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REVIEW ARTICLE
Nucleic Acid Therapeutics: State of the Art and Future Prospects
By Alan M. Gewirtz, Deborah L. Sokol, and Mariusz Z. Ratajczak
A
S WE APPROACH the new millennium, a quick glance
backwards reveals that truly astounding progress has
been made in the identification of genes responsible for cell
growth, development, and neoplastic transformation. With this
knowledge has come a natural desire to ‘‘translate’’ this
information into new therapeutic strategies for many of the
common maladies that afflict humankind. These include in
particular cardiovascular, gastrointestinal, neurologic, infectious, and neoplastic diseases. Attempts at inserting genes into
cells that either replace, or counter the effects of, diseasecausing genes has been one of the primary ways in which
scientists have tried to exploit this new knowledge. This
technically complex, as yet largely unrealized endeavor1,2 is
what most individuals think of when the terms ‘‘gene therapy’’
or ‘‘molecular medicine’’ are discussed. Nevertheless, alternative strategies for treating diseases at the gene level are being
developed. The common goal of these various strategies, which
are turning out to be as technically demanding as more
traditional gene therapy, is to identify disease related genes and
target them for ‘‘silencing.’’ Because the numbers of maladies
that might be treated by this approach are genuinely enormous,
this is clearly a most important field of endeavor. It will be the
goal of this review to describe available strategies for ‘‘silencing,’’ or perhaps more appropriately, perturbing gene expression. Given the expertise and experience of our laboratory, we
will place special emphasis on the use of reverse complementary or so called ‘‘antisense’’ oligodeoxynucleotides (ODN) for
this purpose. Problems associated with the use of antisense
ODN for modifying gene expression are well known and they
will be discussed, along with potential strategies for overcoming these problems. The prospects for ultimately using these
materials successfully in the clinic will also be elaborated upon.
This review is meant to be complete, but not exhaustive. Even a
cursory examination of the literature data base reveals that since
1992 more than 1,300 manuscripts have been published which
list antisense DNA or RNA among its key words. We therefore
apologize in advance to colleagues whose work we do not cite,
but which we nonetheless admire.
NUCLEIC ACID–BASED STRATEGIES FOR PERTURBING
GENE EXPRESSION—A PRIMER
The notion that gene expression could be modified through
use of exogenous nucleic acids derives from studies by Paterson
From the Departments of Internal Medicine, Pathology and Laboratory Medicine, and the Institute for Human Gene Therapy, University of
Pennsylvania School of Medicine, Philadelphia.
Submitted November 12, 1997; accepted April 29, 1998.
Supported in part by National Institutes of Health Grants No.
RO1CA66731, PO1CA72765 to A.M.G., and a Translational Research
Grant from the Leukemia Society of America (A.M.G.)
Address reprint requests to Alan M. Gewirtz, MD, 513B StellarChance Laboratories, University of Pennsylvania School of Medicine,
422 Curie Blvd, Philadelphia, PA, 19104.
r 1998 by The American Society of Hematology.
0006-4971/98/9203-0039$3.00/0
712
et al,3 who first used single-stranded DNA to inhibit translation
of a complementary RNA in a cell-free system in 1977. The
following year Zamecnik and Stephenson4 showed that a short,
13-nucleotide (nt) DNA molecule antisense to the Rous sarcoma virus could inhibit viral replication in culture. The latter
investigators are widely credited on this basis for having first
suggested the therapeutic utility of antisense nucleic acids. In
the early to mid 1980s, Simons and Kleckner5 and Mizuno et al6
showed the existence of naturally occurring antisense RNAs in
prokaryotes and showed that these molecules played a role in
regulating expression of their corresponding genes. These
observations were particularly important because the existence
of naturally occurring antisense RNAs lent credibility to the
belief that the use of reverse complementary nucleic acids was a
‘‘natural’’ mechanism for regulating gene expression, thereby
raising hope that the process could be exploited in living cells to
manipulate gene expression. The work of Izant and Weintraub7
further buttressed belief in this potential by demonstrating that
expression of antisense RNA in eukaryotic cells could also
modulate expression of the complementary gene. These seminal
reports, and many others which quickly followed, have stimulated the rapid development of technologies using nucleic acids
to manipulate gene expression. Virtually all available methods
rely on some type of nucleotide sequence recognition for
targeting specificity but differ where and how they perturb the
flow of genetic information. Most simply, gene expression may
be perturbed at the level of transcription or translation (Fig 1).
Inhibiting Gene Expression at the Transcriptional Level
Inhibition of gene expression at the level of transcription may
be accomplished by at least three different methods. The ‘‘gold
standard’’ exploits homologous recombination.8,9 This approach
is designed to take advantage of naturally occurring cross-over
events during DNA replication. In a typical system, a plasmid
capable of infecting the desired target cells and expressing the
desired sequence is constructed. The construct expresses a
selectable gene marker, such as an aminoglycoside resistance
gene, flanked by sequences complementary to the gene of
interest in the genomic DNA. When the targeting plasmid is
introduced into the cell of interest the vector and complementary portions of genomic DNA undergo rare (,1:1,000 heterologous recombinations) cross-over events during the course of
cell division. The cross-over results in insertion of the targeting
sequence into the genomic DNA at the intended site resulting in
effective destruction of the targeted gene (Fig 2A). Further, the
inserted sequence remains under the control of the targeted
gene’s promoter. Therefore, cells in which the desired event has
occurred are selected by exposure to G418 (geneticin), which is
toxic to cells in the absence of the aminoglycoside resistance
gene. Those cells that survive the exposure must have incorporated and expressed the resistance gene and therefore the
targeting cassette. If resistant cells are injected into a murine
blastocyst, animals expressing the mutated gene will develop,
assuming of course that loss of the targeted gene does not lead
to an embryonic lethal condition. The effect of functional
Blood, Vol 92, No 3 (August 1), 1998: pp 712-736
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NUCLEIC ACID THERAPEUTICS
absence of the targeted gene in a developing animal may then be
discerned, giving important insights into the biologic importance of the gene chosen for elimination. This method, combined with appropriate animal breeding, is quite effective at
generating heterozygous or homozygous loss of function mutants. Further, recent elegant modifications of this basic approach using bacteriophage recombinases such as Cre, which
recognize specific DNA elements known as loxP, have the
capability to significantly increase the efficiency and utility of
this approach.10 Cre, for example, has the ability to excise all
chromosomal DNA between loxP sites and then ligate the cut
ends, a process that can occur even in postmitotic cells.
Constructing a targeting vector with loxP sites and an inducible
promoter allows for temporal and tissue-specific gene targeting
when cells are exposed to Cre. Nevertheless, while homologous
recombination is extremely powerful, it is hampered by that fact
that it remains inherently inefficient, time consuming, and
expensive. It should also be noted that complementation of the
targeted gene’s function by an alternative gene may give a
misleading impression of the targeted gene’s function and
relative importance.11,12 Clearly, this is a method that is
presently restricted to use in cell lines and animal models.
Whether it is likely to have clinical relevance as a therapeutic
modality anytime in the foreseeable future is uncertain.
A second option for disrupting gene expression at the level of
transcription uses synthetic ODN capable of hybridizing with
double-stranded DNA.13-15 Such hybrids are typically formed
within the major groove of the helix, but very recently a strategy
for hybridization within the minor groove has also been
reported.16 In either case, a triple-stranded molecule is produced, hence the origin of the term triple-helix forming
oligodeoxynucleotide (TFO) (Fig 2B). TFOs may act in two
ways. They may prevent binding of transcription factors to the
gene’s promoter and therefore inhibit transcription. Alternatively, they may prevent duplex unwinding and, therefore,
transcription of genes within the triple-helical structure. TFO
sequence requirements are based on the need for each base
comprising the TFO to form two hydrogen bonds with its
complementary base in the duplex. This constrains TFOs to
hybridization with the purine bases composing polypurinepolypyrimidine tracks within the DNA. The bonds formed
under these conditions are also referred to as Hoogsteen bonds
after the individual who first described them. They may form in
the parallel or antiparallel (reverse Hoogsteen) orientation
relative to the 58-38 orientation of the purine strand depending
on the thermodynamics of the specific base interactions involved. An A or a T in the TFO can bond with the A of an A · T
pair in the DNA duplex, while G can bond with the G of a G · C
pair. C can also bond with the G of a GC pair if protonated (C1 ).
Accordingly, TFOs containing C form stable hybrids under
acidic conditions. Although this tendency can be modified
somewhat by methylation of the cytosine at the C-5 position,17
C-containing TFOs are expected to be less active at physiologic
pH. In contrast, G- and T-containing TFOs can form stable
hybrids at physiologic pH.18 However, these TFOs are plagued
by the fact that physiologic concentrations of potassium inhibit
triplex formation, although some very recent studies suggest
that TFOs substituted with 7-deazaxanthine can hybridize
efficiently with their target even in the presence of 140 mmol/L
713
K1.19 G-containing TFOs also require divalent cations such as
Mg21 for stability, while A-containing TFOs appear to require
Zn21.
Stability of triple helices is further dependent on a number of
factors, including the length of the TFO, with ,13 nucleotides
being suggested as a minimum for phosphorothioate compounds,20 and the presence of any base mismatches. Such
mismatches are particularly problematic when they occur in the
middle of a strand because they interfere significantly with
‘‘nucleation,’’ the relatively slow process whereby the initial
base-pair associations between the TFO and the strand being
targeted are brought about. In fact, a single mismatch in the
middle of the strands, or the presence of a single pyrimidine
base in the homopurine run, can decrease TFO affinity by 20- to
30-fold. In addition to these considerations, it has also been
reported that helicases, enzymes which unwind duplexed DNA
for transcription and repair, easily disrupt triple-stranded DNA.21
These problems have significantly hampered the use of TFOs as
reagents for studying gene function in intact cells, and make
them quite problematic as potential pharmaceuticals.14
Despite the problems discussed above, a number of approaches have been investigated for optimizing the activity of
the TFO. One is to use an oligonucleotides that binds to
alternate strands of the duplexed DNA,22 a maneuver that
obviates the requirement for polypurine-polypyrimidine sequence in the target DNA. Another is to increase the binding
affinity of the TFO by covalently linking the DNA to intercalating groups such as acridine23,24 and psoralens.25,26 Incorporation
of strand-cleaving moieties may also increase efficiency of
TFOs.27 Finally, work on expanding the third-strand binding
code may also enhance the utility of this approach. Recent
experiments from Wang et al28 and from Kochetkova et al29,30
have provided evidence that triple-helix formation can occur in
living cells, suggesting that these difficulties may ultimately be
overcome. If shown to be practical, it has also been postulated
that TFOs may prove useful in the treatment of certain genetic
disorders such as sickle cell anemia and hemophilia B, where
their ability to induce mutations might be used to correct single
base–pair mistakes responsible for the disease.28,31-33 Because
this method may also inadvertently introduce undesired mutations into the genome by the same mechanism, concerns have
been raised about using this approach in patients.
One final approach that has not undergone extensive development, at least at the level of transcription, is the use of specific
nucleic acid sequences to act as ‘‘decoys’’ for transcription
factors (Fig 2C).34,35 Since transcription factor proteins recognize and bind specific DNA sequences, the principal upon
which electrophoretic mobility shift assays are based, it is
possible to synthesize nucleic acids that will effectively compete with the native DNA sequences for available transcription
factor proteins in vivo. If effective, the rate of transcription of
the genes dependent on the particular factor involved will
diminish. Unless single gene transcription factors can be
identified, it is difficult to conceive how this approach, though
potentially effective for controlling cell growth, can be made
gene specific. Transcription factor decoys will not be further
considered here, but the decoy concept will be mentioned again
briefly below because RNA decoys have also been used to block
translation.
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Fig 1.
Fig 3.
Fig 2.
Fig 4.
