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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.

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Related in: MedlinePlus

Liver specificity of MTTR promoter, RNA processed from anti-HBV pri-miR cassettes, and effect on HBsAg concentration in supernatants of cultured Huh7 cells. (a) Liver-derived Huh7 or kidney-derived HEK293 cells were transfected with 100 ng of pMTTR-FLuc or pCMV-FLuc, which contained the Firefly luciferase reporter gene under control of MTTR or CMV promoters, respectively. In addition the phRL-CMV (Promega) plasmid, constitutively expressing Renilla luciferase, was cotransfected to normalize the Firefly luciferase measurements. Forty-eight hours after transfection cells were harvested and activity of the two reporter genes was determined independently. Mean Firefly to Renilla luciferase activity is presented and error bars indicate the standard error of the mean (n = 4). (b) Northern blot analyses of 30 μg RNA isolated from Huh 7 cells infected with HD Ads at a multiplicity of infection (MOI) of 100 infectious viral particles per cell. The probes used were complementary to the predicted 5, 8, or 9 guide sequences derived from the panel of vectors expressing antiviral pri-miRs from the MTTR promoter. Equal loading of the lanes was verified by stripping and reprobing the blot with a labelled oligonucleotide complementary to U6 small nuclear RNA. Putative guide (G) and precursor (P) bands are indicated. (c) Inhibition of HBV replication in vitro was determined by measuring HBsAg levels using ELISA. Cell culture supernatants were obtained from Huh7 cells that had been transfected with the pCH-9/3091 replication-competent plasmid and then infected with the indicated HD Ads at MOIs of 100, 250, 500, or 1000. Data is expressed as SEM from three replicates. The statistical significance was calculated using a pair-wise comparison according to the Student t-test. P values less than 0.05 (∗) or less than 0.01 (∗∗) were considered statistically significant.
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fig3: Liver specificity of MTTR promoter, RNA processed from anti-HBV pri-miR cassettes, and effect on HBsAg concentration in supernatants of cultured Huh7 cells. (a) Liver-derived Huh7 or kidney-derived HEK293 cells were transfected with 100 ng of pMTTR-FLuc or pCMV-FLuc, which contained the Firefly luciferase reporter gene under control of MTTR or CMV promoters, respectively. In addition the phRL-CMV (Promega) plasmid, constitutively expressing Renilla luciferase, was cotransfected to normalize the Firefly luciferase measurements. Forty-eight hours after transfection cells were harvested and activity of the two reporter genes was determined independently. Mean Firefly to Renilla luciferase activity is presented and error bars indicate the standard error of the mean (n = 4). (b) Northern blot analyses of 30 μg RNA isolated from Huh 7 cells infected with HD Ads at a multiplicity of infection (MOI) of 100 infectious viral particles per cell. The probes used were complementary to the predicted 5, 8, or 9 guide sequences derived from the panel of vectors expressing antiviral pri-miRs from the MTTR promoter. Equal loading of the lanes was verified by stripping and reprobing the blot with a labelled oligonucleotide complementary to U6 small nuclear RNA. Putative guide (G) and precursor (P) bands are indicated. (c) Inhibition of HBV replication in vitro was determined by measuring HBsAg levels using ELISA. Cell culture supernatants were obtained from Huh7 cells that had been transfected with the pCH-9/3091 replication-competent plasmid and then infected with the indicated HD Ads at MOIs of 100, 250, 500, or 1000. Data is expressed as SEM from three replicates. The statistical significance was calculated using a pair-wise comparison according to the Student t-test. P values less than 0.05 (∗) or less than 0.01 (∗∗) were considered statistically significant.

