Questioning the Utility of RNA-DNA Chimera in Gene Repair
In principle, we should be glad that Eric Kmiec and his colleagues published in Science's STKE (1) a detailed experimental protocol of their gene repair method (2, 3). However, a careful reading of their contribution raises more doubts about the method. The research published in Science five years ago by Kmiec and his colleagues was said to demonstrate that chimeric RNA-DNA oligonucleotides could correct the mutation responsible for sickle cell anemia with 50% efficiency (4). Such a remarkable result prompted many laboratories to attempt to replicate the research or utilize the method on their own systems. However, if the method worked at all, which it rarely did, the achieved efficiency was usually lower by several orders of magnitude.
Now, in the Science's STKE protocol, we are given crucial information about the method and why it is so important to utilize these expensive chimeric RNA-DNA constructs. In the introduction we are told that the RNA-DNA duplex is more stable than a DNA-DNA duplex and so extends the half-life of the complexes formed between the targeted DNA and the chimeric RNA-DNA oligonucleotides. This logical explanation, however, conflicts with the statement in the section entitled "Transfection with Oligonucleotides and Plasmid DNA" that Kmiec and colleagues have recently demonstrated that classical single-stranded DNA oligonucleotides with a few protective phosphothioate linkages have a "gene repair conversion frequency rivaling that of the RNA/DNA chimera". Indeed, the research cited for that result actually states that single-stranded DNA oligonucleotides are in fact several-fold more efficient (3.7-fold) than the RNA-DNA chimeric constructs (5). If that is the case, it raises the question of why Kmiec and colleagues emphasize the importance of the RNA in their original chimeric constructs. Their own new results show that modified single-stranded DNA oligonucleotides are more effective than the expensive RNA-DNA hybrids. Moreover, the current efficiency of the gene repair by RNA-DNA hybrids, according to Kmiec and colleagues in their recent paper is only 4×10-4 even after several hours of pre-selection permitting multiplification of bacterial cells with the corrected plasmid (5). This efficiency is much lower than the 50% value reported five years ago, but is assuredly much closer to the reality.Andrzej Stasiak
Laboratoire d'Analyse Ultrastructurale,
Université de Lausanne
Lausanne Ch-1015, Switzerland
1. H. Parekh-Olmedo, K. Czymmek, E. B. Kmiec, Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors. Science's STKE (2001), http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/73/pl1.
2. K. R. Thomas, M. R. Capecchi, Recombinant DNA technique and sickle cell anemia research. Science 275, 1404-1405 (1997).
3. A. Stasiak, S. C. West, E. H. Egelman, Sickle cell anemia research and a recombinant DNA technique. Science 277, 460-462 (1997).
4. A. Cole-Strauss, K. Yoon, Y. Xiang, B. C. Byrne, M. C. Rice, J. Gryn, W. K. Holloman, E. B. Kmiec, Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273, 1386-1389 (1996).
5. H. B. Gamper, H. Parekh, M. C. Rice, M. Bruner, H. Youkey, E. B. Kmiec, The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts. Nucl. Acids Res. 28, 4332-4339 (2000).
Dr. Stasiak questions the effectiveness and utility of the oligonucleotide-directed DNA repair phenomenon that we reported several years ago (1). In that publication we reported correction of the mutation responsible for sickle cell anemia with a high efficiency, with the caveat that the frequency of correction was variable. Since that time, we and others have endeavored to establish the generality of the phenomenon in numerous systems and have undertaken an investigation of the genetic and molecular basis of the underlying mechanism. Our laboratory and others have found high variability in the efficiency of correction of numerous target genes. In fact, we described variability in the original Science paper and have sought to understand the basis of this ever since. We do not yet fully understand all the components implicated in variability, but we have identified several contributory factors. These include the purity of the chimera, the repair efficiency of the cell type, the presence of p53 and its mutational state, the sensitivity of the assay system used to detect repair, the efficiency of plasmid transfection and the presence of certain suppressor-like proteins, some of which may function in an anti-recombinatory manner.
Dr. Stasiak states that the method rarely works. I provide a list of published papers and new abstracts from my laboratory and from independent laboratories in which the method has been used with success (1-24). There were nine presentations on the success of the chimeric oligonucleotides at the American Society of Gene Therapy meeting on May 30-June 3, 2001 (and published in Mol. Therapy 3, May 2001). None of these investigators have any association with my lab. Two are most illustrative: i) Tran et al. describe gene correction of an Enhanced Green Fluorescent Protein (EGFP) gene mutation in fibroblasts using the original design and modifications of the original design, achieving 2.4% conversion (23), and ii) Dickson et al. used the original chimera design to correct an apoE2 mutation (20). Tagalakis et al. report consistent correction efficiencies of 30% and higher, using a transformed lymphoblast cells line, the same type of cells used in our original Science paper (1).
