1099. RNAi-Based Therapy for the Treatment of HCV
Authors: Linda B. Couto, Amy E. Parker, Gabriel Haniff, David A. Suhy, Alexander A. Kolykhalov, Peter W. Roelvink, Luz Maria Garcia, Rosanna Schroeder, Mark A. Kay, Sara M. Cunningham
Journal: Molecular Therapy (2006)

HCV represents a major world-wide health problem and the current therapies are inadequate due to the robust generation of viral escape mutants. RNAi-based therapy is ideally suited for the treatment of HCV, as the virus contains a single-stranded RNA genome, and anti-HCV RNAs can be designed to simultaneously target multiple regions of the genome. Our strategy for development of this anti-HCV therapeutic was to first identify at least three shRNAs that were each capable of silencing HCV. Since a mouse model for HCV is not available, we developed a surrogate assay using firefly luciferase-HCV fusion reporter plasmids. Co-expression of the shRNA and reporter plasmids in mouse liver was accomplished by high pressure tail vein (HPTV) injections. Liver lysates were evaluated biochemically for luciferase expression and the percentage of silencing was assessed. Three shRNAs targeting 5′ and coding regions of the HCV genome were capable of silencing luciferase activity by 80–99%. These shRNAs were used to create a triple expression cassette capable of simultaneously expressing all three shRNAs. When this plasmid was evaluated in vivo using HPTV injections for its ability to silence the corresponding three HCV targets, greater than 90% inhibition of luciferase activity was observed. This triple expression cassette was cloned into an AAV8 vector and evaluated for both safety and efficacy. Efficacy was assessed by injecting the luciferase-HCV fusion plasmids via HPTV into mouse livers two weeks following a low pressure tail vein administration of rAAV, and harvesting livers two days later. Greater than 90% inhibition of the luciferase-HCV fusion protein was observed at doses of 1×1011 or 3×1011 vector genomes (vg)/ mouse. To further improve the efficacy and safety of this approach, we evaluated the use of a self complementary (sc) AAV8 vector to express the three shRNAs. Three doses of scAAV8, from 5×108 − 5×1010 vg/mouse, were used. All three HCV shRNA targets were silenced by 80–99% using the mid and high doses of scAAV8. High levels of gene transfer were observed by Southern blot analysis and expression of all three shRNAs was confirmed using a novel Q-PCR assay that detects both processed and unprocessed shRNAs. At the highest dose of vector, a transient elevation in alanine aminotransferase levels was observed at day 7, but returned to baseline by 1 month post-injection. These results provide encouraging data for the development of an RNAi-based therapy to treat individuals with HCV.Benitec stock holder. HCV represents a major world-wide health problem and the current therapies are inadequate due to the robust generation of viral escape mutants. RNAi-based therapy is ideally suited for the treatment of HCV, as the virus contains a single-stranded RNA genome, and anti-HCV RNAs can be designed to simultaneously target multiple regions of the genome. Our strategy for development of this anti-HCV therapeutic was to first identify at least three shRNAs that were each capable of silencing HCV. Since a mouse model for HCV is not available, we developed a surrogate assay using firefly luciferase-HCV fusion reporter plasmids. Co-expression of the shRNA and reporter plasmids in mouse liver was accomplished by high pressure tail vein (HPTV) injections. Liver lysates were evaluated biochemically for luciferase expression and the percentage of silencing was assessed. Three shRNAs targeting 5′ and coding regions of the HCV genome were capable of silencing luciferase activity by 80–99%. These shRNAs were used to create a triple expression cassette capable of simultaneously expressing all three shRNAs. When this plasmid was evaluated in vivo using HPTV injections for its ability to silence the corresponding three HCV targets, greater than 90% inhibition of luciferase activity was observed. This triple expression cassette was cloned into an AAV8 vector and evaluated for both safety and efficacy. Efficacy was assessed by injecting the luciferase-HCV fusion plasmids via HPTV into mouse livers two weeks following a low pressure tail vein administration of rAAV, and harvesting livers two days later. Greater than 90% inhibition of the luciferase-HCV fusion protein was observed at doses of 1×1011 or 3×1011 vector genomes (vg)/ mouse. To further improve the efficacy and safety of this approach, we evaluated the use of a self complementary (sc) AAV8 vector to express the three shRNAs. Three doses of scAAV8, from 5×108 − 5×1010 vg/mouse, were used. All three HCV shRNA targets were silenced by 80–99% using the mid and high doses of scAAV8. High levels of gene transfer were observed by Southern blot analysis and expression of all three shRNAs was confirmed using a novel Q-PCR assay that detects both processed and unprocessed shRNAs. At the highest dose of vector, a transient elevation in alanine aminotransferase levels was observed at day 7, but returned to baseline by 1 month post-injection. These results provide encouraging data for the development of an RNAi-based therapy to treat individuals with HCV. Benitec stock holder.