Mentions: Despite the high efficacy and prolonged knockdown of HBV replication by HD Ad HBV pri-miR-31/5-8-9, previous studies have shown that hepatic transgene expression from the CMV promoter is not durable [35, 36]. To address this limitation, HD Ad vectors expressing anti-HBV pri-miRs from a liver-specific MTTR promoter were generated. Initially, liver-specific expression of the MTTR promoter was assessed using a reporter gene assay following transfection of liver-derived (Huh7) and kidney-derived (HEK293) cells (Figure 3(a)). Measurement of Firefly luciferase activity showed reporter gene activity in both cell lines receiving the plasmid containing the constitutively active CMV promoter. However, when cells were transfected with cassettes containing the MTTR transcriptional regulatory element, Firefly luciferase activity was barely detectable in the kidney derived cell line, and activity was significantly higher in the liver-derived cell line. To assess processing of the artificial pri-miRs, RNA isolated from Huh7 cells infected with the MTTR-containing HD Ads at a MOI of 100 infectious viral particles per cell were analyzed by low molecular weight northern blot hybridization (Figure 3(b)). Hybridization using a probe complementary to guide 5 confirmed efficient generation of anti-HBV pri-miRs from each of the MTTR promoter-containing monomeric and trimeric HD Ads. This was evident from the detection of a band of approximately 21 nucleotides in length, which was absent from control cells infected with the empty HD Ad Δ28 vector. Higher molecular weight bands representing unprocessed or partially processed intermediates were present in low amounts. As expected, hybridization to a probe complementary to the guide 8 sequence resulted in detection of a putative guide in RNA samples isolated from cells infected with HD Ad HBV pri-miR-31/5-8-9 but not in HD Ad HBV pri-miR-122/5 or HD Ad HBV pri-miR-31/5. Similarly, the probe detecting guide 9 sequences only detected a band in RNA isolated from cells treated with the trimeric HD Ad vector. The lower concentration of the guide 9 sequence is in accordance with our previous observation that this sequence is the least efficiently processed guide derived from the artificial tricistronic cassette [11]. A band of approximately 21 nucleotides was not detected in RNA samples isolated from cells infected with empty vector (HD Ad Δ28) or uninfected cells (Figure 3(b)).


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)