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NUCLEIC ACID THERAPEUTICS
Inhibiting Gene Expression at the Translational Level
Strategies for inhibiting translation are primarily directed
toward impairing utilization of messenger RNA (Fig 3). Approaches that have this as a goal are what have traditionally
been designated ‘‘antisense’’ strategies because of their reliance
on the formation of reverse complementary (antisense) WatsonCrick base pairs between the targeting construct or vector, and
the mRNA whose function is to be disrupted. It is the specificity
of the Watson-Crick base pairing that allows a particular mRNA
species to be selectively targeted. The antisense strategies rely
on either introducing the reverse complementary nucleic acid
sequence into the target cell, or on expressing the reverse
complementary sequence in the target cell from a transfected
viral or plasmid vector. The reverse complement may be DNA
or RNA. The relative merits of each are discussed below. In
theory, however, if hybridization between the target mRNA and
the exogenous nucleotide sequence occurs, a duplex is created
which, in effect, forms a ‘‘jam’’ that prevents the ribosomal
complex from reading along the message. If the ribosomal
complex can’t read the message, the appropriate tRNAs are not
assembled and the encoded peptide is not made. This would
appear to be a relatively foolproof mechanism for preventing
mRNA utilization but, as was true for triple-stranded DNA
molecules, RNA-RNA or RNA-DNA duplexes can be unwound
by a variety of repair/editing enzymes such as helicase and RNA
unwindase.36 In addition, the ribosomal complex itself has
unwindase activity that likely permits ‘‘reading’’ of the complexly fold mRNA. In the case of ODN that are targeted
downstream of the translation initiation site, it has been shown
that the ribosomal complex can unravel the RNA/DNA duplex,
allowing the complex to read through the block.37 Peptide
assembly is thereby unperturbed.
;
Fig 1. Gene expression may be disrupted, as indicated by the ‘‘X,’’
at the level of transcription, or translation. Oligonucleotides can
inhibit transcription (1) by triple-helix formation with chromosomal
DNA, or by acting as decoy’s for transcription factors (see Fig 2).
Hybridization of an oligonucleotide to mRNA may inhibit translation
(2) by hindering the ability of the ribosome complex to ‘‘read’’ the
mRNA sequence, or by providing a substrate for RNase H (see Fig 3).
Fig 2. Strategies for inhibiting transcription. (A) Homologous
recombination. Cross-over exchange between the targeting vector
and genomic material during cell division is indicated. See text for
more detailed explanation of events. (B) Triple-helix formation in the
major groove between a polypyrimidine oligodeoxynucleotide (open
pattern line) and polypurine sequence in double-stranded (black line)
DNA. Textural representation of this event is indicated below the
cartoon. (C) Decoy strategy. Double-stranded oligonucleotides compete with the native binding site for transcription factor protein (dark
globular structures).
Fig 3. Strategies for inhibiting translation. Diagrammatic representations of (A) hammerhead ribozyme; (B) antisense oligodeoxynucleotide; (C) antisense RNA. Note that targeting specificity is conveyed in
each case by Watson-Crick base pairing between complementary
sequences.
Fig 4. Chemical modifications of oligonucleotides. (A) Common
modifications of the phosphodiester linkage, sugar, and bases moieties are depicted. Note that 28-O methyl sugar modifications confer
stability to single-stranded RNases but not DNases. They do not
allow binding of RNase H either. The propynl pyrimidines demonstrate enhanced binding to RNA but cannot permeate membranes.
(B) N3-5P phosphoramidates and peptide nucleic acid backbone modifications confer enhanced stability and RNA binding. However, the latter do
not permeate membranes and neither activates RNase H.
715
Events that are triggered as a result of duplex formation are
dependent on the nature of the antisense molecules used for
mRNA targeting. Oligonucleotides of many, but not all, types
(see below) support the binding of RNase H at sites of
RNA-DNA duplex formation. Such binding is thought to be an
important effector of antisense actions because once bound,
RNase H, a ubiquitous nuclear enzyme required for DNA
synthesis, functions as an endonuclease that recognizes and
cleaves the RNA in the duplex. Escherichia coli RNase H
requires a minimum of four RNA-DNA base pairs for enzyme
binding,38 while human RNase H may require only one.39 Once
initiated, destruction of the message by cleavage is assured. Of
significant interest also is the fact that the DNA comprising the
duplex is undamaged by the enzymatic attack. Therefore, it is
free, at least theoretically, to hybridize with multiple RNA
molecules, leading their destruction in a catalytic manner. Some
chemical modifications, such as the phosphorothioates, are
thought to activate RNase H very efficiently40 while others do
not support the activity of this enzyme at all (see below). It is
also probably worth noting RNase H may produce unanticipated, non–sequence-dependent effects by cleaving transiently
formed duplexes, or with sites of partial complementarity.41
Although RNase H is generally thought to be critical for
antisense effectiveness,42 not all studies support this contention.
For example, Rosolen et al43 reported that overexpressing
RNase H in U937 cells to a level 10 times greater than normal
did not enhance the antisense effectiveness of a c-myc targeted
ODN. One caveat in interpreting these experiments is that it was
not human RNase H that was overexpressed.43 Similarly,
Moulds et al44 studied antisense inhibition mechanisms using a
microinjection assay and oligonucleotide modifications that
were either permissive of, or did not support, RNase H binding.
Their studies suggested that if a stable RNA-DNA duplex was
formed translation of the targeted mRNA was completely
inhibited. Based on these results they predicted that binding of
RNase H to such a stable duplex would not further increase the
efficiency of antisense inhibition of protein synthesis. These
studies were somewhat artificial in that duplexes were preformed ex vivo and were then injected into the nucleus of the
cell. Therefore, the direct physiologic relevance of these
experiments is uncertain. More indirect is the fact that despite
the apparent importance of RNase H for generating an antisense
effect, few published reports have actually provided direct
evidence of such attack by demonstrating that the predicted
cleavage fragments have been generated.45,46 This may be
because once the mRNA molecule is nicked, it is likely very
rapidly destroyed.
The fate of RNA-RNA duplexes is less certain. As mentioned
above, they may be unwound, in which case an antisense effect
might not be expected. Alternatively, the dsRNA may serve as a
substrate for editing enzymes such as double-stranded RNA
adenosine deaminase (DRADA).47-49 When DRADA deaminates adenosine, inosine is formed. The presence of inosine may
tag the mRNA molecule for destruction. In any case, the
message becomes unreadable. It is straightforward that either of
these eventualities would contribute to an antisense effect. It is
also straightforward that without physical destruction or modification of the targeted mRNA, strand unraveling would abrogate
an antisense effect.
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716
In an attempt to assure destruction of the mRNA target when
using an RNA molecule for targeting, many researchers have
been investigating the utility of ribozymes. Ribozymes are
catalytic RNA molecules whose structures are based on naturally occurring site-specific, self-cleaving RNA molecules50-53
(Fig 3A). The catalytic moiety of ribozymes recognize specific
nucleotide sequence, commonly GUX, where X 5 C, U, or A54
or, in some cases, NUX, where N 5 any nucleotide.55 Four
major classes of naturally occurring ribozymes have been
described, along with many artificially engineered types based
on the folding and cleaving properties of the naturally occurring
types.56-59 When the site-specific cleaving motifs of ribozymes
are incorporated into single-stranded RNA molecules whose 58
and 38 ends have been designed to hybridize with specific
sequence flanking an available catalytic cleavage site within an
mRNA target, a trans-acting and specific mRNA cleaving
molecule results. Such molecules are potentially very efficient
because once they cleave their target, they are released from
their mRNA target and are free to hybridize with another mRNA
molecule. Like ODN then, they can destroy multiple mRNAs in
a catalytic fashion.60,61
The chemical and physical requirements of ribozymemediated catalysis are being carefully studied in hopes that
more efficient molecules can be synthesized.62-64 In common
with oligonucleotides are issues that bear on stability of the
molecules and how to deliver them efficiently to cells. Structure/
function considerations unique to these molecules include their
speed of association with the target.65 Critically important as
well is the ribozyme’s dependence on divalent cations for
binding to, and cleavage of, their substrate.65,66 Hammerhead
ribozymes cleave most efficiently in an environment containing
greater than 500 mmol/L magnesium while the intracellular
environment has been estimated to have a magnesium concentration of ,500 µmol/L.67 Length of the flanking antisense
guide sequences are also important.62 For example, it has been
shown that if flanking antisense recognition sequences extend
beyond a certain length, ribozyme turnover is slowed and
specificity of the reaction is decreased.68 Finally, the intracellular localization of the ribozyme has also been shown to be
critical for activity because the ribozyme and its mRNA target
clearly need to be in physical proximity.69-71 However, even
physical colocalization of target mRNA and ribozyme is
insufficient for complete cleavage as shown recently by Jones
and Sullenger,72 who found only ,50% modification of an
mRNA target by a ribozyme expressed is tandem off the same
plasmid construct. The stability and activity of ribozyme
constructs can also be profoundly influenced by secondary
structure73 and protein interactions.74
Finally, the nucleic acid decoy strategy mentioned briefly
above has also been used to inhibit translation. This has been
most extensively investigated in the context of attempts to
inhibit replication of the human immunodeficiency virus (HIV).
In this case, viral mRNAs encode proteins required for expression of viral genes. If expression of these genes can be inhibited,
the virus fails to propagate. With this purpose in mind, a number
of investigators have reported expressing RNA molecules
corresponding to the HIV trans activation response (TAR)
element or the Rev response element (RRE) in different cell
types, and subsequently demonstrating that cells expressing
GEWIRTZ, SOKOL, AND RATAJCZAK
such constructs were protected against infection by HIV-1.75-77
The vectors used for these studies were primarily neo-RRE and
tRNA-RRE fusion gene constructs, suggesting that the carrier
RNA to which the RRE element was fused was not critical for
conveying protection. Rather, it was assumed that it is the RRE
element functioning as a competitor, or decoy, for available Rev
protein was responsible for protecting the cells. In the specific
case of the Rev decoy some data were provided to support this
assumption. Preliminary in vitro binding studies identified a
13-nt RNA sequence within the RRE that effectively bound
Rev. In vivo expression of this sequence in the form of a tRNA
fusion transcript was shown to inhibit HIV replication,75 and
such inhibition was correlated with inhibition of Rev function.
However, it must be noted that direct proof of decoy RNA and
Rev protein interaction was not provided. Accordingly, while
this approach to translational control of gene expression is
certainly interesting, and may potentially work via the mechanism proposed, specificity at the single gene level remains an
important issue that must be resolved.
OLIGONUCLEOTIDES—CONSIDERATIONS ON THE USE
OF MODEL MOLECULES FOR TRANSIENT DISRUPTION
OF GENE EXPRESSION
Chemical Considerations
Whether being used as an experimental reagent or pharmaceutical, ODN need to meet certain physical requirements to make
them useful. First, ODN need to be able to cross cell membranes
and then hybridize with their intended target. The ability of an
ODN to form a stable hybrid is minimally a function of the
ODN’s binding affinity and sequence specificity. Binding
affinity is a function of the number of hydrogen bonds formed
between the ODN and the sequence to which it is targeted. This
is measured objectively by determining the temperature at
which 50% of the double-stranded material is dissociated into
single strands and is known as the melting temperature or TM.
