Antisense Theraputics - S. Agrawal (Humana) WW

270 Pages·2020·22.2 MB·English

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History of Antisense
Oligonucleotides

Paul C. Zamecnik

1. Introduction
and Early Studies
Blologlcal
science 1s a rapidly flowing experlmental stream, at times
encountering a dam that impedes further progress. At such a pomt, a single
crack may induce a maJor breakthrough Dlscovery of the double hehcal struc-
ture of DNA in 1953 (I) caused such an event, with flooding of new mforma-
tlon into the area now known as molecular biology.
At this same time, our laboratory (2,3) developed a cell-free system for the
study of protein synthesis, a domain separate from the DNA world. In 1954,
James Watson and this author examined his wire model of DNA and puzzled
about how the information from the gene became translated mto the sequence
of a protein (4). The histochemlcal studies of Brachet (5) and Caspersson (6)
had shown that m the pancreas, an organ very actively synthesizing protems
for export, the cytoplasm was rich in what became known as rlbonuclelc acid.
But how the DNA of the nucleus unwound its double strand and transcribed
the RNA, the apparent intermediate m protein synthesis found in the cytoplasm,
was unknown (7).
The first example of the versatility of nucleic acid base pairing in the flow of
mformation from DNA to protein was the discovery of transfer RNA (8-1 J)
and perception of its role m translating the language of the gene mto the
sequence of protein (12). Deciphering of the genetic code (13,14) next brought
to light the precision of the tRNA-mRNA
hybridization steps m protein trans-
latlon. tRNA (an antlsense or negatively stranded RNA) acts in four dlstin-
guishable ways, as follows.

1. By base palring with messenger RNA to initiate translation of the message,
2 By base pairing with messenger RNA to propagate translation of the message;

From
Methods
m Molecular
Medicme
Anbsense
Therapeutm
Edlted by S Agrawal
Humana
Press Inc , Totowa,
NJ

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Zamecnik

3 By base pairmg with rlbosomal
RNA to position the trmucleotlde
antlcodon
region for optlmal hybridization
with the messenger RNA codon, and
4 By presentmg a terminating
antlcodon (which IS an antisense trmucleotlde)
to
end the nascent protein sequence

Puromycin, a natural nucleotide analog, provided an early example of
antisense mhibrtton of protein synthesis (15).
Further experimentation supported the hypothesis (16) that hybridization of
synthetic exogenously added ohgonucleotides can influence cellular metabo-
lism at three distinct levels: rephcation (17), transcription (l&19), and transla-
tion (20,21). The variety and importance of these steps mvited the thought that
natural ohgonucleottdes might play such roles m living cells (22). In the double
helix, the DNA strand that carries the genetic message has been designated the
sense strand. Its complementary mate, necessary as a template for the synthesis
of a new sense strand, has become known as the antisense strand. Antisense
polynucleotides have, m fact, for over two decades been known to occur natu-
rally in prokaryotes (23), and have recently been found m eukaryotes (24). For
some years synthetic oligonucleotides have also been reported to be capable of
playing varied antisense inhibitory roles (25-28).
Quite separate from the synthetic oligonucleotide field were independent
developments from sphcmg larger segments of negatively stranded DNA into
the genomes of cells, with the help of plasmids and viruses. These antisense
strands of DNA were successfully integrated mto the genomes of relatively
few host cells. Nevertheless, by selection processes these antisense sequences
were picked out and found to be replicated along with the recipient’s genomic
material. Thus, they were capable of blocking or altering the expression of
cellular genes m a hereditary way. An example of the success of this technique
is the permanent alteration of the color of petunias by antisense interference
with synthesis of the flavonoid genes (29). An important difference between
these approaches is that the synthetic, relatively short oligomers enter virtually
all cells m a tissue culture or living animal (3&32) whereas the plasmids carry-
mg much longer antisense polynucleotides generally enter a small percentage
of cells, but may nevertheless have a dramatic genetic therapeutic effect (33).
In the early 1960s the limits of DNA and RNA synthesis were in general
trmucleotides, but by the end of the decade skilled scientists were able to
construct oligomers 10-15 U m length, and to ligate such segments together
(34). The message encoded in DNA or RNA, however, remained difficult to
decipher. In 1965, Holley and colleagues (35) sequenced the primary structure
of tRNA,i,, using the new method for scale-up and isolation of the tRNA fam-
ily of molecules worked out m our laboratory by Roger Momer (36). In this
highly competitive quest, several groups accomplished the sequencing of other
particular tRNAs shortly thereafter (3 7-39), getting little credit for their efforts.

