www.elsevier.com/locate/jconrel

Journal of Controlled Releas

Review

Lipidic carriers of RNA/DNA oligonucleotides and

polynucleotides: What a difference a formulation makes!

Michael KellerT

IC-Vec Ltd., Flowers Building, Armstrong Road, London SW7 2AZ, United Kingdom

Accepted 3 December 2004

The recent regeneration of faith into the biotech-

nology industry is largely based on one long neglected

species of biopolymers: ribonucleic acid (RNA).

Although its importance has never been underesti-

mated by the research community, RNA owed its

ambiguous reputation mainly due to a myth hooked in

biological research laboratories: its apparently

binherent instabilityQ led researchers (too easily) to

conclude that RNA, which is a variation of the

biopolymer that holds all genetic information in living

cells (deoxyribonucleic acid; DNA), is too difficult a

choice to work with—especially as a therapeutic tool.

Several types of RNA exist, exhibiting roles in

both structure and function. The most interesting

RNA entity for scientists involved in gene regulation

is the messenger RNA (mRNA) that translates genetic

information encoded by the DNA into protein.

mRNA, as a result, varies in length depending on

the size of the gene that it translates.

mRNA is produced by cells when the cellular

metabolism is in need of a specific protein, and

subject to degradation thereafter. Its structural fea-

tures–the presence of the 2V-hydroxyl group on the

ribose ring–gives the RNA the potential to act not

0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

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only as a mediator of the genetic information into

proteins, but also to have catalytic capabilities [1].

The drawback of this additional hydroxyl group,

however, is an increased instability compared to its

DNA counterpart. In addition, mRNA is produced as

a single-stranded biopolymer only, which is an addi-

tional element of instability. While an enhanced

degree of instability is essential for maintaining

intracellular dynamics in order to readily eliminate

unnecessary mRNA subsequent to translation into

protein, there is a price to pay for the researcher: the

handling of RNA needs much more care than DNA.

This is not only due to their structural differences, but

also due to the omnipresence of RNAse, an enzyme

evolved to degrade RNA [2]. These combined

features appear to be too much of a worry to consider

such a molecule to be applicable for routine use in

research laboratories, not to mention its use as a

therapeutic agent.

So how come that RNA has come out of its

shallow grave and has undergone a dramatic revival,

both in academia and in the biotech industry? The

RNA excitation dates back to the early 1990s, where

plant biologists working with petunias were surprised

to find that introducing numerous copies of a gene

that codes for deep purple flowers led–not as

expected–not to an even darker purple hue, but rather

e 103 (2005) 537–540

M. Keller / Journal of Controlled Release 103 (2005) 537–540538

to plants with white or patchy flowers [3,4]. Similarly,

when plants were infected with an RNA virus that had

been genetically engineered to contain fragments of a

plant gene, the plant’s gene itself became inactivated

[5]. Only when Fire et al. published their work in

1998 were these puzzling observations fully under-

stood [6]. RNA viruses replicate through double-

stranded intermediates that are cleaved by a cytosolic

enzyme (DICER) [7] into short RNAs with a specific

structure: two 21-nucleotide strands of RNA with 19

complementary nucleotides of dsRNA and two

unpaired 3V-nucleotides at either end [8–11]. These

short stretches of viral double-stranded RNA were

dubbed a short interfering RNA (siRNA). The

antisense strand of this siRNA duplex is then

assembled into a multi-protein complex called RISC

(RNA-induced silencing complex) [7]. One of the

RISC proteins (Ago2) catalytically degrades the viral

mRNA, thus eliminating the source of infection [12].

siRNA results when transposons, viruses, or endog-

enous genes express long dsRNA, or when dsRNA is

introduced experimentally into plant and animal cells

to trigger gene silencing, a process known as RNA

interference [6,8,10,13].

Interestingly, synthetic short double-stranded RNA

stretches (siRNA; 21–23 nt) with a specific mRNA

target sequence were also found to have the capacity

to mediate the degradation of mRNA in a specific

way, essentially knocking down a specific gene

function [8]. The length of the siRNA stretch is

important: siRNA sequences exceeding 30 bp increas-

ingly provoke a viral response of the host cell via a

protein kinase R-mediated mechanism, resulting in the

production of interferon [14]. From a chemist’s

perspective, this finding opened the gateway for the

production of gene-specific synthetic siRNA sequen-

ces useful in functional genomics programs and, more

intriguingly, potentially as drugs to treat disease. The

excitement of both the academia and biotech/pharma

industry is visible in the dramatically increased

number of publications dealing with RNAi. Not

surprisingly, a wealth of start-up companies has

established, aiming to exploit the heat of the siRNA

fever commercially.

