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On the mechanism of cationic lipid-mediated delivery of oligonucleotidesShi, Fuxin
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15
Chapter 2
Make sense of antisense oligonucleotides
Fuxin Shi and Dick Hoekstra
Submitted
Chapter 2
16
Abstract
For more than two decades antisense oligonucleotides (ODNs) have been used to modulate
gene expression for the purpose of applications in cell biology, and for development of novel
sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant
strategy, involving the targeting to and association of an ODN sequence with a specific mRNA
via base-pairing. As a result, the translation of the gene into protein will be impaired, thereby
potentially revealing its functional and/or harmful effect in normal and diseased cells/tissue,
respectively. It is still poorly resolved how to design the most efficient antisense
oligonucleotides, but chemical modification of their structure can improve their efficiency of
action, in part due to an enhanced intracellular stability. Appropriate carriers have been
developed to allow efficient entry of ODNs into cells in vitro, and the mechanisms of delivery,
both in terms of biophysical requirements for the carrier and cell biological features of uptake,
are gradually becoming apparent. Remarkably, for some tissues such as liver and brain, it has
been shown that ODNs may acquire intracellular access and delivery into the nucleus, a step
needed for accomplishing the antisense effect, without the need of packaging into a delivery
vehicle. This suggests differences in entry mechanisms, knowledge that is imperative to further
appreciate and bolster antisense technology in its broadest sense. In this context, proper
consideration will be given here to antisense design and chemistry, and the challenge of extra-
and intracellular barriers to be overcome.
Make sense of antisense oligos
17
Introduction
In 1978, synthetic oligonucleotides (ODNs) complementary to Rous sarcoma virus mRNA
were shown to inhibit virus replication (1). This important observation represents one of the
hallmarks in the initiation of the development of antisense technology for therapeutic and cell
biological purposes. In fact, the principle of the approach reflects a naturally occurring
physiological event since endogenously expressed antisense molecules exist which are able to
regulate endogenous gene expression and to defend both prokaryotes and eukaryotes from viral
invasion (2, 3).
Over the last two decades, numerous studies have been carried out to obtain insight into the
mechanism of the action of antisense ODNs, including issues concerning optimal design,
chemical modification, specificity and pharmacology (4, 5). In principle, antisense technology
has a wide perspective of potential applications. At present, prompted by the rapid developments
in genomics, most of these applications are in fundamental research and are largely focussed on
an inhibition of gene expression, which provides crucial insight into the function and potentential
regulation of novel genes (6-8). However, there is a growing interest in developing drugs based
upon an antisense protocol, mostly aimed at interfering with viral infections and cancer (9, 10).
In addition, the antisense approach may be a good alternative to traditional agents for diagnosis
and treatment in nuclear medicine, although relatively few attempts have been made thus far to
evaluate this option (11-13).
Irrespective of the goal of application, a major challenge in antisense technolgy represents the
design of an antisense sequence, which is target-specific, effective and nontoxic (14-19). In spite
of drawbacks, significant progress has also been made in recent years in this area, which has
resulted in the commercial production of antisense therapeutics such as Vitravene, used in the
treatment of CMV retinitis in HIV infections (20, 21) and the undertaking of several promising
phase III trails (22-24).
As with drug development in numerous areas, a detailed understanding of antisense
mechanism and pharmacology are essential for successful application and further improvement
of its effectiveness. Here, we will discuss therefore the understanding thus far of molecular
obstacles and intracellular and extracellular barriers in antisense action and application.
Principle features of the mechanism of antisense action
Crucial toward a prosperous development and widely applied antisense technology is the need
for a detailed understanding of the mechanism of antisense action per se (Fig. 1). Conceptually,
the ability of antisense molecules to bind to complementary mRNA through Watson-Crick base
Chapter 2
18
pairing may sound simple. However, insight into issues as where and how antisense reaches its
target(s) are still largely obscure, especially in case of in vivo studies. Moreover, no evidence is
available which demonstrates the direct binding of ODNs within the cell to the targeted
sequence(s). It is generally believed that antisense either sterically blocks mRNA’s function (Fig.
1A) or promotes enzyme-mediated mRNA degradation (Fig.1B). As an example of the former
possibility, in a cell-free translation system it has been shown that antisense ODNs, such as
peptide nuclei acids (PNAs), which are unable to activate RNase H, can be targeted to mRNA
coding sites. As a result, an interference with polypeptide chain elongation occurs, resulting in
the biosynthesis of truncated protein fragments (Fig.1A; 4, 25, 26). Similarly, steric occupancy
of the 5’cap region with 2’-MOE ODNs interferes with the assembly of the 80S translation
initiation complex, and with the elevation of target mRNA (27). Steric interference of antisense
molecules on distinct RNA regions may also cause an inhibition of pre-mRA splicing (Fig. 1C)
or polyadenylation editing (Fig. 1D), and/or induce mutations of the encoding gene (28, 29).
Antisense-mediated down-regulation of mRNA as a result of the latter’s degradation by
RNase H action, may occur following binding of phosphodiester- or thioate-linked ODNs, as
revealed in cell lysates (30-33). Consequently, the expression of the encoded protein will become
reduced, an effect that will be apparent only after significant turnover of the pre-existing
endogenous protein pool. When the targeted ODN sequences are within the 5’or 3’ non-encoding
regions(Fig. 1 D), the antisense induced cleavage at these non-translation sites accelerates the
degradation of the entire target mRNA. Nevertheless, progress in carefully defining the
intracellular mechanism(s) of processing and action of antisense ODN has been particularly
frustrated by the technical inability to verify the localization of the relatively small amounts of
antisense that gain access within the cells, and the limited recovery of targeted mRNA or protein
fragments (Fig.1).
