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 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 ON for gene correction is the chimeric RNAÆDNA oligonucleotide. CHIMERIC RNA Æ DNA OLIGONUCLEOTIDE FOR TARGETED GENE CORRECTION 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 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; 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) Eur. J. Biochem. 269, 5753–5758 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03299.x RDO 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. 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]. 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 AfiCatSer365offactorIXgeneinrat 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. 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 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. THE MAJOR DELIVERY BARRIERS AND PROBLEMS FACING RDO TRANSFER 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. FUTURE DIRECTIONS: OPTIMIZING RDO DELIVERY SYSTEMS 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. Ó FEBS 2002 Optimizing RNAÆDNA oligonucleotide delivery systems (Eur. J. Biochem. 269) 5755 The 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 nanospheres 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. 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 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 (pK a 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]. 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 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. ACKNOWLEDGEMENTS 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. 5756 L. Liang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 REFERENCES 1. Richardson, P.D., Kren, B.T. & Steer, C.J. (2001) Targeted gene correction strategies. Curr. Opin. Mol. Ther. 3, 1464–8431. 2. Ye, S., Cole-Strauss, A.C., Frank, B. & Kmiec, E.B. (1998) Tar- geted gene correction: a new strategy for molecular medicine. Mol. Med. Today 4, 431–437. 3. Inoue, H., Hayase, Y., Iwai, S. & Ohtsuka, E. (1987) Sequence- dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H. FEBS Lett. 215, 327–330. 4. Monia,B.P.,Lesnik,E.A.,Gonzalez,C.,Lima,W.F.,McGee,D., Guinosso, C.J., Kawasaki, A.M., Cook, P.D. & Freier, S.M. (1993) Evaluation of 2¢-modified oligonucleotides containing 2¢-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268, 14514–14522. 5. Yoon, K., Cole-Strauss, A. & Kmiec, E.B. (1996) Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc. Natl Acad. Sci. USA 93, 2071–2076. 6. Lai, L.W. & Lien, Y.H. (2002) Chimeric RNA/DNA oligonucle- otide-based gene therapy. Kidney Int. 61, 47–51. 7. Parekh-Olmedo, H., Czymmek, K. & Kmiec, E.B. (2001) Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors. Sci. STKE 13, PL1. 8. Lai, L.W. & Lien, Y.H. (2001) Therapeutic application of chimeric RNA/DNA oligonucleotide based gene therapy. Expert Opin. Biol. Ther. 1, 41–47. 9. Lebedeva, I., Benimetskaya, L., Stein, C.A., & Vilenchik, M. (2000) Cellular delivery of antisense oligonucleotides. Eur. J. Pharm. Biopharm. 50, 101–109. 10. Gamper, H.B., Parekh, H., Rice, M.C., Bruner, M., Youkey, H. & Kmiec, E.B. (2000) The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts. Nucleic Acids Res. 28, 4332–4339. 11. Cole-Strass, A., Yoon, K., Xiang, Y., Byrne, B.C., Rice, M.C., Gryn, J., Holloman, W.K. & Kmiec, E.B. (1996) Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273, 1386–1389. 