The TM depends on the concentration of the oligonucleotide, the
nature of the base pairs, and the ionic strength of the solvent in
which hybridization occurs. In the case of phosphodiesters, this
may be estimated from the following formula: TM 5 n(2°C) 1
m(4°C), where n 5 number of dA · T pairs and m 5 number of
dG · C base pairs. Thus, it may be seen that stability will
increase directly with the proportion of dG · C base pairs. This
is because G · C pair with three hydrogen bonds as opposed to
the two bonds formed by dA · T pairs. At physiologic conditions
(37°C, low salt) it is estimated that at least 12 bp need to form in
order to form a stable hybrid with a phosphodiester backbone,78
although more recent studies from Wagner et al79 suggest that 7
nt is sufficient under certain conditions. It is worth noting that a
single base mismatch, depending on its location, type, and
surrounding sequence, can decrease binding affinity as much as
500-fold. mRNA associated proteins and tertiary structure also
govern the ability of an ODN to hybridize with its target by
physically blocking access to the region being targeted by the
ODN.80,81 Finally, it is also clear that ODN should exert little in
the way of non–sequence-related toxicity, and should remain
stable in the extracellular and intracellular milieu in which they
are situated. Meeting all these requirements in any one molecule
has turned out to be a very difficult task because, as might be
expected, satisfying one criterion is often accomplished at the
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NUCLEIC ACID THERAPEUTICS
expense of another. If the object is to create a pharmaceutical
agent, the more complex the molecule, the more expensive its
synthesis. In an age of increasing cost consciousness, this too
becomes an important consideration in the design of these
molecules. For in-depth information and additional references
on any of these issues the reader is referred to one of several
outstanding reviews.82,83
It is probably easiest to approach this subject from the point
of view of a DNA molecule and to consider the various
modifications that might be made to satisfy the criteria mentioned above. Figure 4 shows two nucleotides of a hypothetical
natural oligomer and the phosphodiester bridge that joins them.
While many studies, including some from our own laboratory,
have reported using natural DNA for antisense investigations,12,84-88 it is becoming increasingly common to use material
that has been stabilized against attack from endonucleases and
exonucleases. These omnipresent enzymes attack DNA molecules at the phosphodiester bridges and break them down to
mononucleotides. First-generation antisense molecules were
designed to make the internucleotide linkages more resistant to
attack. This was accomplished primarily by replacing one of the
nonbridging oxygen atoms in the phosphate group with either a
sulfur or a methyl group. This type of modification forms a
phosphorothioate89 or a methylphosphonate ODN, respectively.90
Methylphosphonates are neutral in charge and therefore
lipophilic.91 Accordingly, in addition to being nuclease resistant, it was postulated that they may be taken up by cells in a
more efficient manner, but this is controversial. Nevertheless,
despite these properties, the methylphosphonates have not been
widely used for at least three reasons. First, and perhaps most
importantly, it has been found methylphosphonates do not allow
RNase H–mediated cleavage of the mRNA to which the
molecule may be hybridized. This appears to result in loss of
significant antisense effect. Second, because they are hydrophobic they are difficult to get into solution. Third, the molecules
are chiral at the methylphosphonate bridge and are therefore a
mixture with respect to any given conformation. This likely
lowers target mRNA affinity. Therefore, despite their otherwise
useful properties, other modifications will be required to make
this modification more useful. Some promising leads have been
identified, including the use of methylphosphonate, diester
chimeric molecules.92
In distinct contrast to the methylphosphonates, phosphorothioates are very widely used in both the laboratory setting and the
several clinical trials with antisense molecules that are now
ongoing.93-95 This situation is a result of several desirable
properties imparted to oligonucleotides synthesized with this
modification. First, phosphorothioates are relatively nuclease
resistant. In addition to their relative nuclease resistance, the
negative charge imparted to the phosphate backbone renders the
molecule hydrophilic and, therefore, water soluble. It is also
noteworthy that phosphorothioates permit RNase H activity in
the duplex. Nevertheless, this modification is also problematic.
First, the polyanionic nature of these molecules impairs uptake
because of the negative charge at the cell surface. Second, these
molecules also have a chiral center at the internucleoside
phosphorothioate. DeLong et al have synthesized dithioates in
an attempt to solve this problem. Here both nonbridging oxygen
717
atoms are sulfur substituted.96 Dithioates appear to have lower
binding affinity for RNA but are capable of inhibiting HIV
growth in culture.96 Third, many non–sequence-dependent
effects of ODN are attributed to charge interactions between the
phosphorothioate ODN and proteins found in the extracellular
environment, on the cell surface, and intracellularly.97-99 Fibroblast growth factor (FGF) is a well-studied example. Guvakova
et al100 have shown that phosphorothioates bind bFGF present
on extracellular matrix, and block the binding of FGF to its
surface receptor. Phosphorothioates have also been reported to
bind DNA polymerases, numerous members of the protein
kinase C family, and transcription factors. In addition to these
considerations, phosphorothioates are known to activate complement and may impair clotting by binding to factors such as
thrombin.101,102 Finally, high concentrations of phosphorothioates inhibit RNase H, thereby decreasing their effectiveness.103
A number of strategies have been introduced to overcome the
limitations of phosphorothioates at the same time that their
useful properties are preserved. Using end-capped phosphorothioates, where the 58 and 38 linkages are sulfated, appears to be
a reasonable compromise in that exonuclease stability is
conferred on the molecule and side effects associated with fully
thioated molecules are correspondingly reduced.104,105 Mixedbackbone oligonucleotides (MBOs) are another example.106
Compounds of this type contain phosphorothioate moieties at
the 38 and 58 ends for nuclease stability but with modified
oligodeoxynucleotides or oligoribonucleotides in the central
portion of the molecule to decrease the number of sulfur groups
in the molecule. MBOs of this type have been reported to have
improved properties compared with phosphorothioate oligodeoxynucleotides with respect to affinity to RNA, RNase H
activation, and biological activity. Such molecules are also
claimed to demonstrate more favorable pharmacological, in
vivo degradation, and pharmacokinetic profiles.106
The chemical modifications that can be made to the phosphodiester linkage are essentially without limit, and many have
been made. Two of the more interesting modifications currently
under development are the N38 = P58 phosphoramidates and
the peptide nucleic acids (PNAs). The phosphoramidate modification consists of substituting every 38-oxygen for a 38-amino
group.107 This creates a highly nuclease-resistant molecule with
an ability to form very stable duplexes with single-stranded
DNA, and RNA, by Watson-Crick base pairing. The structure of
complexes formed by phosphoramidates are quite similar to
those of RNA oligomers. In contrast to natural phosphodiesters,
the N38 = P58 phosphoramidates also form stable triplexes
with double-stranded DNA under near-physiological conditions. Although the ability of the phosphoramidates to activate
RNaseH is weak in comparison to natural DNA/RNA complexes, they do effectively block translation because of the
stability of the DNA/RNA hybrids formed.108 Recent studies
suggest that this modification may be useful for controlling cell
proliferation109 and HIV viral replication.
In addition to modifications to the internucleoside bridge,
examples of sugar and base alterations may also be cited.
Changing the sugar’s glycosidic linkage from the naturally
occurring b form, to the a anomeric form, where the base is
projected in the opposite direction, has been found to increase
nuclease stability significantly. However, this compromises
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718
hybridization stability and ability to activate RNase H.110
Whether this is a fatal flaw appears to depend on the system
being explored. Lavignon et al111 have reported that a 20-nt
a-oligonucleotide targeted to the primer binding site (PBS) of a
murine retrovirus inhibited viral spreading if cells were first
permeabilized in the presence of the oligonucleotide. They
speculated that antisense activity resulted from a decrease in
initiation or inhibition of extension of the minus or plus DNA
strands. Chimeric a, b anomeric oligodeoxynucleotides have
also been reported to be effective antisense compounds, as
judged by the ability to inhibit in vitro translation of the pim-1
proto-oncogene because of restoration of the ability of the
molecules to activate RNase H.112
Sugars are also typically modified at the 28 position with
O-methyl, fluoro, O-propyl, O-allyl or other groups. These
modifications increase affinity for RNA and impart some
nuclease resistance. Nevertheless, these molecules do not
support RNase H activity and, for this reason, do not appear to
have significant activity in some assays.113 Therefore, a number
of groups have used the 28-O-methyl modification to flank
natural diesters.113,114 Such chimeric molecules do activate
RNase H if there are at least five internal natural nucleotides.113
Alteration of the C5 position of the pyrimidine bases producing
the C5 propynyl substitutions have attracted notice because of
their affinity for RNA, the stability of the hybrids formed, and
their ability to activate RNase H.115 Whether the latter property
is critical for their activity is in fact uncertain since some have
reported that the tight hybrids formed by these compounds are
very efficient at blocking translation.44 Again, however, these
molecules must be used in conjunction with modified bridges
because these modifications do not protect against nucleases. In
addition, they require a carrier to get them across cell membranes or direct physical injection.116
The PNAs represent a more radical approach to the nuclease
resistance problem. Here, the phosphodiester linkage is completely replaced with a polyamide (peptide) backbone composed of (2-aminoethyl) glycine units.117 Such compounds are
achiral and are completely nuclease resistant as they have no
phophodiester linkages. Since the bases attached to the PNA
backbone are projected in space as they would be on a native
backbone, the PNAs retain their ability to Watson-Crick base
pair with single-stranded DNA or RNA. In addition, homopyrimidine PNAs can form triplexes with double-stranded DNA,
and can also displace a duplexed DNA strand to bind with its
complement.117,118 All of these properties are clearly useful for
antisense gene inhibition. Nevertheless, compounds of this type
also have problems. Because they cannot move freely across
cell membranes they must be injected into cells or delivered
with artificial vectors such as phospholipids.119 In addition to
these problems, the PNAs do not activate RNase H. Accordingly, they most likely exert their antisense effect by blocking
RNA elongation which, as in the case of methylphosphonates,
may not be as efficient as destruction of the mRNA. Finally,
PNAs are also sensitive to local ionic concentration and do not
hybridize as well under physiologic conditions.
One additional chemical strategy that is also of interest is the
use of circular DNA molecules. Circular DNA and RNA
molecules are in fact quite abundant in nature, but it has only
been recently that technical problems associated with their
GEWIRTZ, SOKOL, AND RATAJCZAK
synthesis have been solved.120 Kool et al, the leaders in this
area, have noted that these molecules have a number of
attractive qualities which merit their development. First, since
they have no 58 or 38 end they are resistant to exonuclease
attack. In addition, they appear to have excellent binding
affinity, sequence specificity, and are capable of activating
RNase H. A circular molecule of ,20 to 30 nucleotides in
length should be able to target a linear sequence of ,12 to 14
bases. If the circular DNA is composed predominantly of
pyrimidines, it will not self-anneal and will therefore remain an
open circle. Further, if sequence targeted is a purine-rich area,
the circular DNA will be able to form Watson-Crick base pairs
with one portion of the circle as well as a triple helix with the
resulting duplex as the circular molecule winds around the DNA
target. Therefore, an extremely stable structure is formed.
Finally, the circular DNAs have excellent strand displacement
activity which would, in theory, help them hybridize in areas of
RNA that are folded. This could greatly increase ‘‘targetable
sequence.’’ Whether this approach will work as well for mRNA
targeting remains to be seen. Other, circular RNA/DNA chimeric ODN have been constructed.121-124 Experience with these
modifications remains limited.
Picking the Right Tool for the Right Job—Oligo DNA
Versus Oligo RNA
To inhibit translation, one must make a choice of whether to
use a DNA or RNA molecule. Several factors may help
facilitate this decision. DNA is inherently more stable than
RNA, and is therefore much easier to apply to cells externally in
the absence of a delivery vehicle. DNA is also easy to make on
automated equipment, especially since the antisense ODN used
for this purpose are typically from ,12 to 25 bases long.
Placing functional groups on the DNA molecules to facilitate
binding to, or destruction of, the mRNA and for tracking the
oligonucleotide is also relatively easy. Finally, there appear to
be few restrictions of the sequence that can be targeted.125 This
is in marked contrast to antisense RNA, which must be
delivered by vector; ribozymes, which must be targeted to
cleavable sites; and parenthetically to TFOs, which must, at
least for the moment, be targeted to polypurine-polypyrimidine
stretches of duplexed DNA. Nevertheless, while native DNA is
clearly more resistant to nucleases present in serum or cells than
RNA, it is still very much subject to degradation in either
environment and must often be rendered more resistant by
modifying the phosphodiester bridges between nucleosides, or
the sugar moieties as was discussed above. In addition, the
problems associated with transporting DNA into cells, and
getting it to the proper locations for interacting with its target,
are not at all trivial and are only now becoming understood.