As noted, in the antisense field, RNase H is thought to play a pivotal role. However, it is
essential to take into account that only phosphodiester, phosphothioate and chimeric ODNs (cf.
Fig. 3) can activate RNase H. Any (chemical) modification of the nucleotide and/or changes in
the backbone will reduce or eliminate RNaseH recognition of the DNA-RNA duplex(31, 32).
Given the potentially effective role of RNases in antisense technology, further insight into the
presence of other intracellular RNases may prove worthwhile in fully exploiting the potency of
this technology (34). For example, the 2’, 5’oligoadenylates bind to and activate the latent RNase
L, cleaving viral and cellular RNAs at the 3’side of UpNp sequences, thus leading to an
inhibition of protein synthesis (35). In fact any RNase, which recognizes the double stranded
RNAs or RNA/DNA duplexes could play a key role in the overall action of antisense ODN. In
Make sense of antisense oligos
19
both plants and animals, RNAi is present which consists of about 22 nucleotides in length that is
homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as a guide
sequence that instructs a multicomponent nuclease, RNA-induced silencing complex (RISC), to
destroy specific messenger RNAs (36-38). The RISC contains a helicase activity that unwinds
the double RNA strand, thereby allowing the antisense to bind to the targeted RNA sequence and
causing the ensuing activation of an endonuclease activity that hydrolyzes the targeted RNA at
the site where the antisense strand is bound. Recently, RNase III, which is involved in the
maturation of prokaryotic and eukaryotic RNA, has emerged as a key player in this new and
exciting biological field of RNA silencing or RNA interference (39). In fact RNase III appears
to contain very high affinity RNA binding sites, which readily interact with the double-stranded
RNA binding domain (dsRBD), thus initiating rapid and efficient cleavage.
Figure 1. Major sites of the actions of antisense oligonucleotides. A. Translationalarrest. Antisense oligonucleotides bound to complementary mRNA inhibit polypeptideselongation. B. RNase H activation. Hybridized mRNA is degraded by RNase H that isactivated upon the binding of antisense oligonucleotide on the mRNA. C. Inhibition ofthe splicing of pre-mRNA. Antisense oligonucleotides bound on the splicing site of pre-mRNA interfere with the maturation of mRNA by preventing the binding of spliceosomeon pre-mRNA. D. Pre-mRNA destabilization. Antisense oligonucleotides bound onencoding or non-encoding regions on pre-mRNA can accelerate the RNase-mediateddegradation of the mRNA, or interfere with polyadenylation or cap formation.
DNA
Pre-RNA
AAACAP
AAACAP
spliceosomes
AAACA
AAACA
CAP AAA
CAP AAA
CAP AAA
RNase
CAPAAA
proteinB. RNase H activationA. Translational arrest
C. Inhition of splicingD. Pre-mRNA destabilization
nucleus
cytosol
RNase
Nuclear pore
Chapter 2
20
Protocols for designing proper antisense sequences
Obviously, the proper selection of an appropriate antisense sequence is a crucial step and
remains a major challenge in the successful application of antisense technology (Fig. 2).
Knowledge of the RNA secondary structure, antisense affinity and antisense chemistry are key
factors in antisense design. mRNA is not a single stranded random coil but displays a secondary
or tertiary structure, which plays a significant role in determining the efficacy of ODN antisense
activity (40-47). Usually, a stretch of 10-30 nucleotides on the mRNA is selected as a potential
target for the antisense. This preselected mRNA fragment may engage in intramolecular base
pairing, thus constituting stable secondary and tertiary structures, which may render large parts
of the mRNA inaccessible towards interaction with the matching antisense ODNs. The number
of potential conformational states grows exponentially with the chain length. For example, the
number of hairpin conformations increases from 138 for a 10-nucleotide chain to 24,666 for a
16-nucleotide chain. RNA hairpins are stabilized predominantly by base-stacking interactions
(48).
mRNA folding and stem loops can be predicted by certain computer programs (49, 50), such
as mfold (51-52). However, precise prediction of these structures remains difficult. In addition,
cellular factors may influence the mRNA structures as well. For example, the association of
AUF1, one of A + U-rich binding factors, with RNA substrates induces the formation of
condensed RNA structures (53). The spliceosome (cf. Fig.1C) removes introns from pre-
messenger RNAs by a mechanism that entails extensive remodeling of the RNA structure. The
most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small
nuclear RNAs (snRNAs). In this case, the yeast RNA binding protein Prp24 has been shown to
re-anneal these snRNAs (54). Thus ODN ‘walks’, i.e., spacing ODNs of a given length at
intervals along the RNA and choosing the one with the highest activity, still play an important
role in selecting appropriate antisense molecules. In fact, currently a variety of approaches are
applied to design the most appropriate antisense sequence (Fig.2). Also sequences that are
targeted to initial coding regions are frequently applied since these regions lack secondary
structure. However, although an attractive target, such an approach has been shown to be of little
value in general, since ODNs targeted to these regions have been shown to often generate a poor
downregulation of mRNA content (14). Furthermore, translation initiation sites may display a
shared homology in both related and non-related genes, since ODNs targeted to these sites
displayed both antisense specific effects toward the targeted gene, and non-specific effects on
cell proliferation.
Make sense of antisense oligos
21
Figure 2. Schematic representation of antisense design. A. mRNA walking.Oligonucleotides of a given length, complementary to sequences along the RNAsequence, are synthesized and screened for antisense activity. B. Computer folding ofmRNA. By prediction of mRNA structure, oligonucleotides are designed to accessiblesites on the mRNA. C. Oligonucleotide array. In the scanning array, alloligonucleotides complementary to the target mRNA sites are synthesized andhybridized with mRNA transcripts. Antisense sequences are chosen by selecting theones with high affinity to the mRNA on the array. D. RNase H mapping. RNase H isused to cleave mRNA that hybridizes to a random oligonucleotide library. Appropriateantisense oligonucleotides are selected based on identifying RNase H cleavage sites oftarget mRNA.
