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.

Keywords: WPRE; adeno-associated viral vector; gene therapy; transgene expression.

Figure 1

(A) Schematic representation of viral vectors. ITR, inverted terminal repeat; CAG, combination promoter with CMV-IE enhancer and chicken beta-actin promoter; RFP, red fluorescent protein; BGH, bovine growth hormone derived polyadenylation site; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element. (B) Identification of AAV8 vectors purified by PCR. Gene elements of RFP and WPRE were amplified by PCR from purified viral templates. 1, AAV8-CAG-RFP vectors; 2, AAV8-CAG-RFP-WPRE vectors; neg, negative controls without template.

Figure 2

Enhancement of RFP expression by WPRE in vitro. (A) HEK 293T, Chang liver, SH-SY5Y and C2C12 cells were transduced with the rAAV8 vectors at a dose of 9 × 10¹⁰ vg per well of a 24-well plate. RFP expression was observed under a fluorescent microscope. (B) Relative MFI was calculated from the absolute MFI measured by the Image-Pro Plus 6.0 software (p-values: *<0.05, **<0.005, ***<0.001).

Figure 3

Biophotonic imaging of RFP in different tissues. AAV8-CAG-RFP [WPRE(-)] and AAV8-RFP-WPRE vectors [WPRE(+)] were injected directly into the liver or muscle of mice, while they were administered in the striatum of the brain in rats. Expression of RFP in tissues injected with saline, standard viruses or viruses containing WPRE is shown. Tissues receiving saline were used as negative controls.

Figure 4

Real-time RT-PCR analysis of RFP mRNA levels in tissues transduced with viral vectors. Relative increases in RFP mRNA levels of AAV8-CAG-RFP and AAV8-CAG-RFP-WPRE vectors are shown (p-values: *<0.05, **<0.005, ***<0.001).