scholarly journals The WPRE improves genetic engineering with site-specific nucleases

2017 ◽  
Author(s):  
Jessica M. Ong ◽  
Christopher R. Brown ◽  
Matthew C. Mendel ◽  
Gregory J. Cost
Keyword(s):  
Genetic Engineering ◽  
Mouse Liver ◽  
Hepatitis Virus ◽  
Transcriptional Response ◽  
Fold Increase ◽  
Transformed Cells ◽  
Woodchuck Hepatitis Virus ◽  
Site Specific ◽  
Human And Mouse

AbstractInclusion of the woodchuck hepatitis virus post-transcriptional response element (WPRE) in the 3’ UTR of mRNA encoding zinc-finger or TALE nucleases results in up to a fifty-fold increase in nuclease expression and a several-fold increase in nuclease-modified chromosomes. Significantly, this increase is additive with the enhancement generated by transient hypothermic shock. The WPRE-mediated improvement is seen across several types of human and mouse primary and transformed cells and is translatablein vivoto the mouse liver.

NO IN VIVO EFFECT OF TRISODIUM PHOSPHONOFORMATE ON WOODCHUCK HEPATITIS VIRUS PRODUCTION

2009 ◽  
Vol 90B (1-6) ◽  
pp. 449-451
Author(s):  
Erik Nordenfelt ◽  
Anders Widell ◽  
Bengt GÖran Hansson ◽  
Bengt LÖfgren ◽  
Carsten MÖller-Nielsen ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Virus Production ◽  
Woodchuck Hepatitis Virus

scholarly journals Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

2001 ◽  
Vol 75 (8) ◽  
pp. 3811-3818 ◽  
Author(s):  
Mengji Lu ◽  
Gero Hilken ◽  
Dongliang Yang ◽  
Thekla Kemper ◽  
Michael Roggendorf
Keyword(s):  
Hepatitis Virus ◽  
Viral Population ◽  
Core Antigen ◽  
Woodchuck Hepatitis Virus ◽  
Deletion Mutants ◽  
Wild Type ◽  
Lymphoproliferative Response ◽  
Hepatocyte Cultures ◽  
Liver Tissues

ABSTRACT Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.

scholarly journals In Vitro and In Vivo Infectivity and Pathogenicity of the Lymphoid Cell-Derived Woodchuck Hepatitis Virus

2001 ◽  
Vol 75 (4) ◽  
pp. 1770-1782 ◽  
Author(s):  
Yuan-Yee Lew ◽  
Tomasz I. Michalak
Keyword(s):  
Lymphoid Cell ◽  
Lymphatic System ◽  
Hepatitis Virus ◽  
Lymphoid Cells ◽  
Woodchuck Hepatitis Virus ◽  
Dna Viruses ◽  
B Virus ◽  
Total Pool

ABSTRACT Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (∼103 virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

scholarly journals In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks.

1997 ◽  
Vol 41 (10) ◽  
pp. 2076-2082 ◽  
Author(s):  
J M Cullen ◽  
S L Smith ◽  
M G Davis ◽  
S E Dunn ◽  
C Botteron ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Treatment Group ◽  
Hepatitis B ◽  
Dna Polymerase ◽  
Hepatitis Virus ◽  
Woodchuck Hepatitis Virus ◽  
Normal Ranges ◽  
B Virus

The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.

scholarly journals In Vivo Effects of Mutations in Woodchuck Hepatitis Virus Enhancer II

1998 ◽  
Vol 72 (8) ◽  
pp. 6608-6613 ◽  
Author(s):  
Yu Wei ◽  
Bud Tennant ◽  
Don Ganem
Keyword(s):  
Hepg2 Cells ◽  
Hepatitis Virus ◽  
Woodchuck Hepatitis Virus ◽  
Viral Mrnas ◽  
In Vivo Effects ◽  
Rna Promoter ◽  
Enhancer Ii ◽  
Viral Reverse Transcription ◽  
Transcription And Replication

ABSTRACT Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-myc2gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.