Overall, four distinct approaches have been developed which are currently actively pursued in
the design of antisense molecules, though selection of active antisense molecules still remains
largely a matter of trial and error (Fig. 2). First, mRNA walking or sequence walking (Fig.2A),
which relies on synthesizing a variety of sequences which are targeted to distinct regions on a
given sequence. Usually some 100 different sequences will be tested in such an approach. The
outcome is that often only a few sequences turn out to be effective (55, 56). Obviously, the
advantage of this approach is that given the number of sequences applied an appropriate and
effective sequence may be recovered, but the procedure is costly, time-consuming and laborious.
However, such an approach may suit large-scale pharmaceutical purposes for screening of
optimal antisense drugs. A second procedure relies on computer facilitated screening (Fig.2B),
based on the predicted folding of mRNA in order to identify accessible sites (57-58). As
A: mRNA walking
B: Computer folding of mRNA
C: Oligonucleotide array
D: RNase H mapping
Prediction of mRNA folding
Oligonucleotide
RNase H
Sequencing RNA
Oligonucleotide
mRNA hybridisation
mRNA hybridisation
mRNA
oligonucleotides
mRNA
Scanning the array
Designing oligonucleotidesto the mRNA accessible sites
Analysing the activity of sequences
Chapter 2
22
discussed above, the precise prediction still suffers from structural uncertainties. On the other
hand, this is an inexpensive, fast and easy approach involving the screening of a few sequences.
With this approach usually an effective sequence can be obtained, even though it may not be the
most effective one. On the other hand, it should also be realized that it is not always necessary to
completely inhibit gene expression, particularly in case of functional studies.
In recent years, ODN arrays (Fig.2C) and RNase H susceptibility (Fig.2D) emerged as
important approaches to select antisense molecules. In the scanning array approach, all ODNs
complementary to the target mRNA sites are synthesized, and hybridization is performed with
mRNA transcripts in vitro, i.e., under non-physiological conditions (45, 59). Since these studies
are done in vitro, relevant cellular factors are not present in the system. Nevertheless, with this
approach, a good correlation has been obtained between the eventual antisense effect in cells,
and the binding affinity of antisense ODNs to the mRNA in vitro. With the progress in
automation and miniaturisation of DNA chips, this approach is quite promising and can be
readily adapted to a routine screening in common research laboratories.
Finally, appropriate antisense ODNs can be selected based upon the principle of RNase H-
mediated cleavage of target mRNA after binding of a random ODN library, followed by
analyzing accessible ODN binding sites on the mRNA by gel electrophoresis (60-62). The
advantage of this method is that the random ODN library is easy to synthesize and suitable to
any target mRNA, although the challenge remains to identify the precise cleavage sites, given
that a multifold of such sites may exist whereas the resolution, as obtained by gel
electrophoresis, is usually limited. Accordingly, further improvement is needed to adapt this
procedure as a generally applicable approach.
On improving Antisense Chemistry
Chemical modification of antisense ODNs (Fig. 3) is aimed at (i) improving stability and
affinity of ODNs to target mRNA, (ii) facilitating recruitment of intracellular enzymes (mostly
RNase H) to efficiently cleave targeted mRNA, and (iii) reducing/eliminating the toxicity of
ODNs as such. Early during development, antisense ODNs contained phosphorodiester (PO-
ODNs) linkages. PO-ODNs are capable to recruit and activate RNase H to ODN/mRNA hybrids.
The PO- ODNs per se are sensitive to nucleases and show relatively short half-lives, both
intracellularly and in circulation. Although still in use, currently most applications rely on the
employment of chemically modified ODNs. The most frequently used chemically-modified
antisense compounds are phosphorothioate-linked ODNs (PS-ODNs), in which unbridged
oxygen is replaced by sulfur. In fact PS-ODNs harbor more advantages than only their ability to
Make sense of antisense oligos
23
resist nuclease-mediated degradation. PS-ODNs not only exhibit a long half-life in vitro and in
vivo, but the modified compound retains the ability to recruit RNase H in order to degrade PS-
ODN/mRNA hybrids (63, 64). Importantly, when applied systemically, PS-ODNs may
spontaneously enter tissues, mostly liver, kidney, spleen, intestine and lung. A disadvantage of
the PS linkage is its affinity for proteins, which on the one hand prolongs the circulation time of
ODNs in vivo by protecting them from removal by filtration, but on the other hand frustrates the
interpretation of the antisense effect since non-sequence specific inhihition of cell proliferation
may occur (65, 66). For example, PS-ODNs may bind in a length and to some extent in a
sequence-dependent manner to heparin-binding proteins, such as fibroblast growth factors,
platelet-derived growth factor (PDGF), vascular endothlial growth factor (VEGF) and its
receptor, as well as to the epidermal growth factor receptor (EGFR) (17, 67, 68).
Figure 3. Oligonucleotide analogues and chemical modifications.
