Journal Article

Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

Nickerson, Michael L., Léger, Adrien, Le Guiner, Caroline, Snyder, Richard O., Ferry, Nicolas, Penaud-Budloo, Magalie, Moullier, Philippe, McGee Im, Kate

2011

Overview

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (i.m.) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2-3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the i.m. or the intravenous (i.v.) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Analysis / Animals / Biology / Bisulfite / Chromatin / Chromatin remodeling / CpG islands / Deoxyribonucleic acid / Dependovirus - genetics / DNA / DNA binding proteins / DNA Methylation / Epigenetic inheritance / Epigenetics / Expression vectors / Gene Expression / Gene regulation / Gene silencing / Gene therapy / Gene transfer / Genes / Genetic engineering / Genetic Vectors - genetics / Genomes / Genomics / Histones / Injection / Intravenous administration / Liver / Liver - metabolism / Macaca fascicularis / Methylation / Muscle, Skeletal - metabolism / Muscles / Musculoskeletal system / Nuclei / Primates / Promoter Regions, Genetic - genetics / Proteins / Rous sarcoma virus - genetics / Sarcoma / Skeletal muscle / Sodium / Sulfites / Tissues / Transcription / Transcription (Genetics) / Transcription factors / Transgenes / Transgenes - genetics / Tumors / Virions / Viruses