scholarly journals Immunization with the Gene Expressing Woodchuck Hepatitis Virus Nucleocapsid Protein Fused to Cytotoxic-T-Lymphocyte-Associated Antigen 4 Leads to Enhanced Specific Immune Responses in Mice and Woodchucks

2005 ◽  
Vol 79 (10) ◽  
pp. 6368-6376 ◽  
Author(s):  
Mengji Lu ◽  
Masanori Isogawa ◽  
Yang Xu ◽  
Gero Hilken
Keyword(s):  
Immune Responses ◽  
Antibody Response ◽  
T Lymphocyte ◽  
Nucleocapsid Protein ◽  
Dna Vaccines ◽  
Cytotoxic T Lymphocyte ◽  
Hepatitis Virus ◽  
Woodchuck Hepatitis Virus ◽  
Th Cell

ABSTRACT A number of options are available to modify and improve DNA vaccines. An interesting approach to improve DNA vaccines is to fuse bioactive domains, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to an antigen. Such fusion antigens are expressed in vivo and directed to immune cells by the specific bioactive domain and therefore possess great potential to induce and modulate antigen-specific immune responses. In the present study, we tested this new approach for immunomodulation against hepadnavirus infection in the woodchuck model. Plasmids expressing the nucleocapsid protein (WHcAg) and e antigen (WHeAg) of woodchuck hepatitis virus (WHV) alone or in fusion to the extracellular domain of woodchuck CTLA-4 and CD28 were constructed. Immunizations of mice with plasmids expressing WHcAg or WHeAg led to a specific immunoglobulin G2a (IgG2a)-dominant antibody response. In contrast, fusions of WHcAg to CTLA-4 and CD28 induced a specific antibody response with comparable levels of IgG1 and IgG2a. Furthermore, the specific IgG1 response to WHcAg/WHeAg developed immediately after a single immunization with the CTLA-4-WHcAg fusion. Woodchucks were immunized with plasmids expressing WHeAg or the CTLA-4-WHcAg fusion and subsequently challenged with WHV. CTLA-4-WHcAg showed an improved efficacy in induction of protective immune responses to WHV. In particular, the anti-WHsAg antibody response developed earlier after challenge in woodchucks that received immunizations with CTLA-4-WHcAg, consistent with the hypothesis that anti-WHs response is dependent on a Th cell response to WHcAg. In conclusion, the use of fusion genes represents a generally applicable strategy to improve DNA vaccination.

scholarly journals Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jian-Ping Zhang ◽  
Xin-Xin Cheng ◽  
Mei Zhao ◽  
Guo-Hua Li ◽  
Jing Xu ◽  
...  
Keyword(s):  
Mouse Liver ◽  
Hemophilia A ◽  
Fold Increase ◽  
Therapeutic Effects ◽  
End Joining ◽  
Promising Strategy ◽  
Non Homologous End Joining ◽  
Integration Efficiency

Abstract Background Hemophilia A, a bleeding disorder resulting from F8 mutations, can only be cured by gene therapy. A promising strategy is CRISPR-Cas9-mediated precise insertion of F8 in hepatocytes at highly expressed gene loci, such as albumin (Alb). Unfortunately, the precise in vivo integration efficiency of a long insert is very low (~ 0.1%). Results We report that the use of a double-cut donor leads to a 10- to 20-fold increase in liver editing efficiency, thereby completely reconstituting serum F8 activity in a mouse model of hemophilia A after hydrodynamic injection of Cas9-sgAlb and B domain-deleted (BDD) F8 donor plasmids. We find that the integration of a double-cut donor at the Alb locus in mouse liver is mainly through non-homologous end joining (NHEJ)-mediated knock-in. We then target BDDF8 to multiple sites on introns 11 and 13 and find that NHEJ-mediated insertion of BDDF8 restores hemostasis. Finally, using 3 AAV8 vectors to deliver genome editing components, including Cas9, sgRNA, and BDDF8 donor, we observe the same therapeutic effects. A follow-up of 100 mice over 1 year shows no adverse effects. Conclusions These findings lay the foundation for curing hemophilia A by NHEJ knock-in of BDDF8 at Alb introns after AAV-mediated delivery of editing components.

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