Chapter 2
24
Morpholino ODNs are nonionic DNA analogues, which, compared to PO-ODNs, show less
affinity to proteins, but similar affinity to mRNA. However, they do not recruit RNase H, and
whether these compounds are active as appropriate antisense molecules in vivo, remains to be
determined (69-71). In PNA, the uncharged N-(2-aminoethyl)-glycine linkage replaces the
phosphate deoxyribose backbone. Nonionic PNA binds with a relatively enhanced affinity to
target mRNA, reduces the nonspecific binding to protein, but like morpholino ODNs, does not
recruit RNase H (72-75). It is rapidly cleared from the circulation with an elimination half-life of
about 17 min, in contrast to PS-ODNs, which show a half life of approx. 1 h (76-79). Locked
nucleic acid (LNA) bases are RNA analogues that contain a methylene bridge connecting the 2’-
oxygen of the ribose with the 4’-carbon. This modification renders outstanding affinity of this
nucleotide to complementary mRNA, but reduces RNase H cleavage on the mRNA due to
modification of the sugar. To improve the binding affinity and activation of RNase H, chimeras
of LNA and PO-ODNs have been developed (80, 81). The in vivo activity of such chimeras has
been demonstrated recently (82). 2’-O-methoxyethyl ODNs(2’-MOE) modification conveys
nuclease resistance, high affinity to the target RNA, similar pharmacokinetics as PS linked
ODNs, and importantly, causes activation of RNaseH (83-88).
Although some comparative work on the biostability, the antisense effeciency and in vivo
pharmacokinetics of the various chemically modified ODNs has been done, there is no clear-cut
picture as to a single best design for an antisense structure. For example, it has been reported
that the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher
than that of negatively charged 2'-O-Me and 2'-O-MOE congeners in a cell culture model (89).
However, the same laboratory also reported that 2'-O-methoxyethyl (2'-O-MOE)-
phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C
terminus) exhibited sequence-specific antisense activity in a number of mouse organs.
Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-
1K) were completely inactive (90). LNA-DNA ODNs were found to be the most efficient single-
stranded antisense ODN, when compared to PS-ODNs and 2’-ME ODNs, the former in turn
being more active than the latter (91). Claims have also been made that the efficiency of an ODN
in supporting RNase H cleavage correlates with its affinity for the target RNA. One such a
comparative study established an order of efficiency of LNA > 2'-O-methyl > DNA >
phosphorothioate (81). In summary, no conclusive data have been presented which would
support the general superiority and versatility of a specifically preferred chemically-modified
antisense construct. Rather, data currently available suggest that apart from some obvious
parameters such as biostability, the preferred antisense structure for efficient down-regulation of
Make sense of antisense oligos
25
a given mRNA target can only be revealed by direct comparison in a given experimental model
system, and no prior prediction can be made.
Overcoming cellular barriers in ODN delivery
Crossing the plasma membrane.
When naked ODNs are added to cells, they usually do not permeate across plasma membrane.
A small fraction can be endocytosised via adsorption (Fig.4A). Yet, this fraction is usually very
low since adsorption of the negatively charged ODNs to the net negatively charged plasma
membranes is rather poor. Hence, attempts have been made to chemically modify ODNs to
eliminate the negative charge in order to promote adsorption. However, an enhanced adsorption
does not necessarily improve the antisense effect, because endosomal escape (Fig.4B) poses as a
next barrier in ODN delivery into the cytosol, a minimal requirement for accomplishing an
antisense effect. In some studies, some antisense activity has been reported upon addition of free
antisense ODNs, provided a relatively high concentration was applied, i.e., 10-20 µM. Often,
significant escape of oliogonucleotides from the endosomal compartments was not observed in
these studies, implying that arrival of ODNs at the nucleus was not detectable. Yet, substantial
accumulation of administered naked ODNs into the nucleus has been reported to occur in
cultured bovine adrenal cells (92). Whether a cell type-dependent ODN translocation mechanism
may exist is at present unclear. One such a possibility could be a cell-type dependent expression
of a nuclei acid transporting channel, as identified on rat renal brush border membranes (Fig. 4
A2, 93, 94).
Bypassing the plasma membrane can be accomplished experimentally by microinjection,
electroporation or membrane permeabilization with chemical agents(Fig. 4 A2). Microinjection
has generated valuable insight into the understanding of intracellular antisense processing and
the potential correlation with efficiency. For example, both microinjection and permeabilization
lead to nuclear localization of antisense ODNs (95-98), a localization now thought to be crucial
in order to acquire a potential antisense effect. For practical purposes however, these procedures
are too laborious (microinjection) or harmful to cells (permeabilization and electroporation),
requiring careful adjustment for each cell type employed.
Vector-facilitated delivery of ODNs, relying on the use of liposomes, polymers and peptides,
appears an appropriate means to overcome the plasma membrane barrier in an efficient manner.
In essence, liposomes and polymers are capable of efficiently complexing ODNs and enhancing
the adsorption of complexed ODNs to the plasma membrane. Together, both features promote a
strong enhancement in the intracellular concentration of ODNs as accomplished by endocytosis
Chapter 2
26
of such complexes (Fig.4. A1). Some plasma membrane proteins have been claimed to be
involved in liposome-mediated delivery of oliogonucleotides, which trigger endocytosis (99,
100). The exact function of these membrane proteins still needs to be clarified, but the evidence
to support a specific role of distinct proteins is scanty. More likely, these complexes enter cells
by non-specifically exploiting the endocytic mechanism, presumably mainly involving clathrin-
mediated endocytosis, as has been well-defined for complexes of plasmids with cationic lipids
(lipoplexes; 101, 102) and polymers (polyplexes; 102, 103).
Figure 4. Cellular entry and subcellular trafficking of oligonucleotids. A.Oligonucleotides associate with the cell membrane via absorption or charge attraction.Folowing their binding, the ODN complexes are internalized by endocytosis (A1);alternatively, oligonucleotides may become translocated across cell membranes vianucleic acid channels, fusion or pore formation on the plasma membrane (A2); viacoupling of specific ligands to the complex, targeted internalization may beaccomplished by receptor-mediated endocytosis (A3). B. Following endocytosis,oligonucleotides are either released from endosomes and/or are transported tolysosomes. C. After endosomal release, oligonucleotides are transferred into thecytosol, and acquire access to the nucleus and eventually to mRNA. See text fordetails.