RNA molecules are attractive because they form more stable
duplexes with their mRNA targets. In theory, this might lead to
more efficient antisense effects. Nevertheless, because of the
stability issues discussed above, antisense RNAs and ribozymes
are typically expressed inside the cell from a vector designed for
this purpose. This is problematic for all of the reasons that are
now widely appreciated, including efficiency of transfection,
expression, host cell range, and vector persistence.2 Despite
these concerns, expressing antisense RNA or ribozymes from a
vector is often the only practical approach one can take when
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NUCLEIC ACID THERAPEUTICS
long-term presence of the antisense sequence is desired, ie,
when attempting to target an mRNA that encodes an abundant
and long-lived protein. It should be noted that antisense RNA,
especially in the form of ribozymes, has been delivered to cells
externally.126 For this approach, the RNA molecules must be
protected, eg, by stabilizing the phosphodiester bonds, and by
packaging the material in liposomes.
Size Does Matter
Given the considerations discussed above, an oligonucleotide’s size becomes an important consideration. Antisense ODN
are typically synthesized in lengths of 13 to 30 nucleotides. The
origin of this convention arises from the fact that there are
approximately three to four billion base pairs in the human
genome. Statistical calculations based on this number suggest
that the minimum ODN size needed to recognize a specific gene
is between 12 and 15 bases in length.78,127 This number is
convenient because as mentioned above, a phosphodiester
oligomer needs to be ,12 nucleotides long to form a stable
hybrid under physiologic conditions. Nevertheless, these basic
considerations need to be modified based on a number of
factors, in particular the chemistry of the oligonucleotide. The
sulfur modification, for example, lowers the TM so in comparison to a natural diester, a phosphorothioate targeted to the same
sequence should be made longer by several bases to compensate. Unexpectedly, then, it has been reported that phosphorothioates may be effective and retain specificity with sequences only
8 nt in length.128 This finding may be explained in the following
way. First, the experiments were performed in a frog oocyte
system where the temperature is lower by several degrees in
comparison to mammalian cells. Second, it has also been
reported that sequence adjacent to the targeted region is also
very important in allowing hybridization because such sequence
dictates folding and, therefore, secondary and tertiary structure
of the molecule.129-131 How targetable sequence may be found
will be discussed in more detail below. Sequences longer than
the minimal length to guarantee specificity and formation of a
stable hybrid are also problematic. Longer sequence may form
more stable hybrids through more extensive base pairing, but
they are more expensive to synthesize and, somewhat paradoxically, may also suffer from lack of specificity. This is because
short runs of complementary bases may hybridize if larger
intervening sequences are looped out. Once duplexes form, any
of the events discussed above can occur, leading to unintended
loss of expression of a nontargeted mRNA. It is obvious then
that simple rules, like many factors governing antisense experiments, are only a starting point for individualizing these factors
to a particular set of experimental conditions one encounters in
the system under study.
Targeting mRNA-Sequence Selection
It is straightforward that in order for an antisense molecule to
perturb utilization of the mRNA to which it is targeted, the
mRNA and the oligonucleotide have to hybridize with each
other. As discussed above, hybridization efficiency is primarily
dependent on affinity of the ODN for its complementary
sequence. Nevertheless, other considerations also apply. It has
been reported, for example, that ODN targeted to the 58 end of a
single-stranded loop have orders of magnitude higher affinity
719
for their target than those targeted to the 38 end.129 This
observation may be explained by structural considerations, an
intuitively obvious factor if one considers the highly complex
folding that mRNA molecules may undertake. Such folding
represents a major problem because it is largely unpredictable in
vivo and can clearly render sequence inaccessible to the
targeting ODN. A straightforward consequence of this situation
is that the identifying sequence which is not buried in higher
order structure and, therefore, which is accessible to the ODN,
is a matter of chance.
A number of strategies have evolved to address the problem
of oligo targeting. Because many investigators have reported
success targeting around the initiator codons, or the transcriptional start site, this is often the initial target for many
experiments. If this approach is unsuccessful, or if it is deemed
desirable to target other regions of the mRNA, randomly
selected sequence is then resorted to. This approach can be
extremely frustrating because chance alone appears to dictate
success. In response, it has been suggested that an mRNA
‘‘walk’’ be used as a means for identifying accessible sequence.132 In this approach a series of oligonucleotides are
synthesized from the 58 end of the molecule to the 38 end. These
are then tested for their ability to elicit an antisense effect.133
This method is effective, but because it is trial and error based is
not particularly efficient (,25% success rate) and is potentially
expensive if many sequences have to be tested before a useful
one is found.
Several in vitro model systems for picking mRNA target
sequence are being developed. For example, Mishra and
Toulme134 use an in vitro selection procedure designed to select
random ODN sequences capable of hybridizing with a known
structure, such a stem loop. Such sequences were called
‘‘aptastrucs’’ because they were likely, or apt, to bind to the
structure. In this approach, a population of randomly synthesized oligonucleotides were mixed with the structure of interest
and ODN sequences bound to it were selected and amplified.
Selection was based on enzymatic digestion of nonduplexed
ODN and polymerase chain reaction (PCR) amplification of
those that survived. It is of interest that a DNA hairpin structure
was used as the model example. Although the procedure was
said to be appropriate for either DNA or RNA targets, there is no
doubt that its effectiveness might be limited with the latter since
predicting structure, as was just noted, is problematic. Computer modeling may be of some utility for predicting accessible
RNA sites,135 but more recent studies support the notion that
such structural predictions are of little of no use for picking
target sequence.136
Another strategy of interest has been reported by Rittner et
al65 and is based on the observation that, at least in prokaryocytic systems, the in vitro rate of hybrid formation between
antisense RNA and its complement correlates with the antisense
molecules effectiveness in vivo. Using HIV as a model, these
workers synthesized a set of HIV-1–directed antisense RNAs
with the same 58-end but successively shortened 38-ends
produced by alkaline hydrolysis. The mixture was used to
determine hybridization rates for individual chain lengths with a
complementary HIV-1–derived RNA in vitro. They found that
second order binding rate constants of individual antisense
RNAs differed by more than 100–fold. Of interest, slow-
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720
hybridizing and fast-hybridizing antisense RNAs differed by
only two or three 38-terminally located nucleotides in some
cases. Of most importance, the binding rate constants determined in vitro for individual antisense RNA species correlated
with the extent of inhibition of HIV-1 replication in vivo.
Similar studies have been performed on bcr/abl mRNA with
similar conclusions.137 A more complicated approach based on
predicted structure and hybridization thermodynamics and been
reported by Stull et al.138 It is of interest that the duplex
formation kinetics were the most accurate predictors of ODN
efficiency in this model, perhaps because this variable must be a
function of target sequence availability.
Recently, Milner et al139 used a novel hybridization strategy
to find oligodeoxynucleotides capable of hybridizing with
specific mRNAs. An array of 1938 oligodeoxynucleotides,
which ranged in length from monomers to 17 nt, was synthesized on the surface of a glass plate and used to determine the
potential of any of the oligonucleotides to form heteroduplexes
with rabbit b-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 58 UTR
and bases 1 to 69 of the first exon. These investigators reported
that very few oligonucleotides showed significant heteroduplex
formation with the target. Antisense activity, measured in a
RNase H assay and by in vitro translation, correlated well with
yield of heteroduplex on the array. The investigators point out
that their results help to explain the variable success that is
commonly experienced in the choice of antisense oligonucleotides. It is of interest that there were no obvious features in the
mRNA sequence, or predicted secondary structure which adequately explained the variation in heteroduplex formation. The
investigators suggest that their method may provide a simple
though empirical method of selecting effective antisense oligonucleotides. However, the true test of the predictive value of this
method must rest on the ability of the selected oligonucleotides
to effectively interact with their target in vivo. Because RNA
folding in vivo is likely to be quite different than that
encountered in vitro, this is a critical point. An attempt to
address this problem was recently reported by Ho et al,136 who
used semi-random oligonucleotide libraries to probe a candidate
mRNA molecule for RNase H cleavable sites. Oligos predicted
to be effective were tested in a biological system where
generally good correlation was found.
To address the problem of identifying accessible sequence in
mRNA in vivo, we have synthesized reporter ODN composed
of a stem-loop structure complexed to a fluorescent (F) moiety
on one arm (EDANS) and a nonfluorescent quenching (Q)
moiety (DABCYL) on the other.140 When these molecules
hybridize with a complementary nucleotide sequence, the stem
loop opens, the fluorophore and quenching moieties separate,
and fluorescence is observed at 490 nm when the EDANS
moiety is excited by UV light (336 nm). Such ODN have been
dubbed ‘‘molecular beacons’’ (MB). We have investigated the
utility of MB for demonstrating ODN-mRNA duplex formation
in living cells. MB targeting myb or vav mRNA through
complementary sequences in the loop region were constructed,
and then initially tested in vitro. A threefold molar excess of
target sequence was incubated with AS-myb-MB, AS-vav-MB,
or their respective control (sense; 6-nt mismatch; complete
mismatch) MB in a cell-free system. Quantitative fluorimetry
GEWIRTZ, SOKOL, AND RATAJCZAK
showed that AS-MB generated a greater than 50-fold increase in
signal intensity when compared with control MB. The specificity of hybridization in the presence of competing RNA was then
tested. MB were incubated with 10 µg of K562 cell-derived
total cellular RNA. An ,15-fold greater fluorescence was
observed with AS MB than with any of the controls. Potential
sensitivity of duplex detection in living cells using fluorescence
microscopy was determined by microinjecting preformed MBmRNA duplexes into K562 cells. Signal could be observed with
as little as ,1 3 1024 fg of complex when using a UV fluoride
lens–equipped microscope. Accordingly, detection of MBmRNA hybridization for many genes should be possible. To test
this directly, 550 µmol/L of each MB was microinjected into
living K562 cells. Cellular fluorescence was detected with
AS-MB but not with any controls. Accordingly, MB may well
prove useful for studying the temporal and spatial kinetics of
ODN/mRNA interactions in living cells.
DELIVERY AND SUBCELLULAR TRAFFICKING
OF OLIGONUCLEOTIDES
Delivery and trafficking of oligonucleotides needs to be
considered from both a cellular and subcellular point of view.
Cellular delivery may be nonspecific, ie, all cells may have an
opportunity to take up material or they may be targeted to a
particular population. Subcellular trafficking depends on how
the oligonucleotide molecule used is sorted within the cell. At
the moment, factors that regulate sorting of these molecules is
not well understood, but work with ribozymes at least suggests
that this issue is critical for cleaving the mRNA target.
Nucleic Acid Uptake and Traffıcking
It is probably best to first consider the uptake mechanism of
naked nucleic acids. Although few studies have been performed
with unmodified DNA, uptake of DNA does appear to be a
natural phenomenon that has been postulated to represent a
nucleic acid salvage mechanism for material excreted by
apoptotic cells.141 A number of laboratories have examined
oligonucleotide uptake using either native, methylphosphonate,
or phosphorothioate DNA142-150 (Fig 5). Methylphosphonate
derivatives are uncharged molecules that have been reported to
enter cells via passive diffusion,91 although this concept may
represent an oversimplification. In contrast, native and phosphorothioate oligodeoxynucleotides are polyanionic molecules.
