P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA
Conflict of interest: No Introduction and Objectives: INTRODUCTION: Chronic hepatitis B represents a significant global health challenge. Current treatments often fail due to the persistence of HBV DNA as covalently closed circular DNA or integrated into the host genome, leading to viral reactivatio...
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| Language: | English |
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Elsevier
2024-12-01
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| Series: | Annals of Hepatology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1665268124003983 |
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| author | Giovana Angelice Paula Pedro Henrique da Conceição Roque Rafaela de Sousa Moraes Pereira Vanessa Salete de Paula Fábio Luís Monteiro Lima Lendel da Costa Correia Amilcar Tanuri Elen de Souza Mello Livia Villar Melo Elisabeth Lampe Francisco Mello Campello do Amaral Bárbara do Lago Vieira |
| author_facet | Giovana Angelice Paula Pedro Henrique da Conceição Roque Rafaela de Sousa Moraes Pereira Vanessa Salete de Paula Fábio Luís Monteiro Lima Lendel da Costa Correia Amilcar Tanuri Elen de Souza Mello Livia Villar Melo Elisabeth Lampe Francisco Mello Campello do Amaral Bárbara do Lago Vieira |
| author_sort | Giovana Angelice Paula |
| collection | DOAJ |
| description | Conflict of interest: No Introduction and Objectives: INTRODUCTION: Chronic hepatitis B represents a significant global health challenge. Current treatments often fail due to the persistence of HBV DNA as covalently closed circular DNA or integrated into the host genome, leading to viral reactivation. RNA interference (RNAi) emerges as a promising approach for treating chronic hepatitis through post-transcriptional gene silencing. OBJECTIVE: To design and evaluate the efficacy of RNAi lentiviral vectors targeting key HBV proteins (HBsAg, HBcAg, HBeAg) and pre-genomic RNA (pgRNA) for silencing. Patients / Materials and Methods: Silencing vectors were designed to target overlapping open reading frames, allowing simultaneous silencing of multiple viral proteins and pgRNA with a single RNAi construct. Three vector candidates (siHBV-1 to 3) underwent rigorous in silico testing to minimize off-target effects, evaluating stability and secondary structures. Lentiviral vector production was assessed using flow cytometry to detect green fluorescent protein expression in cell culture. Huh7 cells were transfected with 1 μg of purified HBV genotype A circular monomers, followed by infection with the first lentiviral candidate (siHBV-1), targeting S/Pol genes of HBV (108 TU/mL), three days later. Quantification of HBV proteins using chemiluminescence and HBV DNA using quantitative PCR (qPCR) was performed throughout the post-transfection period. Results and Discussion: Effective silencing of HBsAg expression was observed in cells infected with siHBV-1, with undetectable levels from the third day post-infection compared to untreated controls (p<0.002). Furthermore, HBV DNA was undetectable by qPCR, indicating successful silencing of HBV genotype A in vitro. The production of lentiviral candidates siHBV-2 and siHBV-3 is currently under evaluation. Flow cytometry will be used to determine the transduction rates in Huh7 cells once the tests are completed. Future investigations will explore combinations of these three siHBV vectors to optimize HBV silencing. Conclusions: Lentiviral vector-mediated RNAi offers a promising approach for sustained suppression of HBV replication and gene expression, potentially promoting HBV clearance in chronic carriers. |
| format | Article |
| id | doaj-art-2ec56a5f09244b66a9d44a6901930a39 |
| institution | Kabale University |
| issn | 1665-2681 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Annals of Hepatology |
| spelling | doaj-art-2ec56a5f09244b66a9d44a6901930a392024-12-07T08:25:24ZengElsevierAnnals of Hepatology1665-26812024-12-0129101615P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNAGiovana Angelice Paula0Pedro Henrique da Conceição Roque1Rafaela de Sousa Moraes Pereira2Vanessa Salete de Paula3Fábio Luís Monteiro Lima4Lendel da Costa Correia5Amilcar Tanuri6Elen de Souza Mello7Livia Villar Melo8Elisabeth Lampe9Francisco Mello Campello do Amaral10Bárbara do Lago Vieira11Laboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Molecular Virology, Federal University of Rio de Janeiro, Rio de Janeiro, BrazilLaboratory of Molecular Virology, Federal University of Rio de Janeiro, Rio de Janeiro, BrazilLaboratory of Molecular Virology, Federal University of Rio de Janeiro, Rio de Janeiro, BrazilLaboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, BrazilConflict of interest: No Introduction and Objectives: INTRODUCTION: Chronic hepatitis B represents a significant global health challenge. Current treatments often fail due to the persistence of HBV DNA as covalently closed circular DNA or integrated into the host genome, leading to viral reactivation. RNA interference (RNAi) emerges as a promising approach for treating chronic hepatitis through post-transcriptional gene silencing. OBJECTIVE: To design and evaluate the efficacy of RNAi lentiviral vectors targeting key HBV proteins (HBsAg, HBcAg, HBeAg) and pre-genomic RNA (pgRNA) for silencing. Patients / Materials and Methods: Silencing vectors were designed to target overlapping open reading frames, allowing simultaneous silencing of multiple viral proteins and pgRNA with a single RNAi construct. Three vector candidates (siHBV-1 to 3) underwent rigorous in silico testing to minimize off-target effects, evaluating stability and secondary structures. Lentiviral vector production was assessed using flow cytometry to detect green fluorescent protein expression in cell culture. Huh7 cells were transfected with 1 μg of purified HBV genotype A circular monomers, followed by infection with the first lentiviral candidate (siHBV-1), targeting S/Pol genes of HBV (108 TU/mL), three days later. Quantification of HBV proteins using chemiluminescence and HBV DNA using quantitative PCR (qPCR) was performed throughout the post-transfection period. Results and Discussion: Effective silencing of HBsAg expression was observed in cells infected with siHBV-1, with undetectable levels from the third day post-infection compared to untreated controls (p<0.002). Furthermore, HBV DNA was undetectable by qPCR, indicating successful silencing of HBV genotype A in vitro. The production of lentiviral candidates siHBV-2 and siHBV-3 is currently under evaluation. Flow cytometry will be used to determine the transduction rates in Huh7 cells once the tests are completed. Future investigations will explore combinations of these three siHBV vectors to optimize HBV silencing. Conclusions: Lentiviral vector-mediated RNAi offers a promising approach for sustained suppression of HBV replication and gene expression, potentially promoting HBV clearance in chronic carriers.http://www.sciencedirect.com/science/article/pii/S1665268124003983 |
| spellingShingle | Giovana Angelice Paula Pedro Henrique da Conceição Roque Rafaela de Sousa Moraes Pereira Vanessa Salete de Paula Fábio Luís Monteiro Lima Lendel da Costa Correia Amilcar Tanuri Elen de Souza Mello Livia Villar Melo Elisabeth Lampe Francisco Mello Campello do Amaral Bárbara do Lago Vieira P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA Annals of Hepatology |
| title | P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA |
| title_full | P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA |
| title_fullStr | P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA |
| title_full_unstemmed | P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA |
| title_short | P- 1 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS, VIA SMALL INTERFERING RNA |
| title_sort | p 1 development of lentiviral vectors for inhibition of hepatitis b virus via small interfering rna |
| url | http://www.sciencedirect.com/science/article/pii/S1665268124003983 |
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