Limits...
Inhibition of hepatitis B virus replication by helper dependent adenoviral vectors expressing artificial anti-HBV pri-miRs from a liver-specific promoter.

Mowa MB, Crowther C, Ely A, Arbuthnot P - Biomed Res Int (2014)

Bottom Line: HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks.We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect.Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

View Article: PubMed Central - PubMed

Affiliation: Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Johannesburg 2050, South Africa.

ABSTRACT
Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

Show MeSH

Related in: MedlinePlus

Anti-HBV pri-miR expression in livers of HBV transgenic mice following treatment with HD Ads expressing artificial mono- or trimeric pri-miRs under control of the CMV or MTTR promoters. Northern blot analyses of 30 μg (a) and 60 μg (b) of RNA isolated from mouse livers 1 week after infection with HD Ads. Blots hybridized with probes complementary to predicted guides 5, 8, or 9 were stripped and reprobed for U6 snRNA to confirm equal loading. (c) Quantification of indicated anti-HBV guide sequences generated from CMV and MTTR promoters was determined using a phosphorimager. Data were normalized to U6 snRNA expression and a representative example is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4066856&req=5

fig4: Anti-HBV pri-miR expression in livers of HBV transgenic mice following treatment with HD Ads expressing artificial mono- or trimeric pri-miRs under control of the CMV or MTTR promoters. Northern blot analyses of 30 μg (a) and 60 μg (b) of RNA isolated from mouse livers 1 week after infection with HD Ads. Blots hybridized with probes complementary to predicted guides 5, 8, or 9 were stripped and reprobed for U6 snRNA to confirm equal loading. (c) Quantification of indicated anti-HBV guide sequences generated from CMV and MTTR promoters was determined using a phosphorimager. Data were normalized to U6 snRNA expression and a representative example is shown.

Mentions: To assess expression in vivo of anti-HBV pri-miRs derived from CMV and MTTR promoters, groups of mice were treated intravenously with 5 × 109 HD Ad particles from each of the panel of HD Ad vectors. Seven days after vector administration, RNA was isolated from the livers and 30 μg (Figure 4(a)) or 60 μg (Figure 4(b)) was analyzed by northern blot hybridization. Detection of putative guide sequences correlated with analysis of RNA isolated from cultured liver-derived cells (Figure 3). Probing for guide 5 showed efficient expression and processing of anti-HBV pri-miRs in livers of animals that had been treated with mono- and trimeric HD Ads containing CMV or MTTR promoters. The guide 8 band was only detectable in RNA extracted from mice that received the HD Ads that contained the pri-miR-31/5-8-9 sequence. However the guide 9 sequence was not detectable in murine liver RNA samples and confirms inefficient processing of this component of the anti-HBV trimer. Production of guide strands from CMV- and MTTR-driven pri-miR cassettes was compared by determining guide strand band intensities following probe hybridization to 30 μg (Figure 4(a)) or 60 μg (Figure 4(b)) of RNA. Northern blot analysis of 30 μg of RNA isolated from cells infected with HD Ad carrying cassettes containing the CMV promoter revealed a very faint band corresponding to the expected guides. Increasing the amount of RNA loaded onto the northern blots to 60 μg improved detection of processed transcripts. Importantly, guide strands from RNA isolated from mice that had been treated with MTTR-containing plasmids were more easily measurable. Ratios of the guide strand to U6 snRNA loading control band intensities were used as an indicator of intrahepatocyte guide RNA concentrations (Figure 4(c)). This analysis revealed pri-miR expression and processing from the MTTR promoter was more efficient than that achieved with the CMV promoter.


Inhibition of hepatitis B virus replication by helper dependent adenoviral vectors expressing artificial anti-HBV pri-miRs from a liver-specific promoter.

