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Persistent failures in gene repair Persistent failures in gene repair

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Persistent failures in gene repair - PPT Presentation

71 Gerrit van der Steege Petra HL SchuilengaHut Hendri H PasCharles HCM Buys Hans Scheffer and Marcel F Jonkman Dept of Dermatology University Hospital Groningen Groningen The Nether ID: 940081

rdo rna dna gene rna rdo gene dna chimeric cell 1999 yoon correction 2000 oligonucleotides cells mutation sequence technology

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71 Persistent failures in gene repair Gerrit van der Steege*, Petra H.L. Schuilenga-Hut*, Hendri H. PasCharles H.C.M. Buys, Hans Scheffer, and Marcel F. Jonkman Dept. of Dermatology, University Hospital Groningen, Groningen, The Netherlands Dept. of Medical Genetics, University of Groningen, Groningen, The Netherlands *The authors contributed equally to this study. Published as a correspondence in Nature Biotechnology (2001) 19:305-306 Chapter 5 To the editor: Several recent reports describe the use of chimeric RNA/DNA oligonucleotides(RDOs) to alter DNA sequences. This targeted gene correction strategy, also calledchimeraplasty, initially was shown to change episomal sequences (Yoon et al.,1996), but various examples of altering genomic sequences in both mammalian(Alexeev & Yoon, 1998; Cole et al., 1996; Kren et al., 1998; Kren et al., 1997) andplant cell systems (Beetham et al., 1999; Zhu et al., 1999) have since beendescribed. DNA sequence alterations have also been achieved in nuclear or cell-freeextracts (Cole et al., 1999; Igoucheva et al., 1999). This novel RDO technology holdspromise as a means to correct point mutations in disease genes and would haveseveral advantages over conventional gene therapy strategies relying on geneaddition. Although the number of papers reporting s

uccessful usage of the RDOtechnology is slowly growing, the number of independent groups from which thesestudies derive does not. The basic design of a chimeric oligonucleotide is the same in all studies: double-hairpin folded 68-mers with a chimeric DNA and 2’-O-methyl RNA backbone. Theability to form intramolecular hybrids should protect the RDOs against cellularexonucleases; the RNA residues are methylated, which also prevents degradation.Once transported into the nucleus, the RDO is thought to bind to the DNA target onthe basis of a homology region 25 base pairs in length. It is postulated that thepresence of the RNA residues makes base pairing more effective. Recombinaseactivity may then form intermediate structures, and non-matching base pairs areassumed to attract the mismatch-repair protein machinery. The exact mechanism ofRDO-mediated sequence exchange, however, is still unknown and needs to be Two recent reports describe modifications of the original RDO design and itseffects as measured by in vitro reactions in nuclear extracts (Gamper et al., 2000;Igoucheva et al., 1999). These studies indicate that a mismatching base in the all-DNA strand alone is capable of inducing sequence exchange, whereas a sole Failures in gene repair mismatch in the RNA residue-containing strand is

not. It was also observed that 68-mers only consisting of DNA residues could alter sequences in vitro, whereas thesame constructs failed To investigate the potential of chimeric oligonucleotides in the therapy ofheritable skin diseases, we have studied RDO technology in immortalisedkeratinocytes derived from two patients with epidermolysis bullosa who hadhomozygous mutations in the keratin 14 () and the type XVII collagen geneCOL17A1), respectively (Fig. 1). Both mutations result in absence of thecorresponding proteins. Therefore, our immunofluorescence microscopy-basedassay system, which uses specific monoclonal antibodies for detecting correctedcells, is of very high sensitivity. For both lines, we established efficient transfectionprotocols by testing several transfection agents and monitoring the nuclear uptake offluorescently labelled oligonucleotides by laser-scanning fluorescence microscopy.Over an extended period of time, we carried out several RDO transfection-correctionexperiments with both the keratinocyte cell lines. These also included experimentswith UVB-irradiated cells in an attempt to activate the DNA repair machinery. To date, no mutation corrections have been observed. Attempts to alter thesame epidermolysis bullosa genes in lymphocytes also failed. In addition, eff

orts toreproduce RDO experiments described in the literature, such as -globin inlymphocytes and coagulation factor IX in liver cells, have also been unsuccessful. Inthese latter cases, however, the less sensitive PCR/restriction-fragment-lengthpolymorphism analysis system was used to detect sequence alterations. Chapter 5 Figure 1 Sequences of the genomic targets and the RDOs used in the keratinocyte correctionexperiments. The cell line with the COL17A1 mutation (upper panel) is homozygously deletedfor a GC basepair at position 2342 (GenBank accession no. M91669), leading to absence oftype XVII collagen. The 68-mer C17-RDO sequence is designed to align with the genomicsequence around the mutated position and to re-introduce the deleted base pair. The keratin14 cell line carries a homozygous mutation in the 3’ splice site of intron 1 of KRT14, leading toaberrant splicing and truncated, if any, protein. The K14-RDO should correct the mutatedbase pair. Intronic sequences in the genomic targets are presented in lower case, exonicsequences in higher case. The 2’-O-Methyl-RNA residues in the RDOs are given inlowercase; DNA residues are in higher case. Nevertheless, during a working visit to Kyonggeun Yoon’s laboratory at ThomasJefferson University (Philadelphia, PA), one of us (G. van der St