This charge state makes it very difficult for them to passively
diffuse across cell membranes. Not surprisingly then, ODN
uptake appears to be primarily an active process dependent on
time, concentration, energy, and temperature.91,151,152 Studies
from our own laboratory suggest that the uptake mechanism is
at least partially concentration dependent and that below a
concentration of 1 µmol/L, uptake of phosphorothioate oligodeoxynucleotides is predominantly via a receptorlike mechanism,
while at higher concentrations a fluid-phase endocytosis mechanism appears to predominate.147 Direct physical evidence that
ODN may be found within clathrin-coated pits on the cytoplasmic membrane has also been reported.147 Several receptorlike
proteins responsible for uptake have been described.125,143,145,147
For example, Loke et al143 reported using affinity chromatography to isolate an 80-kD surface protein that appeared to be
responsible for transport. Geselowitz et al145 used photoactivat-
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NUCLEIC ACID THERAPEUTICS
721
This figure not available online; please see print version
Fig 5. ODN Uptake. (A) ODN is found in clathrin coated pits indicative of receptor-mediated endocytosis. (B and C) ODN can be found in
lysosomal (clear vesicles) or endosomal compartments (vesicles filled with darker material); however, some ODN is free and (D) crosses the
nuclear membrane to presumably form hybrids with target mRNA. (E) Control panel labeled with Biotin alone. Oligodeoxynucleotides have been
decorated with gold beads that appear has black dots in the photomicrograph. Their location is pointed to by black arrows. (Reproduced from
The Journal of Clinical Investigation, 1995, vol 95, p 1814 by copyright permission of The American Society for Clinical Investigation.147)
able cross-linkers to study oligonucleotide binding to HL60
cells and found that several proteins were labeled, the predominate species being a 75-kD membrane-associated protein. A
least five major classes of receptorlike binding proteins were
described by Beltinger et al.147
Although there are suggestions that uptake of ODN may be
more efficient in vivo that in vitro,153 a great deal of work is
being done to increase uptake because, as detailed above, ODN
uptake is relatively inefficient. Increased cellular delivery will
likely lead to augmentation of antisense effectiveness, and
several different strategies have been developed to enhance
delivery of these compounds. Microinjection has been used
successfully by many laboratories but is of little use clinically.42,154,155 Other commonly employed strategies may be
classified as those which seek to physically modify the target
cell, typically by permeabilizing the cell’s membrane, and those
which seek to directly or indirectly modify the permeation
properties of the ODN.
Physical disruption of target cell membranes may be accomplished by electroporation156,157 or by use of agents such as
streptolysin, which permeabiize cell membrane.46,156,157 These
methods are clearly only appropriate for ex vivo use, such as
bone marrow purging. Both methods are physically destructive
to cells and their ultimate clinical utility remains uncertain.
Calcium has been used in recent studies to assist uptake of
AS-ODN into the cellular milieu.158 Attempts to determine the
mechanism of Ca21/AS-ODN interactions are currently being
examined because double-stranded DNA is known to bind
Ca21, while inverted repeat hairpin conformations that are
capable of base pairing (palindromes) seem to bind magnesium
and zinc.159
An alternative strategy to physical permeation of the target
cell is to package the DNA in an artificial ‘‘viral vector’’ which
delivers the oligonucleotide into the cell. Such vectors are
typically cationic lipids, which may either be manufactured to
contain the DNA molecule within a lamellar structure (lipo-
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722
some), or mixed with the DNA, allowing the material to coat the
DNA by charge interaction.116,160-162 Cationic peptides are being
developed for a similar purpose.163 Whether packaging the
DNA in a liposome is superior to merely coating it with cationic
lipid is uncertain. In either event, application of the cationic
amphiphile condenses the DNA, and imparts an overall positive
charge that facilitates attachment to the anionic animal cell
membrane while the lipid tails enhance subsequent passage
through the lipid bilayer of the cell membrane. At the same
time, the DNA is also protected from nuclease attack. While it
had been originally assumed that DNA delivered by cationic
lipids were ‘‘dumped’’ immediately into the cytoplasm by
fusion of the lipid with the cell membrane, this is now known to
be incorrect. Rather, it appears that these materials are taken up
by endocytosis but, once inside a vesicle, the cationic lipid
destabilizes the endosomal membrane. How this is accomplished remains uncertain and is likely complex.
Recent structural studies by Radler et al164 revealed that upon
addition of either linear l-phage or plasmid DNA to cationic
lipids, an unexpected topological transition from liposomes to
optically birefringent liquid-crystalline condensed globules is
observed. Study of such structures is expected to shed light on
how DNA is inserted through cell membranes. Other studies
have suggested that vesicle destabilization induces flip-flop of
anionic lipids from the cytoplasmic-facing monolayer, which
diffuse into the DNA/lipid complex. DNA is displaced from the
cationic lipid and released into cytoplasm.165,166 In the case of
polyamines, water is attracted into the vesicle causing it to swell
and burst. Once free in the cytoplasm the DNA diffuses into the
nucleus. Numerous strategies for increasing the efficiency of
cationic lipid delivery of antisense vectors have been reported.
These include fusogenic peptides from appropriate virus in the
lipid complex,167 other conjugates such as cholesterol,168,169 or
by complexing with a specific antibody to allow targeting to a
particular cell type.170 In recent studies on uptake into primary
hematopoietic cells, Kronenwett et al171 reported on the utility
of cationic lipids to enhance delivery of oligodeoxynucleotides.
They found a cell-type–dependent delivery of oligodeoxynucleotides, with greatest uptake in monocytes and smallest uptake in
T cells. CD341 cells, B cells, and granulocytes took up
oligodeoxynucleotides at an intermediate level. Uptake of ODN
into isolated CD341 cells could be increased 100- to 240-fold
using cationic lipids compared with application of ODN alone.
Of note, stimulation of CD341 cells by interleukin-3 (IL-3),
IL-6, and stem cell factor did not significantly improve cationic
lipid-mediated ODN delivery.
Finally, the oligonucleotide itself may be modified. The
strategy most frequently used has been to conjugate the
oligomer, either directly or indirectly, to a ligand specific for a
receptor resident on the cell type of interest. For example, ODN
have been conjugated to folate, mannose, asialoglycoproteins,
and tumor-specific antibodies to target hematopoietic, pulmonary alveolar172 hepatic, and a variety of tumor cells,173
specifically. While such modifications have reported to be
effective in certain controlled circumstances, the general applicability of this approach remains to be determined. Technical
problems, such as dissociation of the ODN and the carrier,
which in the case of ODN linked to asialoglycoprotein was
,85% within 1 hour,174 rapid clearance of antibody conjugated
GEWIRTZ, SOKOL, AND RATAJCZAK
material, or biologic sequestering in the endosome lysosome
compartment172,175 also limit effectiveness of this approach and
still need to be overcome.
Subcellular Traffıcking
Once across the membrane, the intracellular distribution of
oligonucleotides is still somewhat controversial and is no doubt
at least somewhat dependent on the chemistry of the molecules
being studied. Fluorescent-labeled ODN microinjected into the
cytoplasm of cells are found to rapidly accumulate in the
nucleus.155 However, when fluorescent-labeled ODN are placed
in tissue culture media, the labeled molecules accumulate in
vacuoles, presumably endosomes and lysosomes, within the
cytoplasm forming a punctuate perinuclear pattern.176 These
results are in accord with most studies examining uptake of
oligonucleotides from the extracellular environment. In contrast
to ODN localization when the compounds are introduced by
microinjection, there is no visible fluorescence in the nucleus
itself, indicating that the release of ODN from these vacuoles is
an inefficient process. Our own studies have visualized this
process at the ultrastructural level147 and show that oligonucleotides progress through the lysosomal, endosomal compartments.
If the oligonucleotides escape the endosome/lysosome compartment, it appears that they migrate rapidly into the
nucleus.146,147,177 The mechanism appears to be one of diffusion,147,177 with subsequent trapping of the oligonucleotide,
apparently by binding to a unique set of nuclear binding
proteins.177 The fate of oligonucleotides localized to the nucleus
is an important issue in terms of the bioavailability of these
compounds. Should the majority of the oligonucleotide become
tightly complexed to nuclear protein they may become biounavailable and, therefore, inactive. Alternatively, modifications
designed to prevent nuclear import might also have favorable
qualities because such molecules would likely reach higher
cytoplasmic concentrations and should, therefore, have a greater
chance of associating with ribosomal mRNA. This, of course,
presumes that molecules with the desired modifications will be
released intact from the vesicles in which they were imported.
Accordingly, strategies aimed at increasing release of ODN
from endosomal structures, such as the use of fusogenic
peptides derived from the influenza hemagglutinin envelope
protein, may also be successful for similar reasons.178-180
Recent studies with ribozyme molecules support the hypothesis that intracellular sorting and localization of the antisense
molecules may be critical for their activity. In addition to
targeting to a cell type, it is likely that subcellular targeting may
also be an important consideration.181 Sullenger and Cech69
critically examined this possibility by determining whether
colocalization of ribozymes with their intended mRNA target
would increase their efficiency. To perform their experiments,
the investigators used a packaging cell line infected with a
retrovirus (B2A) containing a copy of the lacZ gene. This line
was then infected again with a B2A retrovirus engineered to
express a lacZ ribozyme. It was then hypothesized that B2A
transcripts destined to be packaged in replicating virus would be
processed together in the same nuclear tracts while those that
were fated to be distributed in the cytoplasm as mRNAs would
be processed in different tracts. If colocalizing the ribozyme
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NUCLEIC ACID THERAPEUTICS
with its target (lacZ mRNA) was important for activity of the
ribozyme, this could be determined by measuring a decrease in
titer of B-gal virus while B-gal activity, the result of translating
lacZ mRNA, would be relatively unaffected. This, in fact,
proved to be the case as the ribozyme reduced the titer of
infectious virus containing lacZ by 90%, but had no effect on
B-gal activity in the cells. These results convincingly argued
that ribozymes needed to be in physical proximity of their
target, and there is little reason to suspect that this would not be
true for oligonucleotides as well.
Bertrand et al70 have also performed similar experiments and
have reached similar conclusions. Their study was also designed to better understand the influence of RNA transcript
context on RNA localization and catalytic RNA efficacy in vivo.
Towards this end, they constructed and characterized several
expression cassettes useful for transcribing short RNAs with
well-defined 58 and 38 appended flanking sequences. These
cassettes contain promoter sequences from the human U1
snRNA, U6 snRNA, or tRNA Meti genes, fused to various
processing/stabilizing sequences. The levels of expression and
the subcellular localization of the resulting RNAs were determined and compared with those obtained from Pol II promoters
normally linked to mRNA production, which include a cap and
polyadenylation signal. The tRNA, U1, and U6 transcripts were
nuclear in localization and expressed at the highest levels, while
the standard Pol II promoted transcripts were cytoplasmic and
present at lower levels. The ability of these cassettes to confer
ribozyme activity in vivo was tested with two assays. First, an
SIV-growth hormone reporter gene was transiently transfected
into human embryonic kidney cells expressing an anti-SIV
ribozyme. Second, cultured T lymphocytes expressing an
anti-HIV ribozyme were challenged with HIV. In both cases, the
ribozymes were effective only when expressed as capped,
polyadenylated RNAs transcribed from Pol II cassettes that
generate a cytoplasmically localized ribozyme that facilitates
colocalization with its target. The inability of the other cassettes
to support ribozyme-mediated inhibitory activity against their
cytoplasmic target is very likely due to the resulting nuclear
localization of these ribozymes. These studies show that the
ribozyme expression cassette determines its intracellular localization and, hence, its corresponding functional activity.
PHARMACODYNAMICS OF
OLIGODEOXYNUCLEOTIDE DRUGS
To examine the pharmacokinetics and metabolism of ASODN, extensive investigations have been carried out in a
variety of animal systems, and in a few human trials as well.
Most reports examine the kinetics of intravenous or intraperitoneal administration. Of interest is that the material appears to be
absorbed from the gastrointestinal tract as well as other
routes.182 Using a phosphorothioate modified (20 nt) ODN,
Agrawal et al114,183 examined the effects of intravenous and
intraperitoneal administration in mice. In these studies, approximately 30% of the administered dose was excreted in the urine
over the first 24-hour period while ODN accumulated preferentially in the liver and kidney. Similar results were reported in
subsequent studies.184-187 ODN accumulate in the kidney and
liver at concentrations that exceed plasma levels. In mice, the
brain apparently is a privileged site into which very little ODN
723
accumulates. In monkeys, the plasma half-life of ODN is
similar to that of rodents.101 The organs showing the highest
accumulation of ODN were kidney, liver, thymus, bone marrow,
lymph nodes, salivary glands, lungs, and pancreas. Moderate
accumulation was found in muscle, gastrointestinal tract, and
trachea. The lowest accumulations were found in brain, spinal
cord, cartilage, skin and prostate.
