REVIEW ARTICLE
Optimizing the delivery systems of chimeric RNA
.
DNA oligonucleotides
Beyond general oligonucleotide transfer
Li Liang, De-Pei Liu and Chih-Chuan Liang
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, People’s Republic of China
Special oligonucleotides for targeted gene correction have
attracted increasing attention recently, one of which is the
chimeric RNAÆDNA oligonucleotide (RDO) system. RDOs
for targeted gene correction were first designed in 1996, and
are typically 68 nucleotides in length including continuous
RNA and DNA sequences (RNA is 2¢-O-methyl-modified).
They have a 25 bp double stranded region homologous to
the targeted gene, two hairpin ends of T loop and a 5 bp GC
clamp, that give the molecule much greater stability [Fig. 1].
One mismatch site in the middle of the double-stranded
region is designed for targeted gene therapy. RDOs have
been used recently for targeted gene correction of point
mutations both in vitro and in vivo, but many problems must
be solved before clinical application. One of the solutions is
to optimize the delivery vectors for RDOs. To date, few
RDO delivery systems have been used. Therefore, new
vectors should be tried for RDO transfer, such as the use of
nanoparticles. Additionally, different kinds of modifications
should be applied to RDO carrier systems to increase the
total correction efficiency in vivo. Only with the development
of delivery systems can RDOs be used for gene therapy, and
successfully applied to functional genomics.
Keywords: chimeric RNAÆDNA oligonucleotides; gene

single-stranded chimeric oligonucleotides containing both
DNA and RNA sequences [3]. Monia et al. also reported
that such chimeric oligonucleotides improved the efficiency
of antisense therapy [4]. In 1996, Kmiec’s group constructed
a novel chimeric oligonucleotide for targeted gene correc-
tion [5]. RDO for targeted gene correction is a single-
stranded molecule, typically 68 nucleotides or more in
length, including continuous RNA and DNA sequences
(RNA is 2¢-O-methyl-modified). It has a 25 bp double
stranded region homologous to the targeted gene, two
hairpin ends of T loop and a 5 bp GC clamp that gives the
molecule much greater stability. One mismatch site in the
middle of the double-stranded region is designed for
targeted gene correction [6–8] [Fig. 1]. The RDO is different
from other oligonucleotides in several respects. First, it is a
self-complementary oligonucleotide that folds into a dou-
ble-hairpin configuration, different from plasmids that are
circular, or general oligonucleotides that are linear. Second,
it is chimeric, with both RNA and DNA sequences. Third,
its length is different from most oligonucleotides used for
antisense therapy, which are usually 12–40 bp in length [9],
but RDO is 68 nucleotides or more in length.
Correspondence to D P. Liu, National Laboratory of Medical
Molecular Biology, Institute of Basic Medical Sciences, Chinese
Academy of Medical Sciences & Peking Union Medical College,
5 Dong Dan San Tiao, Beijing 100005, People’s Republic of China.
Abbreviations: BPS, biodegradable pH-sensitive surfactants; CHO,
Chinese hamster ovary cells; DMRIE, dimyristyloxypropyl
dimethylhydroxyethylammonium bromide; DOPE, dioleoyl-
phosphatidylethanolamine; DOPS, dioleoylphosphatidylserine;

genomics and used in human gene therapy.
RECENT PROGRESS IN RDO DELIVERY
Several strategies have been attempted to deliver RDOs.
Microinjection and microparticle bombardment are effi-
cient methods to be used in vitro or on stem cells, but can
not be used in vivo. Two delivery systems have been used for
RDO transfer in vivo. One is the use of liposomes.
Liposomes were believed to encapsulate nucleic acids within
their aqueous core in the past [18–20]. However, some
cationic lipids may attract DNA by electrostatic charges
[21]. Lipofectin (a commercial liposome) was the first
transfection reagent used in RDO transfer. When the
lipofectin–RDO complex was transferred into Chinese
hamster ovary (CHO) cells containing extra-chromosomal
plasmids, 30% correction rate was accomplished at the
episomal targets in CHO cells [5]. Because this gene
correction was in episomal DNA, not nuclear DNA, it
cannot be compared with other experiments. A variety of
liposomes have now been used for RDO delivery, especially
commercially available liposomes [13]. Dioleoyloxypropyl
trimethylammonium methyl-sulfate (DOTAP) was used to
transfer RDOs to lymphoblastoid cells, and corrected point
mutations of the a-hemoglobin gene. DOTAP also deliv-
ered RDOs to MEL-D7 cells and corrected the a
E
gene,
which was introduced into the MEL cells, to the normal a
gene. In HeLa cells and CMK cells transfected with two
other types of liposomes, DMRIE-C and FuGene 6,
detectable corrections can be achieved by RDOs [22].