Liver specificity of MTTR promoter, RNA processed from anti-HBV pri-miR cassettes, and effect on HBsAg concentration in supernatants of cultured Huh7 cells. (a) Liver-derived Huh7 or kidney-derived HEK293 cells were transfected with 100 ng of pMTTR-FLuc or pCMV-FLuc, which contained the Firefly luciferase reporter gene under control of MTTR or CMV promoters, respectively. In addition the phRL-CMV (Promega) plasmid, constitutively expressing Renilla luciferase, was cotransfected to normalize the Firefly luciferase measurements. Forty-eight hours after transfection cells were harvested and activity of the two reporter genes was determined independently. Mean Firefly to Renilla luciferase activity is presented and error bars indicate the standard error of the mean (n = 4). (b) Northern blot analyses of 30 μg RNA isolated from Huh 7 cells infected with HD Ads at a multiplicity of infection (MOI) of 100 infectious viral particles per cell. The probes used were complementary to the predicted 5, 8, or 9 guide sequences derived from the panel of vectors expressing antiviral pri-miRs from the MTTR promoter. Equal loading of the lanes was verified by stripping and reprobing the blot with a labelled oligonucleotide complementary to U6 small nuclear RNA. Putative guide (G) and precursor (P) bands are indicated. (c) Inhibition of HBV replication in vitro was determined by measuring HBsAg levels using ELISA. Cell culture supernatants were obtained from Huh7 cells that had been transfected with the pCH-9/3091 replication-competent plasmid and then infected with the indicated HD Ads at MOIs of 100, 250, 500, or 1000. Data is expressed as SEM from three replicates. The statistical significance was calculated using a pair-wise comparison according to the Student t-test. P values less than 0.05 (∗) or less than 0.01 (∗∗) were considered statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Liver specificity of MTTR promoter, RNA processed from anti-HBV pri-miR cassettes, and effect on HBsAg concentration in supernatants of cultured Huh7 cells. (a) Liver-derived Huh7 or kidney-derived HEK293 cells were transfected with 100 ng of pMTTR-FLuc or pCMV-FLuc, which contained the Firefly luciferase reporter gene under control of MTTR or CMV promoters, respectively. In addition the phRL-CMV (Promega) plasmid, constitutively expressing Renilla luciferase, was cotransfected to normalize the Firefly luciferase measurements. Forty-eight hours after transfection cells were harvested and activity of the two reporter genes was determined independently. Mean Firefly to Renilla luciferase activity is presented and error bars indicate the standard error of the mean (n = 4). (b) Northern blot analyses of 30 μg RNA isolated from Huh 7 cells infected with HD Ads at a multiplicity of infection (MOI) of 100 infectious viral particles per cell. The probes used were complementary to the predicted 5, 8, or 9 guide sequences derived from the panel of vectors expressing antiviral pri-miRs from the MTTR promoter. Equal loading of the lanes was verified by stripping and reprobing the blot with a labelled oligonucleotide complementary to U6 small nuclear RNA. Putative guide (G) and precursor (P) bands are indicated. (c) Inhibition of HBV replication in vitro was determined by measuring HBsAg levels using ELISA. Cell culture supernatants were obtained from Huh7 cells that had been transfected with the pCH-9/3091 replication-competent plasmid and then infected with the indicated HD Ads at MOIs of 100, 250, 500, or 1000. Data is expressed as SEM from three replicates. The statistical significance was calculated using a pair-wise comparison according to the Student t-test. P values less than 0.05 (∗) or less than 0.01 (∗∗) were considered statistically significant.
Mentions: Despite the high efficacy and prolonged knockdown of HBV replication by HD Ad HBV pri-miR-31/5-8-9, previous studies have shown that hepatic transgene expression from the CMV promoter is not durable [35, 36]. To address this limitation, HD Ad vectors expressing anti-HBV pri-miRs from a liver-specific MTTR promoter were generated. Initially, liver-specific expression of the MTTR promoter was assessed using a reporter gene assay following transfection of liver-derived (Huh7) and kidney-derived (HEK293) cells (Figure 3(a)). Measurement of Firefly luciferase activity showed reporter gene activity in both cell lines receiving the plasmid containing the constitutively active CMV promoter. However, when cells were transfected with cassettes containing the MTTR transcriptional regulatory element, Firefly luciferase activity was barely detectable in the kidney derived cell line, and activity was significantly higher in the liver-derived cell line. To assess processing of the artificial pri-miRs, RNA isolated from Huh7 cells infected with the MTTR-containing HD Ads at a MOI of 100 infectious viral particles per cell were analyzed by low molecular weight northern blot hybridization (Figure 3(b)). Hybridization using a probe complementary to guide 5 confirmed efficient generation of anti-HBV pri-miRs from each of the MTTR promoter-containing monomeric and trimeric HD Ads. This was evident from the detection of a band of approximately 21 nucleotides in length, which was absent from control cells infected with the empty HD Ad Δ28 vector. Higher molecular weight bands representing unprocessed or partially processed intermediates were present in low amounts. As expected, hybridization to a probe complementary to the guide 8 sequence resulted in detection of a putative guide in RNA samples isolated from cells infected with HD Ad HBV pri-miR-31/5-8-9 but not in HD Ad HBV pri-miR-122/5 or HD Ad HBV pri-miR-31/5. Similarly, the probe detecting guide 9 sequences only detected a band in RNA isolated from cells treated with the trimeric HD Ad vector. The lower concentration of the guide 9 sequence is in accordance with our previous observation that this sequence is the least efficiently processed guide derived from the artificial tricistronic cassette [11]. A band of approximately 21 nucleotides was not detected in RNA samples isolated from cells infected with empty vector (HD Ad Δ28) or uninfected cells (Figure 3(b)).

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