More recent studies in nude mice have confirmed these
earlier reports, and made comparison between the pharmacokinetics and tissue distribution of phosphorothioate (PS), methylphosphonate (MP), and phosphorodithioate (PS2) oligonucleotide analogs.96 In these experiments, radiolabeled 15-nt
oligonucleotides complementary to the AUG region of K-ras
were used in nude mice bearing a K-ras–dependent human
pancreatic tumor. The oligonucleotide analogs were administered as a single dose in the tail vein. A rapid distribution phase
with t1/2 a values of 1 minute or less and an elimination phase
with average t1/2 b values of 24 to 35 minutes was observed.
Volumes of distribution (Vd) were 3.2, 4.8, and 6.3 mL for PS2,
MP, and PS, respectively, in comparison to 3.6 mL for sucrose, a
fluid-phase marker. In general agreement with previous studies,
relative tissue drug levels obtained at 1 and 24 hours after
administration were kidney . liver . spleen . tumor .
muscle. Total kidney and liver oligonucleotide accumulation
was approximately 7% to 15% of the initial dose, with tumor
accumulating 2% to 3%. Intact compound was recovered from
all tissues, including tumor, as assessed by high-pressure
reversed-phase HPLC coupled to radiometric detection. Importantly, integrity of the oligonucleotides ranged from 73% in
blood to 43% to 46% in kidney and liver. Kidney and liver
appear to be the primary sites of metabolism.
In humans, studies have been performed on patients with
leukemia101,188 and acquired immunodeficiency syndrome
(AIDS).189 In six HIV patients, pharmacokinetic and toxicology
data were gathered after 2-hour intravenous infusions (0.1
mg/kg) of 35S-labeled phosphorothioate oligonucleotide.189
Plasma disappearance curves could be described by the sum of
two exponential, with half-life values of 0.18 6 0.04 and
26.71 6 1.67 hours. The radioactivity in plasma was further
evaluated by polyacrylamide gel electrophoresis, showing the
presence of both intact oligonucleotide and lower molecular
weight metabolites. Urinary excretion represented the major
pathway of elimination, with ,50% of the administered dose
excreted within 24 hours and ,70% eliminated over 96 hours
after dosing. Intact as well as degraded material was found in
the urine. Of interest, the half lives of the oligonucleotide were
shorter than those observed in experimental animals. The
mechanisms responsible for the differences were not clear but
could obviously be caused by species differences in metabolic
capacity, renal clearance, and serum protein binding. In the first
reported case where phosphorothioate ODN were infused into a
patient with ALL, only slight increases in g-glutamyl transpeptidase (gGTP) values, transient metallic taste, nausea, and
vomiting were described.188
As with any new pharmaceutical agent, the potential side
effects of antisense ODN are important to consider and determine. ODN, depending on their target, may disturb the expression of genes not only in neoplastic cells but also in normal,
healthy cells. This raises the issue of differential sensitivity
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724
GEWIRTZ, SOKOL, AND RATAJCZAK
between normal and diseased cells. It is clear that if a gene
common to both populations is being targeted, the normal cells
must be more resistant to loss of the target gene’s function than
the diseased cell. We might also expect some nonspecific side
effects of infused ODN which are not related to their ability to
interact specifically with nucleotides or proteins. For example,
one potential side effect of treatment with phosphorothioate
ODN may be thrombocytopenia. Mice receiving high doses of
phosphorothioate ODN manifest a decrease in platelet counts. It
is hypothesized that the thrombocytopenia is related to the
polyanionic charge of the ODN.190 Cardiovascular toxicity and
deaths in monkeys after intravenous infusions of phosphorothioate ODNs has also been reported.191 Adverse events were noted
only at large doses (5 to 10 mg/kg) and after rapid bolus
administration. Slow infusion of similar doses appeared to be
well tolerated. The toxic effects were believed likely to be due
to complement activation, but release of anaphylatoxins, autocoids, eicosanoids, leukotrines, or cytokines are also possibilites.
POTENTIAL APPLICATIONS OF THE ANTISENSE
STRATEGY IN CLINICAL HEMATOLOGY
Although oligonucleotides have been reported as having
activity against HIV,189 cytomegalovirus (CMV) infections of
the eye,192 and most recently Crohn’s Disease,193 this portion of
the review will focus on potential uses of oligonucleotides in the
treatment of hematologic disorders. These applications are of
interest because they capitalize on ‘‘antisense,’’ as well as
aptameric effects. The former have found the greatest application in the treatment of hematologic neoplasms, while the latter
have been used as thrombin inhibitors for anticoagulation
purposes.
Chronic Myelogenous Leukemia (CML) as a Paradigm
for Nucleic Acid Therapeutics
As of this writing, oligonucleotides are being used as ex vivo
bone marrow purging agents, an approach pioneered by my own
group,84,194 and as potential drugs for direct in vivo administration.195 Most development and clinical experience has been
gained with the ex vivo application, largely because of cost
considerations. Marrow purging requires relatively small
amounts of material in comparison to direct in vivo administration. It is also true that most development has occurred in the
context of attempting to treat CML. The latter disease has been
a lightning rod for development of this approach because of the
presence of a tumor-specific mRNA target that all agree is
important in the pathogenesis of the disease. However, as will
be detailed below, choice of gene target is not always obvious
and many factors must be taken into account in developing an
effective therapeutic oligonucleotide in this and other diseases.
If one considers the molecular pathogenesis of CML, a
number of potentially interesting gene targets to which oligonucleotides might be directed present themselves. First and
foremost is the bcr/abl mRNA itself. Bcr/abl mRNA is the
product of a neogene created by a reciprocal translocation
involving the c-abl gene on chromosome 9 and the bcr gene on
chromosome 22 (recently reviewed in Melo196 ). This translocation results in the formation of the Philadelphia chromosome
and the neogene bcr/abl. There are three major bcr/abl variants
depending on where the invariant portion of abl is fused with
bcr. These are designated b1a2 (p185), b2a2, and b3a2 (both
encode p210). A fourth variant e19a2 (p230), which is associated with a chronic neutrophilia, has been recently described.
Each of these genes may be considered tumor specific and are
likely both necessary and required for the CML transformation
of hematopoietic cells. Because they are tumor specific, and
pathogenetically important, they are an obvious oligonucleotide
target in this disease.
Szczylik et al197 were among the first to suggest that
oligonucleotides targeting bcr-abl might be of therapeutic
utility. These workers found that when primary leukemic blast
cells were exposed to synthetic 18-nt oligodeoxynucleotides
complementary to b2a2 or b3a2 bcr-abl junctions, leukemic cell
colony formation in vitro was significantly suppressed. In
contrast, granulocyte-macrophage colony formation from normal marrow progenitors was unaffected by the same oligonucleotides. Suppression of colony growth was found to be sequence
specific as well in that mismatched oligonucleotides had no
effect on colony formation and oligonucleotides targeting one
breakpoint did not inhibit growth of cells expressing the other.
Finally, when equal proportions of normal marrow progenitors
and blast cells were mixed, exposed to the oligodeoxynucleotides, and assayed for residual colony formation, the majority of
residual cells were normal. These findings suggested that
bcr-abl targeted oligonucleotides represented a means for
specifically killing CML cells. This work was followed by a
number of reports from the same group, and others, which
reported very similar findings.94,198-203
Not all investigators have been able to reproduce the results
described above.204-210 O’Brien et al,208 for example, examined
the effects of antisense oligonucleotides of various lengths,
sequences, and chemistry on the proliferation of eight different
cell lines, five derived from patients with CML and three from
other sources. They found that phosphodiester oligonucleotides
had little antiproliferative activity in their system, presumably
because of degradation by nucleases present in fetal calf serum
or rapid intracellular breakdown. Phosphorothioate oligonucleotides antisense, but not sense, to the B2A2 and B3A2 breakpoints significantly inhibited the proliferation of three CML cell
lines (BV173, LAMA84, and KYO1). However, the antiproliferative effect appeared to be independent of the type of
breakpoint expressed by each cell line, suggesting that the
inhibition was sequence dependent but not sequence specific.
Other investigators have reported that inhibition of CML cell
growth with oligonucleotides is not sequence dependent, suggesting that an aptameric effect is responsible.210
Ribozymes have been applied against this target and appear
to give rise to more specific, but still imperfect, cleavage of their
target.53,211-213 An explanation for some of the controversy
surrounding the ability of this breakpoint to be specifically
cleaved may be found in recent work from the Sczakiel
laboratory.137 It was previously reported that rapid association
of antisense sequence with its target is the most critical
determinant of antisense efficacy.65 In the course of designing
oligonucleotides and ribozymes directed to the bcr/abl breakpoint, kinetic probing combined with calculations of the local
folding potential indicated that the bcr/abl fusion point sequences are not easily accessible for complementary nucleic
acid hybridization.137 The study suggested that selective and
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NUCLEIC ACID THERAPEUTICS
efficient antisense sequences should be directed against the bcr
portion neighboring the fusion point as well as the first eight
nucleotides of the abl sequence. To effectively inhibit this target
then, relatively high doses of oligonucleotides may be required,
and these in turn may produce nonspecific effects. In addition, if
ribozymes with unfavorable kinetics are used to target the
sequence, inhibition is likely to be inefficient and, therefore,
difficult to measure.
Despite the caveats addressed above, Zhao et al214 reported a
clever and apparently successful strategy for inhibiting growth
of CML cells with an anti-bcr/abl ribozyme delivered by a
retroviral vector that also delivered a methotrexate resistance
gene. The hypothesis underlying these experiments was that
transfer of a vector that combined a drug-resistance gene with
anti-BCR/ABL antisense sequences might allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells
functionally normal. A retroviral vector (LasBD) that combined
a methotrexate-resistance gene and antisense sequences directed at the b3a2 BCR/ABL breakpoint was constructed. Cells
engineered to express bcr/abl were transduced with LasBD and
selected in methotrexate (MTX) for 14 days. Expression of the
antisense sequences was reported to reduce BCR/ABL mRNA
and p210(BCR/ABL) protein levels by 6- to 10-fold in most
cells with apparent normalization of cell growth. LasBD also
rendered 20% to 30% of primary Ph2 and Ph1 CD34(1) cells
MTX-resistant and decreased BCR/ABL mRNA levels in MTX
resistant Ph1 CD34(1) cells by 10-fold. LasBD decreased
tumorigenicity of 32D BCR/ABL cells in vivo by 3 to 4 logs.
Based on these results, the investigators concluded that the
methotrexate resistance gene in the LasBD vector could protect
normal hematopoietic cells from MTX-mediated toxicity,
whereas the AS sequences in LasBD could suppress expression
of the BCR/ABL gene and restore normal function of BCR/ABL
cDNA-containing cells. Although the system described was
highly constrained, these results were nonetheless very encouraging.
Regardless of the controversy over whether bcr-abl mRNA
can be effectively destroyed, and whether such destruction leads
to cell death, several groups have proceeded to determine the
potential therapeutic utility of antisense bcr/abl nucleic acids
for inhibiting CML cell growth. The general strategy has been
to transplant CML blast cells into severe combined immunodeficient (SCID) mice and then to examine the effects of
oligonucleotides on leukemic cell growth and animal survival,
an approach originally reported by our group in 1992.195 For
example, Skorski et al215 injected SCID mice with Ph1 CML
blast crisis cell line (BV173) and monitored expression of
bcr-abl transcripts in bone marrow, spleen, peripheral blood,
liver, and lungs. Once disease was established in these mice,
treatment with a 26-nt bcr-abl AS-ODN at a dose of 1 mg/d for
9 days decreased bcr-abl mRNA levels in mouse tissues and
induced the disappearance of CD101 and clonogenic leukemic
cells. Further, antisense treated mice survived 18 to 23 weeks
while control and sense treated mice were dead 8 to 13 weeks
after leukemic cell injection.