Distinct peptides, which have the ability to penetrate into the cell membrane, thereby forming
membrane-localized channels, may mediate ODN delivery via a non-endocytic pathway (Fig. 4
A2). Thus the uptake of ODNs complexed with the antennapedia homeodomain peptide and Tat
protein showed a temperature-, energy- and receptor-independent pathway of internalization,
characteristics which are typical of a non-endocytic mode of uptake (104-106). Also, proteins
DNA Pre-mRNA
mRNA
B. Endosomal release
C. Transportation to active sites
Targettedcomplexes
complex
A. Cell association and entry
protein
A1 A2A1
A2
receptor
lysosome
A3
Make sense of antisense oligos
27
and ligands, which are known to be processed by receptor-mediated endocytosis, such as
transferrin or certain growth factors, may serve the purpose of ODN delivery (Fig.4 A3). Indeed,
intracellular uptake of antisense ODNs linked to transferrin and folic acid was more effective
than addition of unmodified antisense (107, 108). Similarly, antisense ODNs targeted to cancer
cells via the epidermal growth factor receptor or to macrophages via the mannose receptor were
found to be taken up more efficiently than naked ODNs (109, 110). However, as noted, once the
plasma membrane barrier is overcome by exploiting the endocytic entry path, the next
intracellular barrier constitutes the endosomal membrane (Fig.4B), which can be considered the
crucial limiting step in the overall pathway that eventually elicites the antisense effect. This
conclusion may be inferred from the notion that cytosol-localized ODNs, as readily
accomplished by microinjection, rapidly accumulate in the nucleus.
Release from endosomes.
Naked ODNs are not able to permeate endosomal membranes. Accordingly, a perturbation of
the endosomal membrane stability is a necessary step in the translocation process, and it has
been concluded that cationic liposomes (but also polymers) are able to accomplish such a
destabilization. Cationic liposomes readily accomodate DNA via electrostatic interactions in a
complex structure, known as lipoplexes (111, 112). Cationic liposomes not only enhance the
uptake of ODNs over 1000-fold, but also promote their translocation across the endosomal
membrane. As a result, the antisense activity as mediated by a cationic lipid vector can already
be revealed in the nanomolar concentration range of the ODN (113-115). However, the exact
mechanism of this translocation is not clear, although some key factors have been revealed,
which interfere with this process. For plasmid release, following lipoplex entry, it has been
proposed that after the cationic lipid/DNA complexes are internalized into cells by endocytosis,
lipid flip-flop occurs at the level of the endosomal membrane. Thus anionic phospholipids, in
particular phosphatidylserine (PS), are thought to translocate from the cytoplasm-facing
endosomal monolayer to the inner monolayer, from where the lipid may laterally diffuse into the
complex and form a charge neutral ion pair with the cationic lipids. The potential ability of the
negatively charged lipid to transfer into the lipoplex may be triggered and/or facilitated by the
non-lamellar phase properties of (part of) the membrane phase in the lipoplex. In addition, the
translocation and mixing with PS may further promote such a non-lamellar structure of the
lipoplex, factors that are likely further potentiating the process of membrane destabilization
(116-118). Moreover, the presence of PS will also cause displacement of the DNA from the
cationic lipids and release of the DNA into cytoplasm (119-120), a phenomenon that can be
Chapter 2
28
readily simulated in vitro (121). An unresolved question in this context is the issue why the
release of DNA (largely, if not exclusively) occurs at the endosomal membrane, and not at the
plasma membrane, particularly since the composition of its outer monolayer is thought to be
reminiscent of that of the inner endosomal membrane.
From observations that several parameters interfere with ODN transport from the endosome
into the nucleus, lipoplex-mediated transfection or release of DNA or ODN from cationic lipid
complexes in in vitro model systems, insight into a potential mechanism of the release of both
(plasmid-) DNA and ODNs from the endosomal compartment has been generated. Endosomal
release of DNA is relatively inhibited when the lipoplexes are consisting of cationic lipids and
dioleoylphosphatidylcholine (DOPC), rather than DOPE. While DOPE displays polymorphic
properties and readily adopts a nonlamellar, i.e. hexagonal HII organization in isolation, DOPC is
a lamellar membrane bilayer-stabilizing lipid, which moreover is much more strongly hydrated
than DOPE (117, 122, 123). The latter parameter will preclude opposing membranes from tight
intermembrane interactions, which are needed in ODN (or plasmid translocation). Indeed, when
including a membrane spacer such as PEGylated lipids, the bulky and extended polyethylene
glycol headgroup separating opposed membranes, ODN delivery from endosome to nucleus is
strongly inhibited and only becomes apparent when the PEG-lipid has diffused from the
complex, the latter being accomplished by incorporating exchangeable PEG-lipids in the
complex (124). Furthermore, structural studies of ODN cationic lipid complexes as well as
lipoplexes, relying on small angle X-ray scattering and cryo-electronmicroscopy have elucidated
that the complex’ ability to adopt a non-lamellar, i.e., hexagonal phase, is crucial for obtaining
efficient plasmid or ODN delivery, respectively. Close intermembrane interactions between
complex and target membrane are instrumental in this event, which is promoted by parameters
such as the nature of helper lipids, as noted above, and the ionic environment. The latter may
modulate the state of hydration of the interphase and thereby govern head group repulsion within
the lateral plane of the bilayer, close head group- head group interactions facilitating nonlamellar
transitions in bilayer organization. PEGylated lipids and strongly hydrated lipids, such DOPC,
may oppose such transitions by maintaining a distinct distance between and within (laterally)
membranes due to steric interference or by means of hydration repulsion and bilayer
stabilization, respectively (117, 123-126). Indeed, protruding PEG-spacers strongly inhibit
endosomal release of ODN (124, 127). Remarkably, PEGylated lipids, incorporated at mole
ratios as low as 1mol% of the total lipid completely eliminated endosomal release of ODNs,
without significantly interfering with the internalization of the complexes. As noted, upon
Make sense of antisense oligos
29
diffusion-mediated removal of polyethelene glycol analogues from the complexes, a time-
dependent release of ODNs was observed in the cells, as reflected by their nuclear accumulation.