Mowa MB, Crowther C, Ely A, Arbuthnot P - Biomed Res Int (2014)

Anti-HBV pri-miR expression in livers of HBV transgenic mice following treatment with HD Ads expressing artificial mono- or trimeric pri-miRs under control of the CMV or MTTR promoters. Northern blot analyses of 30 μg (a) and 60 μg (b) of RNA isolated from mouse livers 1 week after infection with HD Ads. Blots hybridized with probes complementary to predicted guides 5, 8, or 9 were stripped and reprobed for U6 snRNA to confirm equal loading. (c) Quantification of indicated anti-HBV guide sequences generated from CMV and MTTR promoters was determined using a phosphorimager. Data were normalized to U6 snRNA expression and a representative example is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4066856&req=5

fig4: Anti-HBV pri-miR expression in livers of HBV transgenic mice following treatment with HD Ads expressing artificial mono- or trimeric pri-miRs under control of the CMV or MTTR promoters. Northern blot analyses of 30 μg (a) and 60 μg (b) of RNA isolated from mouse livers 1 week after infection with HD Ads. Blots hybridized with probes complementary to predicted guides 5, 8, or 9 were stripped and reprobed for U6 snRNA to confirm equal loading. (c) Quantification of indicated anti-HBV guide sequences generated from CMV and MTTR promoters was determined using a phosphorimager. Data were normalized to U6 snRNA expression and a representative example is shown.
Mentions: To assess expression in vivo of anti-HBV pri-miRs derived from CMV and MTTR promoters, groups of mice were treated intravenously with 5 × 109 HD Ad particles from each of the panel of HD Ad vectors. Seven days after vector administration, RNA was isolated from the livers and 30 μg (Figure 4(a)) or 60 μg (Figure 4(b)) was analyzed by northern blot hybridization. Detection of putative guide sequences correlated with analysis of RNA isolated from cultured liver-derived cells (Figure 3). Probing for guide 5 showed efficient expression and processing of anti-HBV pri-miRs in livers of animals that had been treated with mono- and trimeric HD Ads containing CMV or MTTR promoters. The guide 8 band was only detectable in RNA extracted from mice that received the HD Ads that contained the pri-miR-31/5-8-9 sequence. However the guide 9 sequence was not detectable in murine liver RNA samples and confirms inefficient processing of this component of the anti-HBV trimer. Production of guide strands from CMV- and MTTR-driven pri-miR cassettes was compared by determining guide strand band intensities following probe hybridization to 30 μg (Figure 4(a)) or 60 μg (Figure 4(b)) of RNA. Northern blot analysis of 30 μg of RNA isolated from cells infected with HD Ad carrying cassettes containing the CMV promoter revealed a very faint band corresponding to the expected guides. Increasing the amount of RNA loaded onto the northern blots to 60 μg improved detection of processed transcripts. Importantly, guide strands from RNA isolated from mice that had been treated with MTTR-containing plasmids were more easily measurable. Ratios of the guide strand to U6 snRNA loading control band intensities were used as an indicator of intrahepatocyte guide RNA concentrations (Figure 4(c)). This analysis revealed pri-miR expression and processing from the MTTR promoter was more efficient than that achieved with the CMV promoter.

Bottom Line: HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks.We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect.Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

View Article: PubMed Central - PubMed

Affiliation: Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Private Bag 3, Johannesburg 2050, South Africa.

ABSTRACT
Research on applying RNA interference (RNAi) to counter HBV replication has led to identification of potential therapeutic sequences. However, before clinical application liver-specific expression and efficient delivery of these sequences remain an important objective. We recently reported short-term inhibition of HBV replication in vivo by using helper dependent adenoviral vectors (HD Ads) expressing anti-HBV sequences from a constitutively active cytomegalovirus (CMV) promoter. To develop the use of liver-specific transcription regulatory elements we investigated the utility of the murine transthyretin (MTTR) promoter for expression of anti-HBV primary microRNAs (pri-miRs). HD Ads containing MTTR promoter effected superior expression of anti-HBV pri-miRs in mice compared to HD Ads containing the CMV promoter. MTTR-containing HD Ads resulted in HBV replication knockdown of up to 94% in mice. HD Ads expressing trimeric anti-HBV pri-miRs silenced HBV replication for 5 weeks. We previously showed that the product of the codelivered lacZ gene induces an immune response, and the duration of HBV silencing in vivo is likely to be attenuated by this effect. Nevertheless, expression of anti-HBV pri-miRs from MTTR promoter is well suited to countering HBV replication and development of HD Ads through attenuation of their immunostimulatory effects should advance their clinical utility.

Show MeSH
Related in: MedlinePlus