Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element

Abstract

Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors.

A tropism-transformed Oncolytic Adenovirus with Dual Capsid Modifications for enhanced Glioblastoma Therapy

“…Female Balb/c mice (18-20 g, Liaoning Changsheng Biotechnology) bearing no tumors were used to analyze the biodistribution of Ad37-knob-exchanged adenoviral vectors. Bio-photonic imaging of virus-mediated RFP expression was detected using an Fx Pro Imaging System (Kodak) 29 at 72 h post virus injection (1 × 10 11 VPs per mouse, intravenously (i.v.)).…”

Recombinant AAV8-mediated intrastriatal gene delivery of CDNF protects rats against methamphetamine neurotoxicity

“…The rAAV packaging method has been reported previously 12. The titers of the rAAV vectors, named AAV8-CDNF and AAV8-RFP, were 2.8 × 10 13 and 1.8× 10 13 vg/mL respectively.…”

Therapeutic efficacy of AAV8-mediated intrastriatal delivery of human cerebral dopamine neurotrophic factor in 6-OHDA-induced parkinsonian rat models with different disease progression

“…The processes of AAV packaging and purification were previously reported [25]. The titers of AAV8-CDNF, AAV8-AADC and AAV8-RFP viral vectors were 2.8 × 10 13 viral genomes/mL (vg/mL), 2.2 × 10 13 vg/mL and 1.8 × 10 13 vg/mL, respectively.…”

“…The cDNA encoding the human CDNF gene, AADC gene or red fluorescent protein ( RFP ) gene was cloned into the pAAV-CAG-MCS-WPRE vector. The processes of AAV packaging and purification were previously reported [ 25 ]. The titers of AAV8-CDNF, AAV8-AADC and AAV8-RFP viral vectors were 2.8 × 10 13 viral genomes/mL (vg/mL), 2.2 × 10 13 vg/mL and 1.8 × 10 13 vg/mL, respectively.…”