Efforts to understand the mechanistic basis of the gene correction activity conferred by the RNA-DNA chimeric molecules by systematic dissection of the structure led us to the unexpected finding that protected single-stranded DNA oligonucleotides were also effective in promoting directed correction. The progression of papers from our lab and other independent labs performing related studies outline similar results with single-stranded DNA molecules (25-35). We agree that the RNA-DNA chimeras are relatively expensive.
Dr. Stasiak suggests that the RNA component might be dispensable. However, our results show that the RNA is important for achieving gene repair when a double stranded vector is used because RNA may potentiate the half-life of the reaction intermediate (27). Based on our published work, RNA strands derived from the original chimera are ineffective when used as single-stranded moieties; however, single-stranded modified DNA molecules can facilitate gene repair (29). We find that the number of modifications to the DNA backbone is important (29). The two methods offer similar rates of repair efficiency. Differences in efficiency may arise from the sequence of the vector used and the cell types used.
The STKE publication provides a simple experimental system for those interested in testing this approach. Thus, our protocol simply allows for researchers to test a different method of gene repair which has been thoroughly tested as robust by several laboratories; that when one method doesn't work for researchers, another method of gene repair is available for researchers to utilize.Eric Kmiec
Director of Genomics Research
Department of Biological Sciences
University of Delaware
and Delaware Biotechnology Institute
Newark, DE 19711
1. A. Cole-Strauss, K. Yoon, Y. Xiang, B. C. Byrne, M. C. Rice, J. Gryn, W. K. Holloman, E. B. Kmiec, Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science 273, 1386-1389 (1996).
2. K. Yoon, A. Cole-Strauss, E. B. Kmiec, Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc. Natl. Acad. Sci. U.S.A. 93, 2071-2076 (1996).
3. B. T. Kren, A. Cole-Strauss, E. B. Kmiec, C. J. Steer, Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25, 1462-1468 (1997).
4. B. T. Kren, P. Bandyopadhyay, C. J. Steer, In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides. Nature Med. 4, 285-290 (1998).
5. P. Bandyopadhyay, B. T. Kren, X. Ma, C. J. Steer, Enhanced gene transfer into HuH-7 cells and primary rat hepatocytes using targeted liposomes and polyethylenimine. Biotechniques 25, 282-292 (1998).
6. V. Alexeev, K. Yoon, Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide. Nat. Biotechnol. 16, 1343-1346 (1998).
7. V. Alexeev, O. Igoucheva, A. Domashenko, G. Cotsarelis, K. Yoon, Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat. Biotechnol. 18, 43-50 (2000).
8. B. T. Kren, B. Parashar, P. Bandyopadhyay, N. R. Chowdhury, J. R. Chowdhury, C. J. Steer, 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. U.S.A. 96, 10349-10354 (1999).
9. L.-W. Lai, H. O'Connor, Y.-H. Lien. Correction of carbonic anyhdrase II mutation in renal tubular cells by chimeric RNA/DNA oligonucleotide. Abstract presented at the 1st Annual Meeting of the American Society of Gene Therapy, Seattle, WA., May, 1998.
10. L.-W. Lai, B. Chau, Y.-H. Lien. In vivo gene targeting in carbonic anhydrase II deficient mice by Chimeric RNA/DNA Oligonucleotides. Abstract presented at the 2nd Annual Meeting of the American Society of Gene Therapy. Washington, DC, June 1999.
11. L.-W. Lai, Y. H. Lien, Homologous recombination based gene therapy. Expt. Nephrol. 7, 11-14 (1999).
12. L.-W. Lai, D. Doty, R. Khan, M. Omeara, Y.-H. Lien. Conversion of a Single Base Pair Mutation in Newborn Carbonic Anhydrase II Deficient Mice by Chimeric RNA/DNA Oligonucleotides. Abstract presented at the 4th Annual Meeting of the American Society of Gene Therapy. Seattle, WA, May, 2001.
13. T. Zhu, D. J. Peterson, L. Tagliani, G. St. Clair, C. L. Baszczynski, B. Bowen, Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 96, 8768-8773 (1999).
