Gene Repair by Means of Trans-splicing: A Delicate Balancing Act between Efficiency and Specificity?
Authors: Alessandro Rusconi
Journal: Molecular Therapy (2001)

One of the most elegant options in therapeutic gene transfer is the possibility of transducing gene fragments that can transiently or permanently correct a resident gene or its RNA. The obvious advantage of these methods resides in the sequence-specificity of the therapeutic intervention. These gene transfer methods are particularly attractive as they do not intrinsically require a strict targeting of the transfer or a stringent regulation of expression of the therapeutic gene fragment. The repair is done on resident targets and therefore the specificity of the therapy is given by the natural expression pattern of the target itself. Furthermore, these types of interventions can be designed to control diseases caused by gain-of-function mutations (that is, to knock out dominant mutations), a situation that is much more difficult to address with other gene transfer strategies. During the past few years we have seen reports on oligonucleotides that can interfere with splicing [1Dunckley M.G. Manoharan M. Villiet P. Eperon I.C. Dickson G. Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.Hum. Mol. Genet. 1998; 7: 1083-1090Crossref PubMed Scopus (208) Google Scholar] or prompt permanent gene correction by mismatch repair [2Igoucheva O. Alexeev V. Yoon K. Targeted gene correction by small single stranded oligonucleotides in mammalian cells.Gene Ther. 2001; 8: 391-399Crossref PubMed Scopus (144) Google Scholar]. In addition, the expression of therapeutic trans-splicing RNA has been proposed. These molecules consist of RNA molecules that can be trans-spliced onto resident RNAs. There are two fundamental strategies to attain this goal. In the first approach, the trans-splicer molecule contains a ribozymic function that performs the splicing reaction on the corresponding target [3Lan N. Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA.Mol. Ther. 2000; 2: 245-255Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. In this approach, the specificity is provided by a relatively short guide sequence that hybridizes with the target RNA along 6 to 10 nucleotides. With such a reduced complexity, it was not surprising to observe a rather large number of undesired splicing events. In the second approach, the pre-trans-splicing molecule (PTM) includes a 5’ region that matches, in antisense orientation, the 3’ splice-acceptor region of a target gene [4Puttaraju M. Jamison S.F. Mansfield S.G. Garcia-Blanco M.A. Mitchell L.G. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.Nat. Biotechnol. 1999; 17: 246-252Crossref PubMed Scopus (154) Google Scholar, 5Kikumori T. Cote G.J. Gagel R.F. Promiscuity of pre-mRNA spliceo-some-mediated trans splicing: a problem for gene therapy?.Hum. Gene Ther. 2001; 12: 1429-1441Crossref PubMed Scopus (23) Google Scholar, 6Puttaraju M. DiPasquale J. Baker C.C. Mitchell L.G. Garcia-Blanco M.A. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.Mol. Ther. 2001; 4: 105-114Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. The trans-splicing reaction is thought to occur after hybridization of the guide sequence of the PTM with the target precursor RNA, by the natural machinery (the spliceosome). In the hybridized region, the target's 3’ splice acceptor is usually masked and it is therefore assumed that the spliceosome will connect the 5’ donor of the target with the alternative acceptor, thus generating a chimeric mRNA whose region beyond the trans-splice point can be designed at will for therapeutic or experimental purposes. The group of Lloyd Mitchell (Intronn LLC, Raleigh, NC) has proposed this technique and shown it to be reasonably efficient (up to 15% trans-splicing in a transient model [4Puttaraju M. Jamison S.F. Mansfield S.G. Garcia-Blanco M.A. Mitchell L.G. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.Nat. Biotechnol. 1999; 17: 246-252Crossref PubMed Scopus (154) Google Scholar]), and, in last month's issue of Molecular Therapy, they reported that the reaction is encouragingly specific (about 50-fold preference over a nonspecific, co-expressed target [6Puttaraju M. DiPasquale J. Baker C.C. Mitchell L.G. Garcia-Blanco M.A. Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.Mol. Ther. 2001; 4: 105-114Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]). In this second paper, however, the authors did not examine whether and how many resident mRNAs would be fortuitously or unspecifically trans-spliced. In an almost simultaneous publication, Robert Gagel and colleagues (Univ. Texas, Houston) [5Kikumori T. Cote G.J. Gagel R.F. Promiscuity of pre-mRNA spliceo-some-mediated trans splicing: a problem for gene therapy?.Hum. Gene Ther. 2001; 12: 1429-1441Crossref PubMed Scopus (23) Google Scholar] have recapitulated the experiments of the Intronn team with another PTM/target set. In their setup, trans-splicing was also documented at reasonable levels. However, a closer analysis of the products generated by 5′-RACE indicated that many resident mRNAs were unspecifically co-trans-spliced. The authors could not yet assess the relative frequency of trans-splicing for each unspecific target. Nevertheless, they observed the phenomenon with three different constructs, and their observations are rather disturbing. There are several reasons that could explain the discrepancy between the two reports. The Texas team invoked the possibility that the difference may be due to the fact that, in the cited earlier work of the Intronn group [4Puttaraju M. Jamison S.F. Mansfield S.G. Garcia-Blanco M.A. Mitchell L.G. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.Nat. Biotechnol. 1999; 17: 246-252Crossref PubMed Scopus (154) Google Scholar], the authors used an in vitro transcribed target rather than a PolII transcript. In last month's Molecular Therapy paper, however, Mitchell and colleagues transiently co-expressed both the target and the PTM, analogous to the work of the Texas group. Another possibility, correctly commented on by the Texas authors in their paper, is that the aberrant trans-splicing may be caused by the huge overexpression of the PTM. This is very plausible; however, a reduction of the expression is not necessarily a good solution, because the trans-splicing reaction requires hybridization between the PTM and the target precursor, and the efficiency of the reaction is thermodynamically dependent on the excess of the PTM. Thus, the dilemma: How much is too much, and how little is too little? In other words, what is the proper expression level that will ensure reasonable efficiency while maintaining a high specificity? A similar dilemma applies to the length and structure of the guide sequence, whereby a long stretch may increase stability, but also may offer alternative interactions. Indeed, the simplest explanation of the difference is that the two teams just used different sequences and that the Intronn team had the luck of using a sequence that does not have too many unspecific partners in the transcriptome or in the artificial challenger used in their experiment. In fact, the overall design of the PTM virtually permits trans-splicing even when the hybridization region covers only a few nucleotides. Because introns are relatively large (which means they offer more nonspecific target material) and because their sequences are riddled with relatively simple motifs, it seems plausible that there would be many nonspecific reactions occurring in artificial trans-splicing. Perhaps a convenient strategy would be to design the guide sequence to interact more with the exon part of the target. However, this is rather laborious, as it requires a redesign of the PTM-encoded exon to escape the hairpin interaction with the guide sequence. Overall this means that the design of trans-splicing molecules will have to rely on pure empirical observations for a long time to come, a situation analogous to the use of antisense oligonucleotides. There is another important aspect that still requires investigation. It is not clear whether the PTM requires a 5’ cap structure, which is added in transcripts starting from PolII promoters. Perhaps it would be even better if the PTM did not possess a 5’ cap because, by default, it would not be recognized as a mRNA and thus would be much less readily captured by the spliceosome unless it was hybridized to a genuine mRNA target. There are several ways to introduce cap-less PTMs in cells: in vitro transcription, expression via PolIII, expression via phage RNA polymerase, and so forth. Only systematic experiments will tell the quantitative advantages and disadvantages of these alternatives. In the discussion section of their manuscript, the Texas team seem very worried about their observation of nonspecific products in trans-splicing and make a strong point about the potential hazard posed by fragmented vectors such as the split AAV systems [5Kikumori T. Cote G.J. Gagel R.F. Promiscuity of pre-mRNA spliceo-some-mediated trans splicing: a problem for gene therapy?.Hum. Gene Ther. 2001; 12: 1429-1441Crossref PubMed Scopus (23) Google Scholar]. Indeed, their preoccupation holds for those therapeutic approaches in which particularly powerful protein domains are encoded by the PTM moiety (for example proto-oncogenic sequences, signal transducers, and so on) that could be maintained in the nonspecific fusion products. However, the reality is that splicing is intimately linked to the open reading frame that is defined by upstream exons. This means that there is only one chance in three to obtain an in-frame spliced fusion gene with non-addressed targets. Second, daily laboratory observations strengthen the notion that randomly assembled—and thus unevolved—chimeric proteins are generally much less stable in biological systems than the polypeptides that have passed through evolutionary selection. Therefore, the safety aspect may not be as dire as postulated. Still, the problem persists, and we would all welcome further systematic experiments to assess quantitatively the general validity of the observations of the Texas group and to find the necessary solutions that will bring us out of the empirical era of trans-splicing. Finally, I firmly believe that it would also be extremely important to extend these types of specificity analyses to approaches that use a high excess of oligonucleotides designed for mismatch gene correction, another field from which we may hear more controversial observations in the future.