eege) has obtainedlimited success with a melanocyte cell line derived from an albino mouse and anRDO designed to correct a mutation in the tyrosinase gene. Yoon and colleagues,who are gratefully acknowledged, have successfully applied the RDO technology in C17-RDOCol17A1) delexon 30CCCAGACGGACACCAAGGCCCAAGAGGTTGGTCACGGGTCTGCCTGTGGTTCCGGGTTCTCCAACCAGTGTGCGCG-guccaggacgACGGgucugccuguTT T T TCCCAGACGGACAT K14-RDOKRT14mut 1810 1820 1830 1840 1850 1860 1870TGCGCG-aaagguaggaCGCTaagagugucgT TCGCGC TTTCCATCCTGC Failures in gene repair several cell systems, including this albino melanocyte cell line (Alexeev et al., 2000;Alexeev & Yoon, 1998). The above mentioned correction of the tyrosinase mutationoccurred only once in a particular series of five experiments, as demonstrated bypigmentation of a couple of cells in the culture dish. This success was achieved withan RDO synthesised by Eurogentec (Seraing, Belgium), our regular supplier ofRDOs. This particular experiment thus validated the quality of the RDOs derivedfrom Eurogentec. An unexpectedly high variability of correction frequencies with themelanocyte line has been described but, despite using the ver

y same cell line andRDO, we were in all our attempts thus far unable to reproduce any positive result inour laboratory in Groningen. The reasons for the persistent failure of the RDO technology are unknown.Insufficient quality of the synthesised RDO is unlikely to be the major problem, inview of the tyrosinase correction results. A good RDO quality (e.g., correct synthesislength and purity) is an obvious prerequisite, but poor RDO quality cannot entirelyexplain the lack of success experienced by others and us. It may be that the choiceof keratinocytes as the study system is not optimal. Variation among cell types and alower responsiveness of keratinocytes with respect to RDO-mediated sequencechanges have been described (Santana et al., 1998). However, this does not explainthe failure to be complete, as an ‘all-or-nothing’ principle in this is unlikely. Ourongoing experiments include in vitro reactions using nuclear extracts and thedevelopment of a mutated reporter gene system, enabling sensitive monitoring ofcorrection frequencies in different cell lines and systems. However, preliminaryresults with this latter, sensitive system, used to study episomal correction in CHOcells, also indicate complete failure of the RDO technology. We believe that the persistent failure to implement the

RDO technology isnoteworthy. The complete lack of success hampers further studies and frustrates theusage of this theoretically tempting method. We would like to stress that, despite ourdisappointing experiences, we do not denounce the RDO technology as beinginvalid or objectionable. However, it may be of general concern, that a broadapplication of this technique is still to be awaited, despite the number and the extentof positive reports, especially of some in vivo studies (Bartlett et al., 2000; Kren et Chapter 5 al., 1999; Rando et al., 2000). An international collaboration with free exchange ofresults, cell lines, and RDOs, may not only speed up the elucidation of the stillunknown mechanism behind RDO-mediated sequence change, but also prove (ordisapprove) its applicability. Such a call for a ‘chimeraplasty consortium’ of courseincludes an appeal to ‘the happy few’ who have positive experiences with thistechnology to participate. References Alexeev, V., Igoucheva, O., Domashenko, A., Cotsarelis, G., & Yoon, K. (2000) Localized invivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNAoligonucleotide. Alexeev, V. & Yoon, K. (1998) Stable and inheritable changes in genotype and phenotype ofalbino melanocytes induced by an RNA-DNA oligonucleotide. Nat.Biotec

hnol. 16, 1343. s, M.M., Bartlett, W.T., Inverardi, L., Le, T.T., Man, t.N.,Morris, G.E., Bogan, D.J., Metcalf-Bogan, J., & Kornegay, J.N. (2000) In vivo targetedrepair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNAoligonucleotide. 18, 615. Beetham, P.R., Kipp, P.B., Sawycky, X.L., Arntzen, C.J., & May, G.D. (1999) A tool forfunctional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specificProc.Natl.Acad.Sci. USA 96, 8774. Cole, S.A., Gamper, H., Holloman, W.K., Munoz, M., Cheng, N., & Kmiec, E.B. (1999)Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammaliancell-free extract. 27, 1323. Cole, S.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-DNAoligonucleotide. Science 273, 1386. r, R., & Kmiec, E.B. (2000) A plausiblemechanism for gene correction by chimeric oligonucleotides. Biochemistry 39, 5808. Igoucheva, O., Peritz, A.E., Levy, D., & Yoon, K. (1999) A sequence-specific gene correctionby an RNA-DNA oligonucleotide in mammalian cells characterized by transfection andnuclear extract using a LacZ shuttle system. Gene Ther. 6, 1960. Failures in gene repair Kren, B.T., Bandyopadhyay, P., & St

eer, C.J. (1998) In vivo site-directed mutagenesis of thefactor IX gene by chimeric RNA/DNA oligonucleotides. 4, 285. Kren, B.T., Cole, S.A., Kmiec, E.B., & Steer, C.J. (1997) Targeted nucleotide exchange in thealkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNAoligonucleotide. Hepatology 25, 1462. 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 ratmodel of crigler-najjar syndrome type I with a chimeric oligonucleotide. Proc.Natl.Acad.Sci. 96, 10349. Rando, T.A., Disatnik, M.H., & Zhou, L.Z. (2000) Rescue of dystrophin expression in mdxmouse muscle by RNA/DNA oligonucleotides. Proc.Natl.Acad.Sci. USA 97, 5363. Santana, E., Peritz, A.E., Iyer, S., Uitto, J., & Yoon, K. (1998) Different frequency of genetargeting events by the RNA-DNA oligonucleotide among epithelial cells. 111, 1172. Yoon, K., Cole-Strauss, A., & Kmiec, E.B. (1996) Targeted gene correction of episomal DNAin mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc.Natl.Acad.Sci. 93, 2071. Zhu, T., Peterson, D.J., Tagliani, L., St, C.G., Baszczynski, C.L., & Bowen, B. (1999) Targetedmanipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. 96, 8768. 78