An tisense Oligonucleo tides
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In 1970, the discovery of reverse transcriptase (40,42) made it more feasible
to sequence oligonucleottdes by synthesizing the primary structure of DNA
enzymatically, and then sequencing the nascent DNA so formed. The wander-
mg spot-analysis technique of Sanger and Coulsen (42) at that time made it
possible to determine the sequence of approx 15-25 monomer umts at the 3’
end of a polynucleotide.
The present dtscussion focuses on the role of the synthetic antisense oligo-
nucleotides (20-30-mers) as chemotherapeuttc agents, and omits the splicing
insertion into genomes of the larger (1 ,OOO-2,000 monomer unit) biologically
synthesized polynucleotides. Those of us raised on the principle of Occum’s
razor, which advises making explanations as simple as possible, continue to be
surprised at the unfolding
complexity of this synthetic oligonucleotide
approach to chemotherapy. One was prepared for the nuclease sensittvity of
unmodified oligodeoxynucleotides m a hvmg cell system (16,43), and the
enhancement of therapeutic efficacy by blocking both ends of the ohgodeoxy-
nucleotide (26). The effect of ribonuclease H (44) was generally unexpected,
however, particularly the maJor role it plays in some antisense mhibitions. As
is now known, a stoichiometrically acting oligodeoxynucleotide inhibitor may
activate RNase H during its complementary hybridization with mRNA, then
dissociate from its complement when the mRNA is hydrolyzed at the double-
stranded area, and hybridize with another molecule of mRNA, this repetrtive
action resulting in a catalytic effect.
By 1976, we were able to sequence 2 1 nucleottdes inside the 3’-polyA tail of
the Rous sarcoma vn-us, a terminus similar to that we had previously found on
the avian myelobastosis vn-us (45). Rous sarcoma vu-us was the only purified
vn-us for which a sufficient quantity was available to make a sequencmg effort
feasible At this time, we learned that Maxam and Gilbert (46) had invented a
revolutionary new DNA sequencing technique, and had, unbeknown to us,
begun to decipher the 5’- end of the same Rous sarcoma vu-us. Astonishingly,
both ends of this linear viral genome bore the same primary sequence, and
were m the same polarity (47,48).
It occurred to us that the new piece of DNA synthestzed by reverse tran-
scriptton at the 5’- end of this retrovirus might circularize and hybridize with
the 3’ end, like a dog biting its tail. Electron microscopic studies had suggested the
presence of a circularized intermediate m the rephcative process of this vu-us. Thus,
we considered the possibility of inhibmng viral replication by adding to the rephca-
tion system a synthetic piece of DNA to block the circularizatton step (or alterna-
tively some other step essential for replication), m the former case by hybridtzmg
specifically with the 3’ end of the viral RNA in a competitive way.
It was at this time generally believed that oligonucleotides did not penetrate
the external membrane of eukaryotic cells, to enter the cytosol and nucleus

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(49). Clearly, neither did ATP, except under unusual circumstances, nor Ap,A
(50). Segments of cellular genomes were currently coaxed mto cell entry by an
inefficient calcmm phosphate precipitation procedure. The negative charge of
the oligonucleotide was regarded as presenting a major impediment to traverse
of an ohgonucleotrde through the eukaryotic external cell wall. Nevertheless,
an experiment testing the possibility of synthetic ohgonucleotide cell wall pen-
etration was performed. We added a 13-mer synthetic ollgodeoxynucleotide,
complementary to the 3’ end of the virus, to the medium of chick Iibroblasts m
tissue culture, along with Rous sarcoma vtrus itself. It inhibited the formation
of new vu-us, and also prevented transformation of chick fibroblasts mto sar-
coma cells-both
of these startling observations (16). In a cell-free system,
translation of the Rous sarcoma viral message was also dramatically impaired (20).
Until 1985, little further progress occurred, for three intertwined reasons:
first, there was still widespread disbelief that ollgonucleotides could enter
eukaryotic cells; second, tt was difficult to synthesize an oligomer of sufficient
length to hybridize well at 37°C and of specifictty requisite to target a chosen
segment of genome; and third, there was very little DNA (or RNA) genome
sequence available for targeting m this way. This latter reason determmed the
choice of the Rous sarcoma vuus, which Haseltme et al. (47) and our labora-
tory (48) were sequencmg contemporaneously and whose results were pub-
hshed m tandem.

2. Independent
Complementary
Developments

Two important developments in the late 1970s and early 1980s mcreased
the feasibility
of the synthetic oligonucleotide
hybridization
mhibition
approach. The first were the dramatic improvements m DNA sequencing that
came from the Maxam-Gilbert chemical degradation procedure (46), and the
more convenient dideoxy enzymatic sequencing technique of Sanger’s labora-
tory (52). The second was the solid-phase ohgonucleotide synthetic approach
introduced successfully by Letsmger and Lunsford (52) and Caruthers (53). At
this ttme, as well, there developed a growing acceptance that oligonucleotides
could pass through the eukaryotic cell membrane and enter the cell, and that
they could readily be synthesized and purified. Fmally, an abundance of pot-
ential DNA sequence targets began to appear like fireworks m a previously
darkened genetic sky.
The unmodified antisense oligodeoxynucleotide has proven to be the best
RNase H activator, provided there are at least four or more contiguous hybrid-
izing base pairs. The phosphorothioate
modified oligodeoxynucleotides,
although not so effective, still activate Rnase H, and are quite nuclease resis-
tant (54). These two properties account for the early general preference of the
latter m synthetic oligodeoxynucleotide experiments. In contrast, other varied

