wish to mimic, in
order to see why these early concerns are proving not to be salient. After all, transcription factors
generally are larger in molecular weight than our oligonucleotides, and in some cases much larger, and
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7. yet they wend their way through complex chromatin structure and adherent histones to find their
appropriate DNA binding sites.
As has been well described previously in this conference, historically there have been constraints on
the types of DNA code that can be recognized by triple helix agents. The purine and pyrimidine motifs
both require homopurine tracts within the target DNA. If one does sequence searches of
therapeutically relevant genes, one finds that this homopurine requirement greatly limits the number
of targets that can be addressed. How can we circumvent this code restriction? Certainly a key
objectivemust be to devise novel heterocycles that can engage in hydrogen bonding schemes to recognize
the two base couplets that are currently not part of the triple helix repertoire.
Switchback Triple Helix
FigureS
-7-
Another partial yet highly employable
solution to "extending the code" for the triple
helix was presented by Brian Froehler of
Gilead this past August in Sweden. His idea
was to join two or more oligonucleotide segments
of reverse polarity in such a manner as to allow
one segment to recognize, or "read", one strand
of the double helix, and the second segment to
read the other strand. This construct, shown in
Figure 5, we term a "switchback". The task
remained, however, to shrewdly select,
synthesize and evaluate various linker
moieties, in this case joining the 3' ends of the
two segments, in such a manner that the
complete oligonucleotide molecule bound with

8. an affinity approximating that of a single-polarity oligonucleotide directed against a standard biple
helix target.
Guided in large part by his molecular modeling studies, Froehler tested a series of attractive linker
configurations which were successful in effecting high affinity binding to the completed switchback.
The resulting optimal class of linkers in the 3'-3' orientation is reflected in Figure 6, which shows a
xylene group linking the two deoxyribose termini of two 9-mer oligonucleotide segments directed
against the double-stranded DNA sequence shown in detail at the left and schematically at the right.
Ether-Linked Xylose Diiner
S' 3'
A-T
G-C
A-T
A-T
S' A - T
C+ G - C
T-A-T
T - A - T
T - A - T
T-A-T
T-A-T
C+ G - C
T - A - T
T - A - T
3•~3•
T - A - T
T - A - T
T - A - T
C - G+ C
T - A - T
T - A - T
C - G+ C
T - A - T
T - A - T
T - A S'
T-A
T - A
C-G
T-A
3' - S'
s·~sc:==z=~--5-,-3'
3'----====~-s·
Figure6
-8-
DNA footprinting analyses and thermal
melting studies have demonstrated that this
switchback construct and several variants
perform comparably to the full-length single
polarity oligonucleotide directed against a
standard biple helix site. We believe this is a
clear demonstration that molecular modeling
can be very successfulin the design of improved
triple helix agents, and in prioritizing our
chemical synthesis objectives. Most
importantly, sequence searches for feasible
switchback targets in clinically relevant genes
have revealed an abundance of attractive
targets for prospective triple helix
therapeutics. This further reinforces our
optimism for biple helix methodologies.

9. I would like to return now to the basic structure of the oligonucleotide, and my final topic this evening.
Oligonucleotides are turning out to be far more interesting and versatile than was understood even just a
year ago. Over the past few months, an entirely new pharmaceutical technology using oligonucleotides
has unfolded. This past fall, a handful of scientific reports appeared in Science and Nature. two of
them authored by members of Gilead's scientific advisory group, Harold Weintraub and Jack Szostak,
describing a powerful technique whereby oligonucleotide molecules can be selected and amplified to
yield specific entities that bind very tightly to proteins or small molecules. We refer to these selected
and amplified oligonucleotides as "aptamers", a term coined by Professor Szostak (aptus being the
Latin word for "to fit").
Aptamers are synthesized and identified through a process that can be described as "evolution in a test
tube". Randomly generated oligonucleotide sequences are synthesized, and then selected and replicated
according to their ability to bind to a biological target molecule. In this process, tight binders are
favored over loose binders and, after several rounds of selection and amplification, those
oligonucleotide variants, or aptamers, that bind most tightly can be identified and potentially
employed to interrupt disease processes mediated by the_target to which the aptamer binds. Gilead's
scientists have launched a promising new program in aptamer development, which complements, in
many ways, our efforts in antisense and triple helix oligonucleotides.
I would like to review the aptamer development process in more detail. We begin by generating a
random pool of oligonucleotides via chemical synthesis. Given the enormous number of combinations of
bases that are possible within an oligonucleotide strand of modest length, an extraordinary number of
different chemical entities can be generated, for example 1013
, as in Figure 7. This random pool of
oligonucleotides is then exposed to a biological target molecule whose activity we wish to inhibit.
Those molecules within the pool that bind only loosely to the target are separated from those that, by
chance, bind tightly, and only the tight-binders are subsequently replicated, or amplified, using PCR.
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10. Aptamer Technology
Randomly generate oligos
(-1 x 1013 different molecules)
BiologicalTarget
Molecule
Figure 7
Selectivelyreplicate tight
binders (test tube evolution)
Novel therapeutics having
high affinity and high specificity
This step, wherein only the suitable high affinity molecules are allowed to be replicated to large
numbers, might be called "survival of the fittest".
The process of binding, selection and amplification depicted in Figure 8 is repeated several times, each
cycle resulting in an enrichment of high-affinity sequences within ·the pool of oligonucleotides. After
several cycles, a single oligonucleotide, or a small number of distinct oligonucleotides, can be isolated,
and their precise sequences determined. Subsequently the oligonucleotide, or aptamer, can be assessed
for biological activity, be chemically modified to improve certain pharmaceutical characteristics, and
be produced in larger amounts, via chemical synthesis, for further studies.
This "evolution in a test tube" can be regarded as a molecular screening technique having an
astonishingly high throughput. A rapid biological assay in the conventional pharmaceutical industry,
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11. Aptamer Development
Synthesize random pool-----•~~
of oligonucleotides _.
(-10
13
distinct molecules) _/
Repeat Cycle
Evaluation&; Production "
Sequence
Assay biological activity ◄
Chemically modify , _____ ,:
Scale-up synthesis
Binding
Exposeoligo-
nucleotides to
biologicaltarget
molecule
Selection
Isolate high-affinity
binders from
low-affinity binders
Amplifi,ation/
Amplify selected
pool using PCR
Figure8
when operating at full capacity, might evaluate 10,000 different compounds per year. Using the
aptamer technique just outlined, in a period of weeks approximately 1013
different chemical entities,
all oligonucleotides, can be evaluated. Here, at last, in a screening system, the numbers are on our side.
To summarize, chemical synthesis allows us to generate in a single vessel an enormous diversity of
oligonucleotide molecules. Some small number of discrete chemical entities within this diverse pool
have three dimensional structures that enable them to bind with high affinity to a biological target
molecule. Through so-called evolution in a test tube, the high-affinity species are selectively
amplified. These "evolved" molecules, because of their high specificity and affinity to a particular
biological target molecule, are prospective drug candidates.
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12. It must be stressed that aptamers are as yet at an early stage, and their ultimate pharmaceutical
utility is far from proven. However, they may lead to an array of novel therapeutics, and we are
optimistic about the rapid extension of our oligonucleotide technology into this new arena. The
expanding number of potential oligonucleotide applications - antisense, triple helix, aptamers, and
likely others in the future - provide a solid foundation for pharmaceutical development. As a
consequence of our scientific commitment to the chemistry and biology of this versatile class of
molecules called oligonucleotides, we now find ourselves in a richer, deeper mine than we had
anticipated.
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