Grant: $175,801 - National Institutes of Health - May. 24, 2009
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Award Description: DESCRIPTION (provided by applicant): We have developed an intracellular immunization strategy with a genetically engineered human antibody clone as a novel antiviral therapy against the hepatitis C virus. This antibody clone targets the HCV NS3 protein, which has multiple enzymatic activities (protease, helicase and NTPase) that are crucial for viral genome replication. We have shown that a recombinant human antibody clone reacts with the helicase domain of HCV NS3 and completely inhibits the helicase activity. Intracellular expression of this antibody in either a stable cell line replicating subgenomic RNA, or a transient full-length HCV replication model, reduced both HCV RNA and viral protein expression. During the last review cycle of this application we have developed a mouse xenograft tumor model for HCV replication. We have shown that HCV replication in the subcutaneous tumors in the SCID mice is inhibited by interferon alpha. This provides a reliable animal model for testing intracellular immunization for HCV using the antibody. We also developed methods of encapsulation for plasmid DNA or purified antibodies into nanoparticles by using a biodegradable and FDA approved polymer. In this proposal we will be experimenting with the practical aspects of the intracellular immunization strategy in vitro as well as in vivo using a small animal model for hepatitis C virus. Our focus will be to improve technology for the systemic delivery of nanoparticles carrying the recombinant antibody to liver cells to inhibit HCV replication in a mouse model. Our hypothesis is that intracellular immunization with recombinant antibodies that block the NS3 helicase is an effective strategy for inhibiting hepatitis C virus replication and expression. We propose that encapsulation of the antibody gene into biodegradable nanoparticles will efficiently deliver the antibody gene to hepatocytes and may provide a novel therapeutic strategy for chronic HCV patients who are non-responders to interferon. In Specific Aim 1, we will define the epitope(s) of a recombinant human antibody clone and investigate what proportion of clinical HCV strains are inhibited by the recombinant human antibody clone. In Specific Aim 2, we will investigate the emergence of resistant virus variants that escapes from antibody-mediated inhibition of virus replication in HCV cell culture models. In Specific Aim 3, we will determine whether intracellular expression of recombinant antibody effectively eliminates virus RNA replication using a non-infectious small animal model for hepatitis C. If the experiments proposed in this grant application are successful, then we may have the basis for an alternative antiviral strategy for people with chronic HCV infections who do not respond to current interferon therapy. Public Health Relevance: Chronic hepatitis C virus infection accounts for 27% of liver cancer in the United States. This research proposal intends to develop an intracellular immunization strategy for inhibiting hepatitis C virus. If this project becomes successful then it can lead to a potential therapy to treat chronic hepatitis C infection and prevent liver cancer.
Project Description: First Study: A replication defective adenovirus carrying the heavy and light chain antibody against HCV NS3 helicase gene was prepared to facilitate development of an efficient intracytoplasmic delivery method for the antibody gene to effectively inhibit HCV replication. The success of this intracellular treatment strategy using the recombinant antibody was determined by using subgenomic replicon and a full-length replication model for the HCV JFH-1 clone (HCV 2a). High-level expression of an antibody was achieved in the cytoplasm of Huh-7 cells after infection with the recombinant adenovirus. Western blot analysis revealed that both heavy and light chains of the antibody are accurately processed. Intracellular expression of this recombinant antibody in replicon cell line after adenovirus infection reduced HCV replication, viral RNA levels and virus protein expression. Furthermore, we showed that stable expression of the intracellular antibody in Huh-7.5 did not alter cell growth and viability. The full-length HCV genome replication and the JFH1 virus infection were effectively prevented in Huh-7.5 cell line stably expressing an intracellular helicase antibody. These results suggest that intracellular expression of a human recombinant antibody clone in Huh-7.5 cell line using both viral and non-viral methods inhibit replication as well as infection of highly efficient JFH-1 virus clone.
Infrastructure Description: NA
Jobs Summary: Prime Recipient - Created - Post Doctoral Fellow (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 24, 2009. Help expand these official descriptions using the wiki below.
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