12. Wu, X.S., Liu, D.P. & Liang, C.C. (2001) Prospects of chimeric RNA-DNA oligonucleotides in gene therapy. J. Biomed. Sci. 8, 439–445. 13. Rice, M.C., Czymmek, K. & Kmiec, E.B. (2001) The potential of nucleic acid repair in functional genomics. Nat Biotechnol. 19, 321–326. 14. Lai, L.W. & Lien, Y.H. (2002) Chimeric RNA/DNA oligonucle- otide-based gene therapy. Kidney Int. 61 (Suppl. 1), 47–51. 15. Bertoni, C. & Rando, T.A. (2002) Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides. Hum. Gene Ther. 13, 707–718. 16. Graham, I.R. & Dickson, G. (2002) Gene repair and mutagenesis mediated by chimeric RNA-DNA oligonucleotides: chimeraplasty for gene therapy and conversion of single nucleotide polymor- phisms (SNPs). Biochim. Biophys. Acta 1587,1–6. 17. Kren, B.T., Chen, Z., Felsheim, R., Roy Chowdhury, N., Roy Chowdhury, J. & Steer, C.J. (2002) Modification of hepatic genomic DNA using RNA/DNA oligonucleotides. Gene Ther. 9, 686–690. 18. Liang, E., Ajmani, P.S. & Hughes, J.A. (1999) Oligonucleotide delivery: a cellular prospective. Pharmazie 54, 559–566. 19. Godbey, W.T. & Mikos, A.G. (2001) Recent progress in gene delivery using non-viral transfer complexes. J. Control Release 72, 115–125. 20. Cheung, C.Y., Murthy, N., Stayton, P.S. & Hoffman, A.S. (2001) A PH-Sensitive Polymer That Enhances Cationic Lipid-Mediated Gene Transfer. Bioconjug. Chem. 12, 906–910. 21. Hughes, M.D., Hussain, M., Nawaz, Q., Sayyed, P. & Akhtar, S. (2001) The cellular delivery of antisense oligonucleotides and ribozymes. Drug Discov. Today 6, 303–315. 22. Li, Z.H., Liu, D.P., Yin, W.X., Guo, Z.C. & Liang, C.C. (2001) Targeted correction of the point mutations of b-thalassemia and targeted muatagenesis of the nucleotide associated with HPFH by RNA/DNA oligonucleotides: potential for b-thalassemia gene therapy. Blood Cells Mol. Dis. 27, 530–538. 23. Godbey, W.T., Wu, K.K., & Mikos, A.G. (1999) Poly (ethyleni- mine) and its role in gene delivery. J. Control Release 60, 149–160. 24. De Semir, D., Petriz, J., Avinyo, A., Larriba, S., Nunes, V., Casals, T., Estivill, X. & Aran, J.M. (2002) Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human air- way epithelial cells. J. Gene Med. 4, 308–322. 25. Bandyopadhyay, P., Ma, X., Linehan-Stieers, C., Kren, B.T. & Steer, C.J. (1999) Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor. J. Biol. Chem. 274, 10163–10172. 26. Kren, B.T., Metz, R., Kumar, R. & Steer, C. (1999) Gene repair using chimeric RNA/DNA oligonucleotides. J. Semin. Liver Dis. 19, 93–104. 27. Kren, B.T., Bandyopadhyay, P. & Steer, C.J. (1998) In vivo site- directed mutagenesis of the factor IX gene by chimeric RNA/ DNA oligonucleotides. Nat. Med. 4, 285–290. 28. Kren, B.T., Parashar, B., Bandyopadhyay, P., Chowdhury, N.R., Chowdhury, J.R. & Steer, C.J. (1999) Correction of the UDP- glucuronosyltransferase gene defect in the gunn rat model of crigler–najjar syndrome type I with a chimeric oligonucleotide. Proc. Natl Acad. Sci. USA 96, 10349–10354. 29. Welz, C., Neuhuber, W., Schreier, H., Repp, R., Rascher, W. & Fahr, A. (2000) Nuclear gene targeting using negatively charged liposomes. Int. J. Pharm. 196, 251–252. 30. Liu,L.,Rice,M.C.&Kmiec,E.B.(2001)In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides. Nucleic Acids Res. 29, 4238–4250. 31. Santiago, F.S., Lowe, H.C., Kavurma, M.M., Chesterman, C.N., Baker, A., Atkins, D.G. & Khachigian, L.M. (1999) New DNA enzyme targeting Egr-1 mRNA inhibits vascular smooth muscle proliferation and regrowth after injury. Nat Med. 5, 1264– 1269. 