In order for RNAi to be successful, some pivotal

requirements must be met. Genetically, siRNA

sequences need to be designed to specifically down-

regulate one gene only without non-specifically cross-

interfering with other cellular mRNA (off-target

effect) [15–19]. Chemically, the production of large

quantities of siRNA must be feasible at a cheap price

and within short production times. Technically, the

siRNA sequences are to be delivered into host cells

without compromising cellular viability (i.e., unspe-

cifically interfering in cellular pathways). This crucial

process–the delivery of siRNA– is often neglected. In

order to differentiate the delivery of short double-

stranded (synthetic) siRNA from the delivery of

plasmid DNA (transfection), we suggested the use

of the terminology bsiFectionQ rather than trans-

fection, which is used in the literature for the delivery

of DNA into host cells [20].

siFection of siRNA requires a delivery system

displaying the capability to mediate the RNA

sequence to the intracellular environment. Cationic

liposome carriers have long been used to mediate the

transfer of DNA into cells; since the nature of their

interactions with DNA and RNA is identical (charge/

charge), it seems obvious to adapt formulations that

were developed for (plasmid) DNA to the delivery of

siRNA. However, a closer look at this process reveals

a few cornerstones to be considered. One particularity

of the DNA formulation with a cationic entity such as

peptides/proteins, polymers, or cationic lipids is the

occurrence of the so-called bDNA condensationQprocess. This phenomenon is triggered when 70–

90% of the DNA phosphodiester charge is neutral-

ized, and is manifested by the collapse of DNA into

nanostructures of differential morphology [21,22] due

to the predominant hydrophobic nature of the DNA

nanoparticle. A minimal length of the DNA of about

800 bp is required for a DNA condensation process to

occur [23–25]. This finding has important consequen-

ces: short double-stranded siRNAs are devoid of such

a condensation process. While the volume occupied

by a condensed DNA nanoparticle is about 10,000

smaller than of its uncondensed counterpart, short

double-stranded siRNA stretches retain their initial

volume when complexed to a cationic entity. As a

result, several copies of siRNA are complexed/

incorporated per liposome rather than one single

entity of condensed DNA as found, for example, in

the SPLP system [26]. The delivery aspect of siRNA

is much more related to antisense oligodeoxyribonu-

cleotides (ODN) than to plasmid DNA. Research into

ODN-mediated gene silencing can be traced back to

3’-FITC-siRNApDNA(Cy3)

60mins 60mins

Fig. 1. Intracellular localization of fluorescently labelled pDNA

(Cy3) and 3V-FITC-siRNA in cultured dividing HeLa cells detected

by confocal microscopy 60 min after delivery of the respective

nucleic acids (reprinted with permission from Ref. [20]; copyright

2004, American Chemical Society).

M. Keller / Journal of Controlled Release 103 (2005) 537–540 539

1977 [27] and has been extensively investigated ever

since [28,29]; however, so far, only one antisense-

based drug has been approved by the Federal Drug

Administration (FDA) [30]. Mechanistically, synthetic

siRNA displays the advantage to act solely in the

cytosol on a post-transcriptional level, whereas some

ODNs need to localize to the nucleus in order to

interfere in the gene regulation process since they can

act both on the levels of transcription and translation

[31]. Synthetic siRNAs are most effective when

delivered as double strands, whereas antisense ODNs

are usually delivered as single strands, with some

exceptions [32]. Most interestingly, antisense ODNs

delivered to cultured cells by a transfection reagent

naturally localize to the nucleus within minutes after

transfection, whereas their carrier remains in perinu-

clear areas [33] (Fig. 1).

This phenomenon is also observed with plasmid

DNA complexed with cationic liposomes [34]. In

sharp contrast, synthetic siRNAs localize to perinu-

clear regions and not to the nucleus after siFection,

even after extended periods of time [20,35]. These

differences in uptake, intracellular trafficking, and

storage have important consequences: firstly, the

formulation parameters of synthetic siRNA need to

be carefully established prior to use. In other words,

a delivery reagent developed for DNA is most likely

not suitable for siRNA. Secondly, delivery of siRNA

must be devoid of toxic effects, which come from

the siFection reagent. We have demonstrated that

certain widely used transfection reagents induce a

degree of cellular toxicity that severely interferes

with the outcome of the gene down regulation study.

Thirdly, the formulation parameters of synthetic

siRNA for in vivo use differ dramatically from the

ones described for both ODN and pDNA. It will be

the challenge of formulation chemists to generate

reproducible, stable, non-toxic, and efficient formu-

lations that would enable siRNA to be used as a

therapeutic entity.

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