Víctimas restauradoras: “aquellas que de manera responsable consigo

It becomes more and more clear that it is not feasible to deliver a Dicer substrate by a ribozyme cassette. The data obtained in this thesis therefore are in agreement with recent findings in the RNAi field. Future approaches thus have to apply completely new strategies. One remaining option could be the direct processing of two antiparallel short RNAs, resulting in an siRNA duplex. Of course there will be major difficulties with this strategy. First, to generate two short RNAs instead of one hairpin, 4 internal ribozymes instead of 2 have to be introduced into the RABV genome. The two internal ribozymes were already challenging with respect to the recovery of recombinant RABV. Second, the transcription efficiency most likely will be impaired as well by four ribozymes. The relatively low amounts of the “perfectly processed” hairpin HSmm are considered to be due to factors such as premature termination of RABV transcription, degradation of cleaved products and partially malfunction of the ribozymes in context of a RABV mRNA, as discussed above. In a construct

with 4 ribozymes instead of 2, all these problems most probably will accumulate. An option to deal with this could be the delivery of the two strands required for an siRNA duplex by 2 separate RABV vectors, each transcribing and processing one strand by 2 ribozymes. This system will then depend on coinfection with these two RABV vectors.

For both systems delivering siRNA duplexes, by either one or two RABV vectors, it is an important question, weather the ribozyme derived ends will allow the strands to enter RISC. What has been studied so far with modified ends in siRNA is partially controversial. It was shown that conjugated Alexa-488 was tolerated in the sense strand at both ends and in the antisense strand at the 5’-end, but not at the 3’-end (Harborth et al., 2003). In another work, amino-modifications of siRNA ends were tolerated at all positions, but the antisense 5’-end (Chiu and Rana, 2002). A clear advantage arising from the siRNA duplex strategy is that it allows the duplexes with a perfect match, as no mismatches have to be introduced in order to allow processing by the ribozymes.

A variation of the “perfectly processed” hairpin, HSmm might also be an option to circumvent Dicer. The shRNAs I used contain a stem of 21 nt, the sequence of the anti-GFP shRNAs that were approved to function in the stably transduced A549-LR-siGFP cells, as well as with transient transfection the pSUPER-shGFP plasmid. The use of hairpins longer or shorter than 21 nts has been proposed by different groups.

Synthetically synthesized hairpins with a 29-mer stem have been shown to be slightly more efficient than hairpins with a 19-mer stem (Siolas et al., 2005). They are dependent on Dicer cleavage and therefore could not be delivered adequately from our ribozyme cassette. In the same work it was shown that the 19-mers are not cleaved effectively by recombinant human Dicer in vitro. Although being less potent than 29-mers they show a knockdown phenotype and therefore must be incorporated into RISC. In a more recent study this was analyzed further and the existence of a certain group of short shRNAs (sshRNAs) is proposed (Ge et al., 2010b). They find ssRNAs with stems shortened to a minimum of 16 or 17 nts and minimal loops of 2 – 4 nts (dependent on base pairing of the adjacent 5’ and 3’- nt) also not to be processed by Dicer in vitro, but to be as potent as comparable siRNAs in cell culture. The main difference to the 19-mers from the Siolas study, an R-type shRNA, is that Ge et al. also interchanged the position of sense and antisense strand. By comparison of conventional R-type and exchanged L-type sshRNAs the L-type appears to be more efficient. If indeed no - 115 –

Dicer cleavage is involved, this would make sense, because the hairpin still has to be un- wound and if the antisense strand is at the 5’-end, the only end that can be un-winded, it is more likely to enter RISC. Notably, the in vivo-Dicer activity or the presence of another duplex-specific endonuclease cannot be excluded. Also did they observe that certain sshRNAs could dimerize and these dimers then were cleaved by Dicer. This dimerization however was not a prerequisite for knock-down activity. Interestingly they also analyzed diverse modifications of their L-type sshRNAs. Modifications of the phosphate-backbone were tolerated at the 5’-end of the antisense strand as well as the 3’-end of the sense strand. Longer disulfide-containing groups conjugated to the 3’-end of the hairpin were accepted as well, whereas they abolished the knock-down function, when conjugated to the 5’-end (Ge et al., 2010a).

As it was possible to recover recombinant RABV expressing ribozyme-comprising mRNAs in this thesis, another interesting idea is to directly use the ribozymes in order to cleave and thereby destroy target mRNAs. Trans-splicing as the first ribozymatic reaction has been observed for the T. thermophilae group I intron (Been and Cech, 1986; Inoue et al., 1985) and was shown to be useful to specifically repair defect mRNAs (Sullenger and Cech, 1994). For a trans-cleaving HHRz it is in principle possible to target any RNA sequence by Watson- Crick base pairing of flanking sequences (Haseloff and Gerlach, 1988). The same is true for HDVagRz and other ribozymes. Numerous applications in vitro and in vivo have been proposed for ribozymes targeting mostly mRNAs (reviewed in Mastroyiannopoulos et al., 2010; Scherer and Rossi, 2003). Although established more than 10 years before RNAi, the ribozyme technique never gained the similar impact. Reasons might be that the target sites are more dependent on mRNA structure and that no algorithm is available allowing the prediction of a good target sequence. For their identification empirical screens have to be done that are time-consuming, whereas for siRNAs this is not necessary. Also effects on their target genes seem to be rather moderate compared to the potency of RNAi.

In conclusion, whatever construct will be considered to get a new try, in vitro-analyses of Dicer cleavage or ribozyme processing with synthetic RNAs or in vivo-tests for knock-down, after microinjection into the cytoplasm, can be of advantage to evaluate the probability of success.

4.2Improved rescue for rabies virus

Efficient rescue of RABV from cDNA is dependent on the formation of correct genome ends of the T7-pol derived antigenome-like RABV full-length RNA. As we found the 3’-terminal HDV ribozyme to cleave very inefficiently in the first part of this work, the aim was to replace the HDV sequence by the better cleaving SC1 sequence and thereby improving the rescue efficiency. Since SC1 indeed improved the rescue system significantly, we also applied an HHRz to process the 5’-end of the RABV cRNA. Together, the HHRz and the better cleaving SC1 improved the efficiency of RABV rescue by more than 100-fold and made it a faster and more reliable system (Ghanem et al., 2012).