14. T. Zhu, K. Mettenburg, D. J. Peterson , L. Tagliani, C. L. Baszczynski, Engineering herbicide- resistant maize using chimeric RNA/DNA oligonucleotides. Nat. Biotechnol. 18, 555-558 (2000).
15. P. R. Beetham, P. B. Kipp, X. L. Sawycky, C. J. Arntzen, G. D. May, A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc. Natl. Acad. Sci. U.S.A. 96, 8774-8778 (1999).
16. O. Igoucheva, A. E. Peritz, D. Levy, K. Yoon, A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system. Gene Ther. 6, 1960-1971 (1999).
17. T. A. Rando, M. H. Disatnik, L. Z. Zhou, Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 97, 5363-5368 (2000).
18. R. J. Bartlett, S. Stockinger, M. M. Denis, W. T. Bartlett, L. Inverardi, T. T. Le, N. thi Man, G. E. Morris, D. J. Bogan, J. Metcalf-Bogan, J. N. Kornegay, In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat. Biotechnol. 18, 615-622 (2000).
19. A. D. Tagalakis, I. R. Graham, D. R. Riddell, J. G. Dickson, J. S. Owen, Gene correction of the Apolipoprotein (Apo) E2 phenotype to wild-type ApoE3 by in situ chimeraplasty. J. Biol. Chem. 276, 13226-13230 (2001).
20. G. Dickson, G. Dickson, A. Tagalakis, I. Graham, J. Owen, Z. Mohri, Gene repair for hyperlipidaemia by in situ chimeraplasty. Mol. Therapy 3, S262 (2001).
21. M. S. Andersen, T. Kurihara, B. T. Kren, H. U. Holst, C. J. Steer, L. Bolund, T. G. Jensen, Repair of mutant LDL-receptor genes by chimeraplast-induced gene correction. Mol. Therapy 3, S271 (2001).
22. A. G. L. Urbano, A. G. Porter, Targeted gene knockout mediated by chimeric RNA/DNA oligonucleotides. Mol. Therapy 3, S402 (2001).
23. N. D. Tran, Q. Jiang, J. F. Engelhardt, Optimization of chimeric RNA/DNA oligonucleotides for targeted correction of single base pair mutations using a GFP recovery assay. Mol. Therapy 3, S27 (2001).
24. P. H. Thorpe, B. J. Stevens, D. J. Porteous, Comparing two strategies for functional gene correction. Abstract presented at the 4th Annual Meeting of the American Society of Gene Therapy. Seattle, WA., May, 2001.
25. A. Cole-Strauss, H. Gamper, W. K. Holloman, M. Munoz, N. Cheng, E. B. Kmiec, Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract. Nucleic Acids Res. 27, 1323-1330 (1999).
26. E. B. Kmiec, Targeted gene repair. Gene Ther. 6, 1-3 (1999).
27. H. B. Gamper Jr., A. Cole-Strauss, R. Metz, H. Parekh, R. Kumar, E. B. Kmiec, A plausible mechanism for gene correction by chimeric oligonucleotides. Biochemistry 39, 5808-5816 (2000).
28. H. B. Gamper, Y. M. Hou, E. B. Kmiec, Evidence for a four-strand exchange catalyzed by the RecA protein. Biochemistry 39, 15272-15281 (2000).
29. H. B. Gamper, H. Parekh, M. C. Rice, M. Bruner, H. Youkey, E. B. Kmiec, 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 (2000).
30. M. C. Rice, G. D. May, P. B. Kipp, H. Parekh, E. B. Kmiec, Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides. Plant Physiol. 123, 427-438 (2000).
31. E. B. Kmiec, in The Encyclopedia of the Human Genome, D. N. Cooper, Ed. (Nature Publishing Group, London, UK), in press.
32. M. C. Rice, K. Czymmek, E. B. Kmiec EB. The potential of nucleic acid repair in functional genomics. Nat. Biotechnol. 19, 321-326 (2001).
33. M. Ikejima, E. Nakajima, A. Watanabe, M. Yamamoto, T. Shimada, Interaction between chimeric RNA/DNA oligonucleotides and double-stranded DNA. Mol. Therapy 3, S363 (2001).
34. B. D. Brown, J. L. Fraser, S. Davey, D. Lillicrap, Targeting a single nucleotide exchange reaction by use of a small oligonucleotide in mammalian and yeast cell free extracts. Mol. Therapy 3, S402 (2001).
35. V. Anand, H. Ma, S. L. Diamond, J. Bennett, Cationic M9 peptide increases nuclear uptake of RNA-DNA oligonucleotides. Mol. Therapy 3, S193 (2001).
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