To improve on these results, a strategy using oligonucleotides
with traditional chemotherapy, either in the context of ex vivo
bone marrow purging,216 or treating CML-bearing SCID mice
725
directly with ‘‘low-dose’’ cyclophosphamide and bcr-abl targeted oligonucleotides217 has been reported. With regard to
marrow purging, Skorski et al reported that a 1:1 mix of Ph1
leukemic cells that had been exposed to a combination of
low-dose mafosfamide and bcr-abl AS-ODN appeared to be
effectively (,100%) purged of Ph1 cells by clonogenic assay.
Indeed, bcr-abl transcripts were only found in untreated cells, in
cells treated with mafosfamide, or with AS-ODN alone, but not
in those treated with combination therapy. This strategy was
extended to direct in vivo treatment where CML-bearing SCID
mice were treated intravenously with bcr-abl oligonucleotides
and 25% of what would normally be a therapeutically effective
dose of cyclophosphamide. This treatment strategy was reported to result in cure of 50% of the animals.
Alternative Gene Targets in CML
Although bcr-abl is an obvious target for antigene therapeutics, a number of considerations suggest that it may not be as
ideal a target as first considerations would suggest. It must be
recalled that since anti-mRNA strategies based on the use of
oligodeoxynucleotides are transient interventions, the target has
to be expressed when oligonucleotides are physically present in
the cell. In addition, it is straightforward that the strategy will
only be effective if the target cell fails to tolerate transient
downregulation of the targeted mRNA. There is reasonable
evidence to suggest that bcr-abl mRNA is not expressed in
CML stem cells, despite the fact that the translocation is
present.218 Because CML is a stem cell disorder it is reasonable
to hypothesize that the candidate gene for perturbation must be
expressed at the stem cell level to be effective. If this is not the
case, a constant supply of CML progenitors will be derived
from the untreated stem cell. In addition to this important
consideration, it is not at all clear that transient perturbation of
bcr-abl expression results in cell death.219,220 Finally, given the
known redundancy of signaling pathways, one might also be
concerned that bcr-abl–mediated activation of RAS might have
alternative pathways in primary malignant cells. For all these
reasons then, we chose to pursue nonobvious mRNA targets.
Transcription Factors
c-myb. Myb protein is encoded by the c-myb protooncogene, a member of a transcription factor family composed
of at least two other highly homologous genes designated
A-myb and B-myb.221,222 Located on chromosome 6q in humans, c-myb’s predominant transcript encodes an ,75-kD
nuclear binding protein (Myb) which recognizes the core
consensus sequence 58-PyAAC(G/Py)G-38.223 The protein plays
a major role in regulating G1/S transition in cycling hematopoietic cells, and likely functions as a transactivator of a number of
important cellular genes such as the Kit receptor,85,224 CD4,225
and CD34.226
Attempts to exploit the c-myb gene as a antisense ODN target
in CML was an outgrowth of studies which sought to define the
role of Myb protein in regulating normal human hematopoiesis.227 These studies showed that exposing normal bone
marrow mononuclear cells (MNC) to c-myb antisense ODN
resulted in a decrease in cloning efficiency and progenitor cell
proliferation. The effect was lineage indifferent because c-myb
antisense DNA inhibited granulocyte-macrophage colony-
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726
forming units (CFU-GM), BFU-E (erythroid), and CFU-Meg
(megakaryocyte). In contrast, c-myb ODN with the corresponding sense sequence had no consistent effect on hematopoietic
colony formation when compared with growth in control
cultures. Finally, inhibition of colony formation was also dose
related. Inhibition of the targeted mRNA was also demonstrated. Sequence-specific, dose-related biologic effects accompanied by a specific decrease or total elimination of the targeted
mRNA were strong pieces of evidence to suggest that the effects
we were observing were due to an ‘‘antisense’’ mechanism. As
mentioned above, the mechanism responsible for inhibition of
hematopoietic cell growth appeared to be an inducible block in
G1/S transition.228 In other investigations it was also determined that hematopoietic progenitor cells appeared to require
Myb protein during specific stages of development, in particular
when they were actively cycling,229 as might be expected given
the above functional description of Myb protein. Accordingly,
Myb appeared to be critical for normal hematopoietic cell
development, a finding that was largely confirmed using the
technique of homologous recombination.230
Myb protein is also required for leukemic hematopoiesis.
Evidence to support this statement includes the fact that Myb is
widely expressed in malignant cell lines, and inhibition of Myb
with oligonucleotides inhibits malignant cell line growth.231
Growth of cells derived from primary patient material is also
inhibited.194 However, to be useful as a therapeutic target,
leukemic cells would have to be more dependent on Myb
protein than their normal counterparts, and such data have been
provided in vitro84,194 as well as in vivo in an SCID mouse
model using human leukemic cells.195
Why does downregulating Myb kill leukemic cells preferentially? Our initial studies on the function of the c-kit receptor in
hematopoietic cells suggested that c-kit might be an Mybregulated gene.85 Additional studies have since confirmed
this.224,232,233 Because c-kit encodes a critical hematopoietic cell
tyrosine kinase receptor,234 we have hypothesized that dysregulation of c-kit expression may be an important mechanism of
action of Myb AS ODN. In support of this hypothesis it has
been shown that when hematopoietic cells are deprived of c-kit
R ligand (Steel factor) they undergo apoptosis.235 It has also
recently been shown that when CD56bright natural killer (NK)
cells, which express c-Kit, are deprived of their ligand (Steel
factor), they too undergo apoptosis, perhaps because bcl-2 is
downregulated.236 Malignant myeloid hematopoietic cells, in
particular CML cells, also express c-kit and respond to Steel
factor. Accordingly, we postulate that perturbation of Myb
expression in malignant hematopoietic cells may force them to
enter an apoptotic pathway by downregulating c-kit. Preliminary studies of K562 cells exposed to c-myb antisense ODN
show that such cells do undergo nuclear degenerative changes
characteristic of apoptosis. Of necessity, we must also postulate
that normal progenitor cells, at least at some level of development, are more tolerant of this transient disturbance. Since
neither Steel nor White Spotting (W) mice (which lack the Kit
ligand or its receptor, respectively) are aplastic, this is a tenable
hypothesis.
c-myc. Work from Sawyers et al237,238 has suggested that in
the absence of functional Myc, bcr/abl is unable to transform
primary mouse marrow cells. Based on these studies, Skorski et
GEWIRTZ, SOKOL, AND RATAJCZAK
al have targeted c-myc239 and bcr-abl simultaneously in an effort
to improve on results obtained by targeting bcr/abl alone.240 In
vitro CFU assays and SCID mouse studies were conducted. The
latter were of particular interest because they represent a
potentially relevant treatment model. In these studies, animals
were injected with primary blast crisis CML cells and were then
treated systematically with equal doses of bcr-abl, c-myc, or
bcr-abl 1 c-myc targeted phosphorothioate oligodeoxynucleotides. Compared with mice treated with individual compounds,
the disease process appeared to be better controlled in the group
treated with both oligonucleotides, although the animals were
not cured. The investigators suggested that targeting multiple
cooperating oncogenes might be a useful strategy for increasing
the effectiveness of oligonucleotide therapeutics.
Receptors and signaling proteins. Other potential mRNA
targets for treating CML might also be envisioned. These may
be found among the surface receptors and signaling proteins
constitutive to hematopoietic cells. For example, we have
previously suggested that the Kit receptor might serve this
purpose.85 As noted above, Kit appears to be a c-myb–regulated
gene, and downregulation of Kit in hematopoietic cells is
associated with induction of apoptosis, perhaps an important
mechanistic component of anti-Myb’s ability to kill malignant
cells. Directly comparing the ability of anti-Myb and anti-Kit
targeted oligonucleotides to inhibit the growth of leukemic cells
suggests this possibility while at the same time supporting the
potential of Kit-targeted oligonucleotides in the treatment of
CML and other leukemias.
A newer target that may prove to be of even greater utility in
patients with hematologic malignancies is signaling protein
encoded by the vav proto-oncogene.241,242 Vav is expressed
exclusively in hematopoietic cells where it is assumed to play a
role in signaling. The importance of this role is uncertain, in part
because of conflicting functional studies that have used different
strategies for abrogating Vav gene expression in murine embryonic stem cells.243-246 Our studies with vav-targeted oligonucleotides lend some support to each of the conflicting reports cited
above and imply that vav would be a therapeutically attractive
target in CML.12 We have found that while vav appears to be
required for erythropoiesis in both normal and malignant
hematopoietic cells, malignant myeloid cell growth, in particular myeloid cells derived from CML patients, does appear to be
dependent on Vav expression.12 The rationale for Vav as an
antisense target is therefore anchored in the fact that it is
differentially used in normal and malignant cells.
Early Clinical Experience With Oligonucleotide Drugs in the
Treatment of CML
Early clinical experience with oligonucleotides has been
reported by several groups. In the context of CML, we have
reported, in preliminary communication, results of our clinical
trials to evaluate the effectiveness of phosphorothioate modified
ODN antisense to the c-myb gene as marrow purging agents for
chronic phase (CP) or accelerated phase (AP) CML patients,
and a phase I intravenous infusion study for blast crisis (BC)
patients, and patients with other refractory leukemias.93 ODN
purging was performed for 24 hours on CD341 marrow cells.
Patients received busulfan and cytoxan, followed by re-infusion
of previously cryopreserved ODN purged MNC. In the pilot
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NUCLEIC ACID THERAPEUTICS
marrow purging study 7 CP and 1 AP CML patients have been
treated. Seven of 8 patients engrafted. In 4 of 6 evaluable CP
patients, metaphases were 85% to 100% normal 3 months after
engraftment, suggesting that a significant purge had taken place
in the marrow graft. Five CP patients have shown marked,
sustained, hematologic improvement with essential normalization of their blood counts. Follow-up ranges from 6 months to
,2 years. In an attempt to further increase purging efficiency
we incubated patient MNC for 72 hours in the AS-ODN.
Although PCR and LTCIC studies suggested a very efficient
purge had occurred, engraftment in five patients was poor. In the
phase I systemic infusion study there were 18 refractory
leukemia patients (2 patients were treated at two different dose
levels; 13 had AP or BC CML). Myb AS-ODN was delivered by
continuous infusion at dose levels ranging between (0.3 mg/kg/
d 3 7 days) to (2.0 mg/kg/d 3 7 days). No recurrent doserelated toxicity has been noted, although idiosyncratic toxicities, not clearly drug related, were observed (1 transient renal
insufficiency; 1 pericarditis). One BC patient survived ,14
months with transient restoration of CP disease. These studies
show that ODN may be administered safely to leukemic
patients. Whether patients treated on either study derived
clinical benefit is uncertain, but the results of these studies
suggest to us that ODN may eventually demonstrate therapeutic
utility in the treatment of human leukemias.
Some clinical experience with bcr-abl–targeted antisense
oligonucleotides in CML has also been reported. de Fabritis et
al202 treated a patient with CML in accelerated phase with
autologous bone marrow transplantation. Before reinfusion,
cells were purged in vitro with a 26-nt phosphorothioate
antisense oligodeoxynucleotide specific for the B2A2 junction.
This treatment resulted in a 24% and 41% reduction of
CFU-GM and CD341 cells, respectively. The patient was
successfully engrafted with the purged marrow cells after 17
and 25 days for platelets and neutrophils, respectively. Using
fluorescence in situ hybridization in interphase nuclei, some
Ph2 cells were found after the autograft. The patient was
reported to be in a complete hematologic remission at 9 months
posttreatment.