32. Villa, A.E., Guzman, L.A., Poptic, E.J., Labhasetwar, V., D’Souza, S., Farrell, C.L., Plow, E.F., Levy, R.J., DiCorleto, P.E. & Topol, E.J. (1995) Effects of antisense c-myb oligonucleotides on vascular smooth muscle cell proliferation and response to vessel wall injury. Circ. Res. 76, 505–513. 33. Borchard, G. (2001) Chitosans for gene delivery. Adv. Drug. Deliv. Rev. 52, 145–50. 34. Koping-Hoggard, M., Tubulekas, I., Guan, H., Edwards, K., Nilsson, M., Varum, K.M. & Artursson, P. (2001) Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo. Gene Ther. 8, 1108– 1121. 35. Sato, T., Ishii, T. & Okahata, Y. (2001) In vitro gene delivery mediated by chitosan effect of pH, serum, and molecular mass of chitosan on the transfection efficiency. Biomaterials 22, 2075–2080. 36. Lee, K.Y., Kwon, I.C., Kim, Y.H., Jo, W.H. & Jeong, S.Y. (1998) Preparation of chitosan self- aggregates as a gene delivery system. J. Control Release 51, 213–220. 37. Erbacher, P., Zou, S., Bettinger, T., Steffan, A.M. & Remy, J.S. (1998) Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm. Res. 15, 1332–1339. 38. Bernkop-Schnurch, A. & Kast, C.E. (2001) Chemically modified chitosans as enzyme inhibitors. Adv. Drug Deliv. Rev. 52, 127–137. Ó FEBS 2002 Optimizing RNAÆDNA oligonucleotide delivery systems (Eur. J. Biochem. 269) 5757 39. Lambert, G., Fattal, E. & Couvreur, P. (2001) Nanoparticulate systems for the delivery of antisense oligonucleotides. Adv. Drug. Deliv. Rev. 47, 99–112. 40. Aynie, I., Vauthier, C., Chacun, H., Fattal, E. & Couvreur, P. (1999) Spongelike alginate nanoparticles as a new potential system for the delivery of antisense oligonucleotides. Antisense Nucleic Acid Drug Dev. 9, 301–312. 41. Lambert, G., Fattal, E., Pinto-Alphandary, H., Gulik, A. & Couvreur, P. (2001) Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides. Int. J. Pharm. 214, 13–16. 42. Gorlich, D. & Mattaj, I.W. (1996) Nucleocytoplasmic transport. Science 271, 1513–1518. 43. Aanta, M.A., Belguise-Valladier, P. & Behr, J.P. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl Acad. Sci. USA 96, 91–96. 44. Vacik, J., Dean, B.S., Zimmer, W.E. & Dean, D.A. (1996) Cell- specific nuclear import of plasmid DNA. Gene Ther. 6, 1006–1014. 45. Chen, Z., Felsheim, R., Wong, P., Augustin, L.B., Metz, R., Kren, B.T. & Steer, C.J. (2001) Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide- targeted gene repair. Biochem. Biophys. Res. Commun. 285,188– 194. 46. Weissig, V. & Torchilin, V.P. (2001) Cationic bolasomes with delocalized charge centers as mitochondria-specific DNA delivery systems. Adv.Drug.Deliv.Rev.49, 127–149. 47. Lander,E.S.&Lodish,H.(1990)Mitochondrialdiseases:gene mapping and gene therapy. Cell 61, 925–926. 48. Seibel, P., Trappe, J., Villani, G., Klostock, T., Papa, S. & Reichmann, H. (1995) Transfection of mitochondria: strategy towards a gene therapy of mitochondrial DNA diseases. Nucleic Acids Res. 23, 10–17. 5758 L. Liang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 . 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. with the development of delivery systems can RDOs be used for gene therapy, and successfully applied to functional genomics. Keywords: chimeric RNA DNA oligonucleotides; gene delivery; gene therapy;. ovary cells; DMRIE, dimyristyloxypropyl dimethylhydroxyethylammonium bromide; DOPE, dioleoyl- phosphatidylethanolamine; DOPS, dioleoylphosphatidylserine; DOTAP, dioleoyloxypropyl trimethylammonium