10. Apéndice
10.5 Apéndice E: Técnicas de recolección de datos, formatos y aplicación
In conclusion, our in detail protocol for the fast and less labour intensive, large-scale production of HC-AdVs offers the opportunity to generate these vectors, even in non- experienced laboratories. The advanced cloning procedure, using rare restriction enzymes makes the insertion of the transgene cassette of interest independent of sophisticated and inefficient homologous recombination processes (section 2.2.1) (He et al., 1998; Luo et al.,
2007). In addition, the helper virus AdNG163R-2 provided a pre-condition for a safe HC-AdV production, as it guarantees low helper virus contamination in the final vector preparation (Ng et al., 2001).
The amplification procedure was adapted to HEK293 based cell line 116, which can be grown in suspension culture. Therefore, HC-AdVs production can be performed in a 3 liter spinner flask system, making the entire process less labour and cost intensive and allows amplification of multiple different HC-AdVs, as 5 pole magnetic stirrers (Belco) are already available (Figure 2.1). Moreover, the protocol for the cesium chloride gradient purification provides useful information about the composition of cesium chloride solutions, specifically optimized for Ad5 purification (Figure 2.2), and of course the ingredients of the dialyze buffer, which is in our case a glycerol based buffer. There are also sucrose-based buffers for dialysis available (Offringa et al., 2005), as they should reduce the probability of precipitates within the final virus preparation; however the coherence of glycerol and precipitates has not been confirmed yet.
For calculation of transducing units a protocol for qRT-PCR is provided, whereby one can also perform Southern Blot analysis (experiment was performed by L. Jager within this study), but quantification of the infectious particles is faster and more precise. We used primer directed against the ITR and the packaging signal, which can be subjected to every generated HC-AdV, independent from the introduced transgene expression cassette. Thus, the calculation of different preparations is better comparable (section 2.2.5).
Furthermore, our study demonstrates that HC-AdV deleted for all viral coding sequences is suitable to deliver shRNAs. These shRNAs were recognized and processed by the RNAi enzymes, which render mature miRNAs (Figure 2.4) that have the potential to reduce transient and pre-existing HBV infection significantly. However, the present study also emphasizes that the design of DNA sequences contained in the vector and virus-host interactions during super infection need to be carefully considered.
The effect of shRNA mediated RNA interference against luciferase lasted for two weeks in vivo (data not shown, experiment done by A. Ehrhardt). A long-term study was not
applicable because luciferase expression levels from the first generation (fg) adenoviral vector fgAdluc decreased to undetectable levels 4 weeks post-injection. Persistence of transgene expression levels from fg adenoviral vectors depends on the promoter which controls transgene expression and the mouse strain (Schiedner et al., 1998; Ehrhardt and Kay 2002). In the present work) luciferase expression was driven by the Simian virus 40 (SV40) promoter. However, tissue specific promoters may result in stabilized transgene expression levels with a reduced toxicity profile (Schiedner et al., 1998; Ehrhardt and Kay 2002). Thus, future studies may investigate a HC-AdV with a liver specific promoter driving luciferase expression or transgenic mice for hepatic luciferase expression. With respect to long-term suppression of luciferase expression or therapeutically relevant target transcripts (e.g. viral transcription units or endogenous genes), shRNA may be delivered with recently developed HC-AdV for somatic integration (Ehrhardt et al., 2007). These vectors were shown to result in stabilized transgene expression levels even during rapid cell cycling in vivo (Ehrhardt et al., 2007).
