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 Scienc
Trang 1R E V I E W A R T I C L E
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 delivery; gene therapy; targeted gene correction; nano-particles
Targeted gene therapies are ideal strategies for gene therapy,
which include gene replacement in situ and gene correction
Gene replacement substitutes the mutated gene with a
normal one, but it is a low efficiency method due to the
limitation of homologous recombination Gene correction
is the most putative strategy of targeted gene therapy [1]
Recently, three kinds of oligonucleotides have been used for
targeted gene correction: triplex-forming oligonucleotides
(TFOs), modified single-stranded oligonucleotides and
chi-meric RNAÆDNA oligonucleotides (RDOs)
Triplex-form-ing oligonucleotides are oligonucleotides with high affinity
for polypurine/polypyrimidine sequences, and are capable
of forming a triplex after binding to the major groove of
DNA However, the application of TFOs is limited, because
they can only repair gene defects near homopurine
sequences [2] Modified single-stranded oligonucleotides
(SDOs) are also used for targeted gene correction The most common SDO is phosphorothioate-modified oligonucleo-tide (ON), but unfortunately this is toxic to cells when used
in vivo The third kind of ONfor gene correction is the chimeric RNAÆDNA oligonucleotide
C H I M E R I C R N AÆD N A
O L I G O N U C L E O T I D E F O R T A R G E T E D
G E N E C O R R E C T I O N
Chimeric RNAÆDNA oligonucleotides were first used in antisense gene therapy in late 1980s Inoue et al constructed 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 correccorrec-tion 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;
DOTAP, dioleoyloxypropyl trimethylammonium methylsulfate;
IBCA, poly(isobutylcyanoacrylate); ON, oligonucleotide; PACA,
poly(alkylcyanoacrylate); PEI, poly(ethylenimine); RDO, chimeric
RNAÆDNA oligonucleotide; SDO, single-stranded oligonucleotide;
TFO, triplex-forming oligonucleotide.
(Received 6 March 2002, revised 15 August 2002,
accepted 8 October 2002)
Trang 2RDO is used for targeted gene correction both in vitro
and in vivo Its mechanism is not completely known The
gene correction of RDO is based on the mechanism of
endogenous DNA repair systems after homologous pairing,
rather than homologous recombination Therefore, RDO is
not limited by the frequency of homologous recombination
The two parts of RDO play different roles in gene repair
The DNA region enables gene correction to occur, and the
2¢-O-methyl-modified RNA region stabilizes the structure
[10] Transcription may also be involved in the process of
gene correction RDOs designed for sense strands are much
more efficient than those for antisense strands [Fig 2]
Although RDOs corrected point mutations in animal
models, there is still a long way to go before clinical
applicationis possible The correction efficiency is distinct
(from 1–40%) in different cell lines or tissues [11–17]
Optimizing the length and structure of the RDO is crucial,
such as lengthening the homologous region or changing the
place of the mismatch on the chimeric double-stranded
region Another key step toward clinical application is to
optimize RDO delivery vectors Only with the development
of carrier systems can RDOs be applied to functional
genomics and used in human gene therapy
R E C E N T P R O G R E S S I N R D O D E L I V E R Y
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 aE 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] Anionic liposomes, such as dioleoylphosphatidylserine (DOPS) were also used, and were more effective than the neutral or cationic liposomes for in vitro delivery But all these delivery vectors were all at a low efficiency level Another RDO vector is polyethylenimine (PEI) PEI is a polymer with a backbone of two carbons followed by a nitrogen atom, and can be either linear or branched [23,24] PEI attracts oligonucleotides by electrostatic charge It was used to transfer RDOs into HeLa cells, and the point mutation at the )202 residue of the b-globin gene was corrected
However, all these carrier systems without modifications are not tissue-specific Nowadays, RDO delivery systems with modifications are used for gene correction in vivo and can concentrate RDO within a specific organ Some modified liposomes are under study It was reported that galatocerebroside [25], a type of polysaccharide that can recognize the asialoglycoprotein receptors on hepatocytes, was added to three different liposomes for organ targeting All such decorated anionic, neutral and cationic liposomes delivered RDOs effectively, and promoted AfiC conver-sion at the Ser365 position in the rat factor IX gene [25] Furthermore, PEI was decorated by lactose, another liver-specific ligand PEI (25 kDa) was covalently lactosylated, forming a lactose–PEI complex The complex carrying RDO was administrated by tail vein injection into rats either once or repeatedly at fixed intervals The results showed that RDO converted AfiC at Ser365 of factor IX gene in rat liver specifically Factor IX coagulant activities of rats
Fig 1 Diagrammatic structure of RDO RDOs are typically 68
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.