OLIGONUCLEOTIDES IN THE TREATMENT OF ACUTE
LEUKEMIA AND LYMPHOMA
It is obvious that the number of potential gene targets for the
hematologic and other malignant disorders is enormous, and the
literature is replete with examples of ‘‘antisense knockouts’’
that attempt to demonstrate the utility of such targets under a
variety of experimental conditions. Many of these reports use
cell lines, as opposed to primary cells, and one could also argue
with the completeness of the controls used to demonstrate that
an antisense effect has actually been achieved. Two of these
targets bear particular mention because oligonucleotides designed to inhibit their function have been employed in clinical
trials. These are, respectively, p53 and bcl-2.
p53 is a tumor suppresser gene that is mutated in many forms
of neoplasia, including the blast phase of CML and acute
leukemia.247 Though perhaps a nonobvious target for inactivation, the group at the University of Nebraska has pursued this
gene’s mRNA as an antisense target in a variety of hematologic
malignancies because of results obtained in vitro culture
727
systems. This group has reported that when leukemia cell lines
and primary patient cells are exposed to p53-targeted oligodeoxynucleotides, inhibition of colony growth may be observed.248,249 Treatment of normal bone marrow cells with p53
oligos has no effect on colony formation, therefore suggesting
differential sensitivity. The mechanism for this effect is uncertain, but the preclinical results were believed to be promising
enough to lead these investigators to use a p53-targeted
oligodeoxynucleotide for bone marrow purging249 and for
systemic therapy of treatment refractory leukemias.188,250 In the
most recent study, a 20-nt phosphorothioate oligonucleotide
complementary to p53 mRNA [OL(1)p53] was used in a phase I
dose-escalating trial conducted to determine the toxicity of the
oligodeoxynucleotide after systemic administration to patients
with hematologic malignancies. A total of 16 patients with
either refractory acute myelogenous leukemia (n 5 6) or advanced myelodysplastic syndrome (n 5 10) were treated. Patients were given OL(1)p53 at doses of 0.05 to 0.25 mg/kg/h for
10 days by continuous intravenous infusion. No specific
toxicity directly related to the administration of the compound
was observed. One patient developed transient nonoliguric
renal failure. One patient died of anthracycline-induced cardiac
failure. Approximately 36% of the administered dose of
OL(1)p53 was recovered intact in the urine. Plasma concentrations and area under the plasma concentration curves were
linearly correlated with dose. Leukemic cell growth in vitro was
inhibited compared with pretreatment samples. There were no
clinical complete responses. The investigators concluded that
this particular phosphorothioate oligonucleotide can be administered systemically without complications and speculated that
it might prove useful in combination with currently available
chemotherapy agents for the treatment of malignancies. Whether
any of the effects reported was due to an antisense mechanism is
uncertain because this was not specifically looked for.
The bcl-2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly
found in lymphomas. The translocation results in deregulation
of bcl-2 gene expression and cause inappropriately high levels
of bcl-2 protein production (recently reviewed in Reed et al251 ).
Bcl-2 blocks the apoptotic process that normally contributes to
cell death and thereby facilitates neoplastic cell expansion.
Overproduction of the bcl-2 protein also prevents cell death
induced by nearly all cytotoxic anticancer drugs and radiation,
thus contributing to treatment failures in patients with some
types of cancer. Based on these observations a number of
groups have been interested in blocking bcl-2 function with
antisense oligonucleotides and ribozymes, in particular for the
treatment of lymphomas.
Within the past year Webb et al treated nine patients with
refractory non-Hodgkin’s lymphoma with a bcl-2–targeted
phosphorothioate oligodeoxynucleotide based on preclinical
studies which suggested that the oligonucleotide could cause
disease regression in an animal model.95,252 The oligonucleotide
used was targeted to a portion of the bcl-2 mRNA open reading
frame of the bcl-2 mRNA and was delivered as a daily
subcutaneous infusion for 2 weeks. Toxicity was scored by the
common toxicity criteria, and tumor response was assessed by
computed tomography scan. This study was also more rigorously controlled in terms of mechanism because an antisense
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728
GEWIRTZ, SOKOL, AND RATAJCZAK
effect was specifically assessed by quantification of bcl-2
expression, and bcl-2 protein levels in treated patients as
assessed by flow cytometry. During the course of the study, the
daily dose of bcl-2 antisense was increased incrementally from
4.6 mg/m2 to 73.6 mg/m2. Save for local inflammation at the site
of infusion, no treatment-related toxic effects occurred. In two
patients on the study, computed tomography scans showed a
reduction in tumor size which were stated to be ‘‘minor’’ and
‘‘major,’’ respectively. In two patients, the number of circulating lymphoma cells was also found to be decreased during
treatment. Other indicators of response, such as a decrease in
LDH, were also reported. Bcl-2 protein levels were measured by
flow cytometry in five patients and were found to be decreased
in two. The investigators concluded that in these patients the
antisense therapy led to an improvement in symptoms, and was
accompanied by some objective biochemical and radiological
evidence of tumor response. Based on these favorable responses
the investigators also speculated that bcl-2 antisense therapy
might also prime cells for improved chemotherapeutic response. In common with the experience of others then, the
antisense compounds appear to be relatively nontoxic and there
is at least the suggestion that in some patients useful responses
may be observed. No doubt however, these are likely due to a
combination of aptameric as well as antisense effects.
OLIGONUCLEOTIDES AS ANTICOAGULANT DRUGS
As mentioned above, it is well known that proteins can bind
to DNA via sequence specific recognition motifs and by charge
interactions. Phosphorothioates in particular are noted to have
these properties and one consequence is the prolongation of
clotting times in patients receiving relatively large and rapid
bolus injections of these materials. These facts have been
exploited by some to screen for novel inhibitors of clotting
protein function. An example relevant to this review is the use
of oligonucleotide inhibitors of thrombin.253 The substrate
specificity of thrombin is dependent on two electropositive
surfaces which interact with fibrinogen and heparin, respectively. Knowing this, Tasset et al253 used a combinatorial
screening methodology to identify single-stranded oligonucleotides manifesting high-affinity binding to human thrombin.
With this approach they reported finding a 29-nucleotide DNA
sequence that bound to human thrombin with a kd of approximately 0.5 nmol/L. The antithrombin oligonucleotide inhibited
thrombin-catalyzed fibrin clot formation in vitro likely by
binding to the heparin-binding exosite. Additional inhibitors of
the intrinsic clotting pathway have also been described, the
mechanism for which appear to binding to fibrinogen.254 Given
the relative expense of oligonucleotides in comparison to
heparin or similar drugs it is uncertain whether oligonucleotides
could be made cost efficient, although there are clearly circumstances where the availability of such drugs, for example in
patients with heparin-induced thrombocytopenia, might be of
clinical use.
FUTURE PROSPECTS FOR ‘‘ANTISENSE’’ THERAPY
The prospects for developing effective nucleic acid therapeutics are ultimately dependent on solving the problems that were
alluded to in the preceding discussion. The rationale for moving
forward with this approach is compelling. However, clearly
there is frustration with these techniques as exemplified in
particular with the antisense oligonucleotide strategy.99,255-258
Some of this is caused by unreasonable expectations based on
naive assumptions about how these molecules work. While the
theory is straightforward, the complexity of the task should now
be much more apparent. We have tried to discuss these
problems in a straightforward manner in the course of reviewing this approach to gene therapy. Nevertheless, emphasizing
those issues we believe to be of paramount importance may be
useful for moving the field forward.
First, delivery of oligonucleotides remains an important
problem, and parenthetically, one which is not dissimilar to that
which is faced by investigators who are seeking to deliver viral
vectors into a broad range of cell types. In our view, an efficient
delivery system would likely make available oligonucleotide
chemistries effective and might increase the effectiveness of
these molecules in a dramatic way. In this regard it is useful to
recall that the oligonucleotide must not only cross cell membranes, but must then sort to a location where they may then
begin to interact with their mRNA target. The ability to deliver
ODN into cells and have them reach their target in a bioavailable form must be further investigated.259 Without this ability, it
is clear that even an appropriately targeted sequence is not
likely to be efficient. Recently described strategies for introducing ODN into cells, including various cationic lipid formulations, may address this problem.116,156,260 Colleagues attempting
to deliver vector sequence to cells share this problem.
Second, finding single-stranded mRNA within the bowl of
‘‘spaghetti and meatballs’’ that may serve as a useful analogy
for a highly folded RNA molecule and its associated proteins
remains a major limiting step. It is obvious that if the targeted
sequence is buried within the core of a structurally complicated
mRNA molecule, or rendered inaccessible by an associated
protein, then hybridization of the oligonucleotide and mRNA
may not be possible. Cells have certainly evolved mechanisms
for unwinding and editing RNA, and it may be possible to
exploit these mechanisms for solving this problem. Colleagues
with an interest in RNA biology might make important contributions to this area by addressing this critical issue.
We also need to consider the issue of sequence-dependent
effects which are unrelated to hybridization with a specific
message. These non-antisense, so-called aptameric effects may
be related to the chemistry of the oligonucleotide used and
continue to confound the issue of specific gene inhibition.
Reports that four consecutive guanines (G-4 tract) within a
larger phosphorothioate oligomer have a non-antisense antiproliferative effect is but one example.261,262 In our hands this has
not been an issue,88 suggesting that cell type used may be an
important issue. The products of ODN degradation such as
28-deoxyadenosine or high concentrations of thymidine are
toxic to cells and inhibit cell proliferation.263 Micromolar
concentrations of dAMP or dGMP can have pronounced
cytotoxic or cytostatic effects on cells, particularly those
derived from hematopoietic tissues.264 In addition, phosphorothioate analogs may bind to ribosomes resulting in nonspecific
inhibition of protein synthesis,265 as well as effects on cell
growth, cell morphology, enzyme activities, and even viral
proliferation.103,266-269 Hybridization through partially matched
sequences has also been raised as a problem.41 The latter seems
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
NUCLEIC ACID THERAPEUTICS
a less important consideration because it has been shown that as
few as two base mismatches result in loss of antisense
effectiveness.231 However, having said this, it is useful to point
out that apatmeric effects may be as useful as specific antimRNA effects in the context of achieving a desired biological
effect. In this context, the contribution of an aptamer is
welcome. It is only when investigating the biology of a specific
gene that these effects need to be clearly defined and understood.
Other issues which bedevil this field, such as the problem of
reproducibility of experiments, attest to the need for careful
experimental technique and attention to detail.270 Factors such
as differences in nucleic acid synthesis, handling, storage, and
purification are often overlooked in failed experiments. In many
laboratories verification of the sequence ordered and its stability
under the conditions that it is being used are simply not carried
out. Finally, differences in experimental conditions, cell use,
and familiarity with the methodology may also be contributory
factors. These comments are applicable to all experimental
methods.
Many patients suffering from hematologic, as well as nonhematologic malignancies remain incurable. These conditions, as
well as others alluded to above, afflict a substantial number of
individuals, often in their most productive years. Treating
patients with highly toxic drugs and ionizing radiation is
unpleasant for all connected with this process, especially when
there is little hope for cure. Nucleic acid therapeutics, with their
promise of exquisite molecular specificity and concordant
limited toxicity, continue to be highly attractive pharmaceutical
prospects for this reason. We have no doubt then that the goal of
making useful ‘‘antisense’’ drugs will be achieved. The pace at
which this will occur is dependent, as always, on the ability of
the field to continue to attract talented investigators, and
agencies disposed to funding their research.
ACKNOWLEDGMENT
We thank Drs Arthur Kreig, Cy Stein, and Peter Glazer for helpful
comments during the preparation of this manuscript. The editorial
assistance of Elizabeth R. Bien is also gratefully acknowledged.
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1998 92: 712-736
Nucleic Acid Therapeutics: State of the Art and Future Prospects
Alan M. Gewirtz, Deborah L. Sokol and Mariusz Z. Ratajczak
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