We observed a transient increase in serum HBsAg levels shortly after HC-AdV administration the transgenic mouse model for HBV infection (Figure 2.5B). This was in contrast to studies performed by Uprichard and Carmona (Uprichard et al., 2005; Carmona
et al., 2006). After adenoviral injection there was no transient increase in HBsAg antigen levels. One explanation for these observed differences might be the fact that the two latter studies utilized fg adenoviral vectors. We speculate that the remaining adenoviral genes in fg adenoviral vectors and their gene products in contrast to HC-AdV may have an additional inhibitory effect on HBV infection. In fact, it was demonstrated that inflammatory cytokine induction after systemic administration of HBV transgenic mice with an fg adenoviral vector resulted in reduced HBV replication (Cavanaugh et al., 1998). In contrast to fg adenoviral vectors, HC-AdV lack all viral coding sequences. Thus, immunomodulatory adenoviral gene products are not present after infection with HC-AdV. However, the host response against the incoming adenoviral viral capsid proteins and the subsequent change in the expression profile of the host cells may directly or indirectly result in increased HBsAg levels.
In our first experiment performed in HBV transgenic mice we found that HBsAg levels were transiently increased up to two weeks post-injection of the HC-AdV FTC/HBVU6no.2 (Figure 2.5B). This was in contrast to our second experiment in which we measured a decrease in HBsAg levels shortly post-injection (data not shown, experiment performed by A.Ehrhardt). We speculate that this difference may be related to the HBV viral load of each individual pre- injection of the adenoviral vector. At average HBV transgenic mice used in the first experiment had higher starting HBsAg levels (989 ng/ml, 548 ng/ml, 475 ng/ml, and 365
ng/ml) pre-injection compared to individuals used in the second experiment (103 ng/ml, 323 ng/ml, 276 ng/ml).
Very recently it was reported that injection of high dose of recombinant AAV vectors expressing various short hairpin RNAs caused lethality in mice (Grimm et al., 2006). It was speculated that oversaturation of the shRNA/micro RNA pathways may be responsible. In contrast to superinfection with AAV2/8 vectors expressing the shRNA HBVU6no.2, we only observed one case of lethality in HBV transgenic mice after systemic administration of a high dose of a HC-AdV with the identical shRNA expression cassette (data not shown, experiment was performed by Anja Ehrhardt). However, it is important to point out that the reason for this fatality remains to be investigated and that further studies need to be performed. It was proposed that oversaturation of the micro/short hairpin RNA pathways may be responsible for lethality in AAV8 injected mice. Both, recombinant AAV2/8 and adenoviral vectors have the ability to infect 100% of mouse hepatocytes. However, for sufficient transduction of all mouse hepatocytes using single-stranded AAV2/8 vectors, a high vector dose (7.2 x 1012 viral genomes per mouse) is required. It was demonstrated by Nakai et al. that this dose resulted in up to 1000 vector genome copies per cell (Nakai et al.,
2005). Even for double-stranded AAV2/8 vectors with improved transduction efficiencies, 1x1012 AAV vector genomes per mouse are required to efficiently transduce all mouse
hepatocytes (Grimm et al., 2006). This feature of AAV vectors is in sharp contrast to adenoviral vectors. After transduction of 100% of mouse hepatocytes we only detected up to 12 adenoviral vector genomes per hepatocyte (data not shown, experiment performed by A. Ehrhardt). We believe that this vector genome copy number per cell and subsequent shRNA expression levels were not sufficient to oversaturate the micro RNA/shRNA pathways. To further shed light on the differences between AAV and adenoviral vectors for shRNA delivery against HBV, further studies remain to be performed. One may consider to analyze and to compare additional blood parameters or major factors of the innate immune response. In summary, HC-AdV can be produced at high titers utilizing our detailed protocol. Since helper virus contamination is only negligible, final vector preparation can be used in small and large animal models and in the future potentially also in the clinics. We believe that HC- AdV have the potential to mediate potent shRNA mediated gene silencing in vivo but the design of DNA sequences including shRNAs contained in the vector need to be carefully considered. In addition, we believe that it will be essential to further elucidate biological virus-host interactions during super infection with HBV and adenovirus. Moreover, further understanding of the micro RNA/shRNA pathways will be required before pursuing these gene therapy approaches in a clinical setting.