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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 712-736
REVIEW ARTICLE
Nucleic Acid Therapeutics: State of the Art and Future Prospects
By
Alan M. Gewirtz,
Deborah L. Sokol, and
Mariusz Z. Ratajczak
From the Departments of Internal Medicine, Pathology and Laboratory
Medicine, and the Institute for Human Gene Therapy, University of
Pennsylvania School of Medicine, Philadelphia.
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INTRODUCTION |
AS 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, disease-causing 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.
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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 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).
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| 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).
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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 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.
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| 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).
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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 polypurine-polypyrimidine
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 5 -3 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
(C+). 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
K+.19 G-containing TFOs also require divalent
cations such as Mg2+ for stability, while A-containing TFOs
appear to require Zn2+.
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.
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) Watson-Crick 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.
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| 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.
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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.
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 = C, U, or A54 or, in some cases, NUX, where N = 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 5 and 3 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 ribozyme-mediated 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 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.
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OLIGONUCLEOTIDESCONSIDERATIONS 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 = n(2°C) + m(4°C), where n = number of dA · T pairs and m = 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 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
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| Fig 4.
Chemical modifications of oligonucleotides. (A) Common
modifications of the phosphodiester linkage, sugar, and bases moieties are depicted. Note that 2-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.
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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 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 5 and 3
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 Mixed-backbone oligonucleotides (MBOs) are
another example.106 Compounds of this type contain phosphorothioate moieties at the 3 and 5 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
N3  P5 phosphoramidates and the peptide nucleic acids
(PNAs). The phosphoramidate modification consists of substituting every
3-oxygen for a 3-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 N3 P5 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  form, to the  anomeric form, where the base is projected in the opposite direction,
has been found to increase nuclease stability significantly. However,
this compromises 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 -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 ,
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 2 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 2-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 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 5 or 3 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 JobOligo 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 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 5 end of a single-stranded loop have orders of
magnitude higher affinity for their target than those targeted to the
3 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
5 end of the molecule to the 3 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
5-end but successively shortened 3-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-hybridizing and fast-hybridizing antisense RNAs differed by only
two or three 3-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 -globin mRNA.
The oligonucleotides were complementary to the first 122 bases of mRNA
comprising the 5 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 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 MB-mRNA duplexes
into K562 cells. Signal could be observed with as little as ~1 × 10 4 fg of complex when using a UV fluoride
lens-equipped microscope. Accordingly, detection of MB-mRNA
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 Trafficking
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 photoactivatable
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
View larger version (170K):
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| 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)
|
|
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.46,15 |
Introduction
References