Apart from the overall structural and physical considerations that apply to accomplishing
optimal ODN delivery, endosomal release of ODNs can also be affected by the presence of
serum proteins. When such proteins absorb and/or penetrate into the complexes, the ODNs also
remain trapped in the endosomal compartments, and significant ODN release does not occur
(115, 128). In such cases, serum proteins might stabilize the complex membrane by penetration
or via surface adsorption, and/or may cause a steric interference which precludes the intimate
interaction between complex and endosomal membranes in order to induce the release of ODNs
into the cytosol, as described above.
Passage across the nuclear membrane.
Upon microinjection into the cytosol or following their escape from endosomes, ODNs rapidly
accumulate into the nucleus by passive diffusion through the nuclear membranes, whereas other
organellar membranes pose as an effective barrier. A recent study suggested that the diffusion
rate of ODNs is dependent on their length (129). A fragment consisting of 100 bp appears fully
mobile in the cytoplasm, displaying a diffusion rate compatible to that of similarly sized FITC
dextran, and only 5 times slower than that obtained in water. The diffusion of larger fragments is
remarkably slowed down, with little or no diffusion for nucleotides with a length beyond
2000bp. Small ODNs, up to several hundreds of base pairs, readily acquire access into the
nucleus. However, although ODNs of 500 bp diffuse relatively rapidly through the cytoplasm,
their avid crossing of the nuclear membrane was not observed. In passing, such observations are
also highly relevant in elucidating the mechanism by which (much larger sized) plasmids are
supposed to acquire access towards the nuclear machinery.
Within the nucleus, nucleic acid fragments of all sizes were nearly immobile on a distance
scale of 1 micron over a time interval of several minutes. Interestingly, similarly sized FITC
dextrans i.e., up to 580 kDa, diffused freely in the nucleus. Accordingly, it was speculated that
once entering the nucleus, the ODNs are rapidly recruited and immobilized upon their interaction
with nuclear components, including the positively charged histones(Lukacs,-G-L,2000). Other
studies have shown that intranuclear ODNs may also extensively bind to the nuclear RNA matrix
(130, 131). In deed, some controversy may exist concerning the (fractional) nuclear mobility of
ODNs. In fact, it has been shown, that at least a substantial fraction of the ODNs may shuttle
between the cytoplasm and the nucleus (132). Thus, when ODNs were microinjected into one
nucleus of a binuclear cell, they were found to appear readily in the other nucleus. Presumably,
Chapter 2
30
the extent of shuttling may very much depend on the nature of the ODN (length) and whether or
not appropriate target sites are reached. However, at steady state, fluorescently tagged ODNs are
usually predominantly found in the nucleus, and a significant pool in the cytosol is not observed.
When cationic lipids are used to deliver ODNs, the overall distribution of the ODNs is similar
to that obtained upon microinjection of ODNs in the cytosol, except for a minor fraction that
may become entrapped in endocytic compartments. The lipids of the vector itself remain
associated with endocytic compartments, and in contrast to polymers (103), cationic lipids have
not been observed in association with the nuclear membrane (115, 133). However, when the
complexes are released from artificially ruptured endosomes, the dissociation of cationic lipids
and ODNs can be seen to occur at the nuclear membranes, the ODNs becoming detectable in the
nucleus, while the cationic lipids diffuse into the nuclear membrane (130). As noted, at least
some cationic polymers may directly mediate the delivery of ODNs at the nuclear membrane,
likely as a result of endosomal rupture. A typical example is polyethylenimine (PEI), which is
capable of mediating a cell cycle independent nuclear entry of plasmid DNA (103). Also with
ODNs as cargo, PEI arrives at the nuclear membrane, in contrast to cationic liposomes (134).
Nuclear transport of ODNs, following their deposition into the cytosol, occurs by diffusion since
it is not affected by depletion of the intracellular ATP pool or temperature; neither is the effect of
nuclear association affected by the presence of excess unlabeled oligomers. It has been suggested
that nuclear entry can be accomplished by translocation across the nuclear pores, a conclusion
that was derived from the observation that entry could be inhibited by wheat germ agglutinin, a
nucleoporin inhibitor (135, 136). Although the size-dependent ODN transport as noted above,
may also reflects a size-limiting pore-mediated mechanism of nuclear ODN translocation, more
specific experiments, for example. by antibody-specific inhibition, would be helpful to firmly
establish the role of nuclear pores in entry. Indeed, further work will be needed, as no consensus
has been reached yet (99).
Within the nucleus, accumulation of the oligomers involves at least in part their association
with a distinct set of proteins, as revealed by crosslinking of photosensitive oligomers (95).
These proteins can be extracted in the presence of high salt (0.2 M-0.4 M NaCl). Importantly, all
experimental evidence available thus far supports the view that nuclear accumulation of ODNs is
important for eventually obtaining an antisense activity. In the following section, we will
discuss the potential mechanisms by which antisense activity is acquired.
Make sense of antisense oligos
31
Intranuclear distribution of ODNs; a correlation with antisense activity.
Once ODNs cross the nuclear membrane, they distribute throughout the nuclear lumen.