nucleotides in length, including continuous 2¢-O-methyl-modified
RNA and DNA sequences, a 25 bp double-stranded region homo-
logous to the targeted gene, two hairpin ends of T loop and a 5 bp GC
clamp, which give the molecule greater stability. One mismatch site in
the middle of the double-stranded region is designed for targeted gene
therapy.
Fig. 2. The mechanism of RDO action for targeted gene correction is to
repair mismatch after homologous alignment. The DNA strand is
responsible for the correction, while the RNA strand stabilizes the
structure. The RDO designed for reacting with the sense strand is more
efficient than that for the antisense strand.
5754 L. Liang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
decreased to 40% of normal, showing the effect of RDO
conversion [26,27]. B. T. Kren reported that, for in vivo
transfection, chimeric oligonucleotides were fluorescently
labeled, and then complexed with lactose–PEI at a propor-
tion of 1 : 6 (ON phosphate/PEI amine) in 5% (w/v)
dextrose. The lactose–PEI–RDO complex was distributed
homogeneously throughout the liver as early as 2 h after tail
vein injection, and not in lung, heart and kidney [28]. The
results showed that the G residue at nucleotide 1206 was
replaced and UGT1A1 genetic defect was corrected (the
genetic basis of Crigler–Najjar syndrome type I) in Gunn rat
liver. In addition, the RDOs were complexed with anionic
liposome AVETM-3 after AVETM-3 was coated with
protamine sulfate. The complex delivered RDO to the
nucleus more effectively than those without modifications
[29].
In short, at present the best RDO delivery systems are
modified PEI or liposomes, such as lactosylated PEI or

The following are clues on finding such a delivery system.
Applying novel delivery systems to RDO transfer
Recently, several novel delivery systems have been used
successfully for plasmid DNA transfer or antisense oligo-
nucleotide transfer. These are putative RDO delivery
systems.
Pluronic gel is a substance traditionally used for trans-
dermal injection. One special characteristic of pluronic gel is
that it exists as a liquid when cold, and becomes solid at
body temperature. Recently, pluronic gel has been used for
antisense delivery, especially in blood vessels, and it has the
advantage of prolonged delivery [31,32].
There are several substitutes for PEI. Chitosan, or
poly(
D
-glucosamine), is a natural cationic amino-polysac-
charide [33–38], and attracts oligonucleotides with electro-
static charge. Chitosan may be a substitute of PEI because it
has low toxicity, and is biocompatible and resorbable.
Regarding its efficiency, at least at 96 h after transfection of
HeLa cells, chitosan was found to be 10 times more efficient
than PEI [34]. Dendrimers are a new kind of reproducible
substances, with a hydrocarbon core and charged surface of
amino groups. They have the advantage of having a defined
small size. However, their efficiency and toxicity still need
evaluation.
Nanoparticles are new delivery systems. By the method of
associating with oligonucleotides [39], they can be classified
into encapsulating nanoparticles, complexing nanoparticles
and conjugating nanoparticles [Fig. 4]. The first type is

nanospheres, alginate nanosponges accumulated oligonu-
cleotides in the lung 10-fold more than IBCA when the same
amount was administrated intravenously [40]. This differ-
ence in tissue distribution may be due partially to the
polysaccharide nature of some nanoparticles. Such poly-
saccharides can be recognized by receptors in specific
tissues, such as pulmonary tissue. However, the difference of
metabolism passage may also explain the distribution
difference. Those nanoparticles that are metabolized in liver
certainly accumulate in hepatocytes. Therefore, even though
nanosponges are generally the best, other nanoparticles
should also be developed to find if they are tissue specific.
RDO DELIVERY SYSTEMS WITH MORE
THAN ONE MODIFICATION
Oligonucleotide delivery is a multistep process, and must
pass a series of barriers. Basic carrier systems normally have
difficulties with targeting. To improve this, delivery systems
should be decorated with ligands for a variety of purposes,
such as tissue targeting, endosomal release and nuclear
targeting. Tissue targeting has been realized in RDO transfer,
but other modifications, which have been used in antisense
oligonucleotide delivery, still need testing on RDO transfer.
Endosomal release is a rate-limiting step of gene delivery.
Oligonucleotides must be released from the endosome
before entering the nucleus. However, if oligonucleotides are
released too early, they will have difficulties in cytoplasmic
transport. The best place for endosomal release is the
perinucleus. Many fusogenic or pH-sensitive agents are
attached to delivery vectors, to facilitate endosomal release
[9,20]. DOPE (dioleoylphosphatidylethanolamine) has the

develop mitochondrion-specific delivery systems for RDO.
Two methods used in plasmid transfer show prospects of
delivering oligonucleotides into mitochondria [46–48].
Mitochondriotropic vesicles are cationic amphiphiles con-
taining a hydrophilic charged center and a hydrophobic
core, capable of transferring nucleic acids to mitochondria
[46]. This strategy is easy to handle, and may be used for
RDO delivery. Another strategy is the conjugation of
mitochondrial signal peptides to oligonucleotides, similar to
nuclear signal peptides. By imitating mitochondrial entry of
polypeptides that are synthesized in the nucleus and
function in mitochondria, gene transfer is realized [48].
However, this technology is still in its infancy.
However, one modification usually helps to overcome
only one hurdle. In this regard, modifying the delivery
systems with two or more ligands is a promising method to
construct an all-round delivery system, which can pass all
barriers smoothly. One example is PEI modified with both
polysaccharides and DOPE. The polysaccharide ligand is
for cell targeting, and the DOPE ligand is for controlling
endosomal release. To date, the most efficient decoration for
endosomal release is BPS, while the ligands for tissue
targeting differ in different tissues. Good RDO carrier
systems may combine these two advantages. Additionally,
nanoparticles and nanosponges should also be modified
with specific ligands for tissue distribution and targeting [39].
DESIGNING RDO SPECIFIC DELIVERY
SYSTEM
It is possible that a novel RDO specific delivery system,
which is both safe and efficient, will be invented in the near

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