Antisense Oligonucleotides
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modifications at the mternucleotide bridging phosphate site result m inability
to activate RNase H, and thus present a disadvantage. Included in this category
are methyl phosphonates, a-oligonucleotides, intemucleotide peptide bonds, and
others. Modifications on the ribosyl moiety, such as the 2’-0 methyl group, also
fail to activate RNase H. Hybrid and chimeric ohgonucleotides are coming
mto increasing usage, smce they combine terminal nuclease-resistant segments
of an ohgonucleotide with a central RNase-sensitive portion (see Chapter 14).
Furthermore, if only the central portion of the oligomer is phosphorothioate
modified, whereas the peripheral 3’ and 5’ segments are, for example, 2’-0
methyl-modified oligomer moieties (55), the nonspecific effects of the totally
phosphorothioate oltgomer (56) are to a considerable extent mmimized The
selfstabilized snap-back oligomer is particularly advantageous m providmg
enhanced nuclease resistance with other desirable properties (57).
In addition to the promise of the antisense approach documented m these
pages and elsewhere, there are reasons why the competitive oligonucleotide
hybridization technique may not be successful in attempts to inhibit noxious
genes, wherever they may exist m the animal and plant kingdoms A central
reason is failure to find a smgle-stranded segment of genome that is highly
conserved and accessible. Secondary and tertiary structure of the genome may
prevent hybridization. Protems that have a high association constant with the
area of genome targeted, i.e., promoters, enhancers, and modulators, for
example, may prove to be barriers to ohgonucleotide hybridizations. The
aggregation effect on ohgonucleotides of a G-quartet motif (58,59) is also an
impediment. Zon (60) touches on some of these aspects in a historical review.
It would be advantageous if hybridization inhibition could be achieved at
the transcription level. This would block the amplification step, which results
in numerous copies of mRNA for translation. A favorable site for transcription
hybridization is the transcription bubble, consisting of 12-17 nucleotides of
unwound double-helical DNA. As an example of this approach, we have found
m an in vitro transcription system that specific hybridization mhibition can be
induced using a linearized plasmid segment of HIV for a template With
T7RNA polymerase, a gag RNA of about 640 nucleotides can be synthesized.
This synthesis can be inhibited by a complementary 14-mer unmodified
ohgodeoxynucleotide, lust downstream of the T7 promoter (19). The most
effective inhibitor is a plus-sense oligonucleotide complementary to the nega-
tive DNA strand that serves as a template for pre-mRNA synthesis.

3. Examples of Current
Disease Targets
Let me mention a few current medically related investigations involving our
own laboratory that appear to show promise: HIV, influenza, and malaria. A
study on HIV (61) shows that target selection in the HIV genome is important

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for prevention of development of escape mutants Whereas escape mutants
appeared after 20 d treatment of chronically infected Molt-3 cells with an
antisense phosphorothioate oligomer pmpomtmg a splice acceptor site, contm-
ued inhibition without escape over an 84-d experimental period occurred when
revl-28
or gag-28 were the targets.
A second example is the use of antisense oligomers to inhibit Influenza viral
replication (62). At 10 PM concentration, replication of influenza C vnus was
inhibited
90% m tissue cultures of MDCK cells by a sense oligophos-
phorothioate targeted against the rephcase gene of the negatively stranded
virus. In lo-d-old embryonated chick eggs, phosphorothioate ohgomer injec-
tion also induced marked inhibition of vu-us production (63).
Another example is inhibition of replication of Phsmodzum
fuhparum

malaria by a phosphorothioate oligodeoxynucleotide
targeted against the
dihydrofolate reductase-thymidylate synthase gene of the parasite (64). This
enzyme is essential as a donor of a methyl group in the conversion of
deoxyuridine monophosphate to thymidme monophosphate m the parasite,
which must synthesize its own pyrimidmes, being unable to use exogenous
thymidme for synthesis of DNA. The adult erythrocyte is one of the rare
eukaryotic cells that oligodeoxynucleotides do not penetrate. Fortunately, how-
ever, when a malarial parasite pushes its way into a red cell, it creates a
permeabihzed erythrocyte membrane plus a parasitophorous duct (65), through
either or both of which the oligodeoxynucleotide reaches the parasite inside its
protective erythrocyte envelope. A fluorescently labeled oligodeoxynucleotide
lights up a circular area inside an erythrocyte m which the P. fulczpurum
para-
site resides, surrounded by its own membrane, whereas the uninfected red cells
fail to show evidence of cell entry (66). The above-mentioned antisense ohgo-
mer shows a sequence-specific IDSo for rephcation of the parasite at 2-5 x 1