Examination by light microscopy revealed that ODNs usually do not reach the nucleoli, where
the ribosomal RNAs are transcribed from a number of chromosomes and assembled with
ribosomal proteins into ribosomal subunits. In fact, ODNs can form condensed spherical foci
(150-300nm) in a concentration dependent manner, which are not colocalized with the known
nuclear bodies. The function of these oliogonucleotide bodies is not known. It has been
speculated that the accumulation of ODNs as nuclear bodies is a response of the cell to sequester
excess ODNs in order to reduce their toxic effect (137). The exact localization of the
predominantly nucleoplasma-localized distribution of ODNs is still largely unresolved. We (130)
and others (137) have shown that the diffusely distributed ODNs within the nucleoplasm are
resistant to mild removal of loosely bound nuclear proteins and to removal of chromatin with
DNase. However, they are susceptible to the removal of RNA, following Rnase treatment (137),
and following treatment with high salt (95). These data thus indicate that ODNs are extensively
bound with non-chromatin structures in the nucleus, which is usually designated as the nuclear
matrix. Among others, these structures are composed of heteronuclear RNA (hnRNA), the
fraction of nuclear RNA which contains primary transcripts of the DNA, representing a nuclear
precursor fraction that is processed to messenger RNA, and hnRNPs, a large family of proteins,
implicating in the processing of nascent mRNA transcripts.
In permeabilized cells, ODNs readily migrate into the nucleus at ambient temperature, at
which conditions PS-ODNs, rather than PO-ODNs, give rise to a multitude of large, irregular
aggregates (138). High-affinity PS-ODN, but not PO-ODN, presumably reacts with the nuclear
lamina. Simultaneously, ODNs cause decompaction of chromatin, the PS-ODN aggregates
appearing as compact inclusions in homogeneously dispersed chromatin. After microinjection of
S-ODN into intact cells, these effects were not observed, although the nucleic acids rapidly
moved into the nucleus and condensed into a large number of well-defined, spherical speckles or
longitudinal rodlets (135, 136, 139). A high degree of complex formation between ODNs and
nuclear proteins has also been reported, as demonstrated in a gel shift assay (65). However, the
nature and number of these proteins is unknown, and whether such interaction may also occur
intracellularly, remains to be determined. Even though ODNs presumably bind to nuclear matrix
proteins, this will shorten the spatial and temporal distance to target mRNA. Nuclear matrix
binding of ODNs is not sequence-dependent, implying that nuclear matrix binding per se does
not reflect the efficiency of an antisense effect. Thus it remains to be determined how such
Chapter 2
32
interactions provide insight as to how antisense, but not mismatched sequences, finds their
targets.
Meeting intranuclear targets and identifying the fate of targets.
Obviously, the target of antisense molecules is the complementary mRNA. In spite of the fact
that antisense ODNs can hybridize to in vitro transcribed mRNA or to synthesized
complementary RNA or DNA fragments, the evidence that such an event also occurs within cells
is scanty. In an effort to gain such evidence, we have delivered radio-labeled or fluorescently-
labeled antisense ODNs and mismatched sequences into target cells. Following delivery, both
ODNs become extensively bound to the nuclear matrix, similarly as described above. When total
RNA was isolated from cells treated with the labeled antisense or mismatched ODNs, the
antisense sequence showed the expected high affinity to target mRNA, but not to other mRNAs
whereas the mismatched sequence did not display affinity to target mRNA. These data were
taken to indicate that antisense ODNs hybridize to target mRNA with high affinity over
mismatched control via their initial binding to the nuclear matrix (130). Accordingly, this
scenario would be consistent with the notion that upon hybridization of antisense ODNs to target
mRNA, its induced degradation occurs, and as a consequence protein synthesis becomes
impaired. Importantly, intracellular cleavage of target mRNAs by antisense binding could be
revealed in these studies (30, 32, 130).
In order to appreciate the biological effect of antisense treatment, it should be taken into
account that the existing pool of a target protein will not be affected other than by its natural
turnover. Thus, the antisense can only inhibit or eliminate de novo synthesis of target proteins.
Hence, the time course of the appearance of an antisense effect will vary according to the half-
life of target protein degradation, implying that relatively shortly following treatment, a large
preexisting pool of target proteins may obscure a potential biological effect of the antisense in
causing effective reduction of newly synthesized targets.
In vitro versus in vivo; role of extracellular barriers.
It is becoming apparent that many fundamental issues on the mechanism of nucleic acid
delivery can be conveniently and properly investigated in vitro, including intracellular
processing and intracellular ODN stability. However, it has become equally clear that a direct
extrapolation from in vitro to in vivo should be done but with caution. Apart from factors related
to the circulation, the role of the extracellular matrix is still puzzling, and it seems that thus far
its role has been inadequately mimicked in vitro. One such an example involves studies on the
delivery of ODNs to brain tissue. Thus, ODNs can be readily introduced into a variety of
Make sense of antisense oligos
33
neuronal cell lines, provided that delivery is mediated via an appropriate vector, such as cationic
lipids. By contrast, when injected intracerebrally in rats, the ODNs also acquire ready access to
neuronal cells, accumulating in cytoplasm and nuclei without the need of such a carrier (Shi et
al., unpublished observ.;140). Up to some 4 hr after injection of the free ODNs into whole brain,
the ODNs show a diffuse distribution in the extracellular matrix and in the cells. After 24 hr, the
associated pool of ODNs with the extracellular matrix has strongly diminished while
concomitantly, the pool of intracellular ODNs greatly increases. Another example represents the
uptake of ODNs by liver tissue. Following intravenous administration of naked ODNs, they
become associated with the extracellular matrix, which is followed by internalization in all cell
types of the liver, including appearance of ODNs, albeit in relatively minor amounts, in the
nucleus. Although the mechanism of uptake remains to be determined, it is again apparent that
also in this case nuclear access of ODNs in vivo can be accomplished in a vector- independent
manner (141).
