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On the mechanism of cationic lipid-mediated delivery of oligonucleotidesShi, Fuxin

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Publication date:2004

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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


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.


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


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


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


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


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.


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


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


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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