Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain

Abstract

Recombinant adeno-associated viruses (rAAVs) can transduce several tissues, including the brain. However, in brain the duration of gene expression in different areas is variable, which has been ascribed to viral (CMV) promoter silencing in some regions over time. We have compared expression of enhanced green fluorescent protein (EGFP) in the nigrostriatal pathway of rats mediated by rAAVs containing the CMV or platelet-derived growth factor-␤ chain (PDGF-␤) promoter. In addition, we studied the effects of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on transgene expression in vivo. The rAAV vectors containing the neuron-specific PDGF-␤ chain promoter transduced significantly more dopaminergic neurons than

Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons

“…Our results show that the 2A sequence does not compromise rAAVmediated transgene expression in vivo for at least 1 month, since EGFP and ␣-synuclein levels in the brain were comparable to those achieved with rAAVs lacking 2A. 52 However, whether or not the 2A peptide may increase immunity against other transgene products, or after gene delivery by a different route or by more immunogenic viral vectors, remains to be addressed.…”

“…38 Finally, the 2A system should be useful to co-express therapeutic genes with a readily detectable reporter gene or cell-surface marker, in order to identify and track transduced cells in vivo, and to determine accurately the titer of transducing units of different types and batches of rAAVs by flow cytometry. 52 A potential drawback of the 2A system may be that the 23 amino acids being added to the C-terminus of the protein encoded upstream of 2A might influence the function or activity of some proteins. However, this has not been observed for proteins and enzymes tested to date, including SOD-1 and EGFP (this study), chloramphenicol acetyltransferase, 37,39 38 puromycin Nacetyl transferase, ␤-glucuronidase and alkaline phosphatase.…”

“…psubPDGFsyn-IRES-EGFP (Figure 1k) was obtained by isolating the syn-IRES-EGFP-WPRE fragment from psubCMV-syn-IRES-EGFP, and subcloning it into psubPDGF. 52 All constructs used in expression analyses contained the woodchuck hepatitis post-transcriptional regulatory element 59 after the second coding sequence and before the SV40 polyadenylation signal. All plasmids were sequenced to confirm sequence identity.…”

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

“…Even so, the NSE promoter has been used in AAV vectors for efficient and long-term expression of GFP in neurons in vivo [19]. A hybrid hCMV/PDGFβ promoter and the PDGFβ promoter alone each showed robust and prolonged neuronal gene expression in vivo [24,32]. The hCMV promoter, used here, mediated expression in astrocytes and neurons for weeks in vitro, although it is known to be subject to transcriptional silencing over the course of months in vivo, probably due to methylation [14,19,22,25].…”

Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

“…Utilizing recombinant adeno-associated viral BDNF/WPRE (rAAV/BDNF/WPRE)-mediated overexpression of BDNF 26,27 near the spinal lumbar dorsal horn after partial nerve injury, we hypothesized that chronic spinal BDNF over-expression would alleviate chronic neuropathic pain. This would provide an alternate method to cell transplants that supply the antinociceptive agent.…”