How these remarkable differences between in vivo and in vitro application in terms of cellular
access of ODNs should be accounted for, has not been addressed thus far. Whether the
extracellular matrix harbors means to effectuate and/or to facilitate the mechanism of entry,
remains to be determined. In the context of in vitro observations it is difficult to envision that if
endocytosis would represent a major means of entry in vivo as well how endosome- localized
ODNs would readily acquire access to the cytosol in vivo and not in vitro. This raises the issue of
the existence of alternative (facilitated) mechanisms of entry in vivo, which should be capable of
mediating direct delivery of ODNs into the cytosol, thus allowing rapid nuclear delivery. ODN
transport via a plasma membrane-localized nucleic acid channel as recently described to be
present in rat renal brush board plasma membranes could fulfill such an alternative (93, 94). It is
possible that an enhanced endocytic capacity in vitro and/or a less efficient translocation
mechanism at such conditions, possibly depending on extracellular modulators for optimal
activity, may obscure the seemingly low delivery to nucleus of added free ODNs in vitro.
Even though naked ODNs can enter cells in certain tissues, they permeated very poorly into
others such as brain or tumor after systematic administration. This necessitates the use of
appropriate vectors in vivo, particularly in targeted delivery.
Overcoming the reticuloendothelial system (RES).
Dictated by pharmacokinetic distribution studies in vivo on drug delivery, organs of the RES,
such as liver, spleen, kidney and lungs represent most likely effective clearance sites, following
systemic administration of ODN complexes. Even though a wide tissue distribution of ODNs has
Chapter 2
34
been reported, except for the brain and testes, a major fraction, irrespective of the route of
administration, appears to end up in RES organs. Accordingly, directing ODNs to non-RES
organs remains a challenge in antisense technology.
Free ODNs are cleared from plasma according to a kinetics with two exponentials, showing
half-lives of about 20 min and approx. 24 h, respectively, although it may vary with the chemical
nature of administered ODNs. Provided that the majority of naked ODNs are cleared in the first
few hours, liposomes and other delivery vectors are used to protect and prolong the half-life of
ODNs in the circulation in order to improve the interaction frequency with non-RES organs.
However, to avoid the RES, additional modification of the delivery vehicle per se is needed, like
the inclusion of PEGylated lipids, modulation the surface charge and inclusion of appropriate
targeting devices for tissue/cell-specific delivery. Such systems have proved useful, for example,
in targeted delivery of DNA into the brain. Thus a PEGylated (DSPE-PEG) DNA lipoplex,
consisting of phosphatidylcholine and the cationic lipid didodecylammonium bromide, was
conjugated with transferrin receptor antibodies, OX-26. Following systemic administration, such
complexes were shown to cross the blood brain barrier by transcytosis via transferrin receptors
on brain endothelial cells. The complexes subsequently distributed over a large area in the
central nervous system, including neurons, choroid plexus epithelium, and the brain
microvasculature (12, 142, 143). A priori, in this manner it should thus be possible to specifically
target to defined brain cell populations, following systemic administration. Importantly however,
successful application of such an approach will require the dissociation of the PEGylated lipid
from the complex, as discussed above. Hence rather than the essentially non-exchangeable
DSPE-PEG, this kind of approach requires a controllable release of PEGylated lipid analogues
(124, 144, 145)
Reducing the toxicity of ODNs.
In spite of the fact that successful clinical trials have been reported (22-24), scepticism
remains as to unambiguous proof for accomplishing a genuine antisense effect. Obviously this
will frustrate a more general acceptance and effectuation of antisense therapy. In fact, for any
kind of drug application, including the application of antisense technology, potential toxic side
effects are a primary concern. In cell culture, the main toxicity is due the inhibition of cell
growth as a result of the binding of ODNs to membrane or other intracellular proteins. In vitro
cytotoxicity can be usually controlled by adjusting the proper dosage of ODNs or by their
packaging in delivery vectors. The in vivo toxicity often correlates with the capture and long-
term deposit of ODNs in RES organs, causing harmful side effects as renal tubule
Make sense of antisense oligos
35
degeneration/necrosis, splenomegaly, thrombocytopenia and elevation of liver transaminases
(146, 147). Some of these side effects are similar to those associated with delivery of other non-
related polyanions such as dextran sulfate (148). Improved ODN chemistry, for example by
reducing the negative charge, may at least in part relieve these problems. The potential toxicity
of ODNs with chemically modified linkages has not been addressed thus far. Thus future work
should also be aimed at clarifying the metabolic fate of ODNs. However, in general, from the
clinical toxicity profile of ODNs directed against viruses and tumors, this new generation of
drugs is still more tolerable than conventional chemotherapy and radiotherapy (149-151).
Conclusion and remarks In the past 5 years, considerable progress has been made in
understanding the mechanism(s) of antisense activity, in developing ODN chemistry and in
setting up antisense-related clinical trails, with as landmark the production of the first
commercial antisense drug against CMV retinitis. Evidently, the activities leading to these
prosperous developments have raised a wealth of insight into many fundamental cellular
processes ranging from events related to the regulation of gene function to signal transduction
pathways. Thus therapeutic progress is closely related to that of fundamental development and
the spin off for the latter is far beyond the actual purpose of ODN research as such. In terms of
therapeutic application of antisense technology, the window could be extended beyond treatment
of primarily cancers and viral infections, which may be accomplished by improving therapeutic
index, better specifying the antisense effect and lowering the costs.
AcknowledgmentWork carried out in the authors laboratory was in part supported by a grant from TheNetherlands Organization for Scientific Research (NWO)/NDRF Innovative DrugResearch (940-70-001).
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