Trang 3decreased 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 (ONphosphate/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
polysaccharide modified liposomes [Fig 3] Such
decor-ation not only facilitates targeting, but also improves the
relative efficiency of RDOs However, these modified
delivery systems are not ideal, because of instability in
serum or toxicity to cells
T H E M A J O R D E L I V E R Y B A R R I E R S A N D
P R O B L E M S F A C I N G R D O T R A N S F E R
RDO and its delivery systems must overcome several major
hurdles for in vivo gene transfer This is a multistep process
[9,18,19] First, the delivery systems should have low
immunogenicity Those delivery systems that can trigger
immune responses can only be used to transfer RDOs into
tumor cells as gene vaccines Second, vectors should be
tissue-specific Because most genes only function in specific
tissues, targeted gene therapy of RDOs should only be at
specific organs Gene conversion in all organs is wasteful,
and could even increase undesired side-effects Third, they
should pass the cell membrane, be released from endosomes
and be transported easily in the cytoplasm Fourth, they
should enter the nucleus easily For transfection in vivo, this
is one of the most important hurdles Finally, cytotoxity is
another major problem Only low or nontoxic delivery
systems could be used in humans Therefore, we must test
different delivery systems on RDOs, and try to lower their
cytotoxity and improve their efficiency
F U T U R E D I R E C T I O N S : O P T I M I Z I N G
R D O D E L I V E R Y S Y S T E M S
An ideal RDO delivery system should have little or no toxicity and high efficiency It should be an all-round delivery system that can pass delivery barriers smoothly [30] 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 represented by nanosponges, such as alginate nanosponge [40], which are sponge-like nanoparticles containing many holes that carry the oligonucleotides Additionally, nano-capsules such as poly(isobutyl-cyanoacrylate) (IBCA) are also encapsulating nanoparticles They can entrap oligonu-cleotides in their aqueous core [41]
Fig 3 RDO delivery systems and transfer barriers.
Fig 4 Three types of nanoparticles (A) Nanosponge, an encapsula-ting nanoparticle, which encapsulates DNA within its core (B) Complexing nanoparticle, which attracts DNA by electrostatic charges (C) Conjugating nanoparticle, which links to DNA through covalent bonds.
Trang 4The second type is complexing nanoparticles, which are
coated with cationic polymers Such nanoparticles associate
with oligonucleotides by electrostatic attraction
Third, oligonucleotides can also be conjugated to
nano-spheres by covalent bonds Different nanonano-spheres have
different characteristics Encapsulating nanoparticles,
espe-cially nanosponges, may be the best among all
nanoparti-cles, because they protect oligonucleotides from proteins
and enzymes, and do not change the DNA conformation
through electrostatic force [39] For example, in alginate
nanosponge, 80% of the oligonucleotides were still
unde-graded after 1 h of incubation with fetal bovine serum [39]
Considering tissue specificity, PACA
[poly(alkylcyanoacry-late)] nanoparticles, which are complexing nanoparticles,
can deliver oligonucleotides specifically to the liver In
contrast, alginate nanosponges concentrate oligonucleotides
in the lung, liver and spleen Comparing two encapsulating
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
R D O D E L I V E R Y S Y S T E M S W I T H M O R E
T H A N O N E M O D I F I C A T I O N
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
ability to form nonbilayer phases and promote
destabiliza-tion of the bilayer of the endosome membrane DOPE is
added to cationic liposomes and other delivery systems to
control endosome rupture Biodegradable pH-sensitive
surfactant (BPS) typically has a lysosomotropic head
(pKa 5–7) and a hydrophobic tail [20] BPS is stable at
alkaline conditions However, in the acid environment of
the endosome, as the pH value decreases, BPS will be
ionized and will destabilize the endosome membrane
Therefore, BPS is an ideal strategy of controlling endosomal
release
The nuclear membrane of eukaryotic cells is a barrier for
chemicals more than 9 nm (for macromolecules greater
than 70 kDa) [42] Nuclear signal peptides can be
irrevers-ibly linked to one end of oligonucleotides, forming oligo-nucleotide–peptide conjugate [43,44] The nuclear signal peptide allows effective transfection with minute quantities
of DNA Transfection enhancement (10–1000-fold) as the result of the signal peptide was observed irrespective of the cationic vector or the cell type used Therefore, nuclear signal peptides are a putative strategy for nuclear location and penetration
RDO is also reported to correct point mutations in mitochondria isolated from hepatocytes, indicating that mitochondria have the machinery required for the repair of single-point mutations [45] In order to understand the mechanism of RDO for targeted gene correction and apply them to mitochondrial gene therapy, it is essential to 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]
D E S I G N I N G R D O S P E C I F I C D E L I V E R Y
S Y S T E M
It is possible that a novel RDO specific delivery system, which is both safe and efficient, will be invented in the near future This may be a new polysaccharide-based delivery system or RDO conjugated to a short peptide with a special function Particluar attention should also be given to new encapsulating nanoparticles By these methods, not only transient transfection, but also prolonged and controlled-release transfection may be realized A delivery device especially suitable for RDO transfer is also possible Because RDO has an RNA sequence, molecules that can bind with both RNA and DNA oligonucleotides may be putative complexing agents for RDO delivery
A C K N O W L E D G E M E N T S
The authors would like to thank Dr Xue-Song Wu, and Chang-Mei Liu for suggestions on the manuscript This work is supported by the Chinese High-Technology (863) Program 2001AA217171 and NSFC/ RGC 3991061991.
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