Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain

AbstractThe blood–brain barrier is the main obstacle to efficient delivery of therapeutic reagents, including viral vectors, into the central nervous system (CNS) for treating global CNS diseases. In this study, the effects of mannitol infusions on global brain gene expression of a novel AAV vector were examined after intravenous (iv) or intracisternal injection. Initially, a self-complementary adeno-associated virus serotype 2 vector (scAAV) was compared to traditional single-stranded AAV2 vector for reporter gene expression in the brain of adult mice with or without pretreatment of an iv mannitol infusion. One to two months postinjection, analysis of vector-transduced green fluorescent protein (GFP) expression in the brain revealed that vector delivery to the CNS via iv injection required pretreatment with mannitol. This expression was observed only when scAAV vectors were used. Using these conditions, transgene expression was observed in various neurons and glial cells throughout the brain. The peripherally administered scAAV vectors also transduced the cells in multiple somatic tissues with efficient expression in liver (20–30% of hepatocytes), but was less efficient in other somatic tissues. Intracisternal injection of scAAV vector produced a broad and intense transgene expression in both neurons and glial cells in the CNS of injected mice ranging from the olfactory area to the brain stem and spinal cord. More than 50% of the Purkinje cells in the cerebellum expressed GFP. Intravenous infusion of mannitol before intracisternal injection of the scAAV vector enhanced the dispersion of the vector in the CNS. Further optimization of these steps combining peripheral and intracisternal scAAV gene delivery should facilitate the development of treatments for global CNS diseases, especially diseases involving both the somatic system and the CNS, such as lysosomal storage disorders

A study of alterations in DNA epigenetic modifications (5mC and 5hmC) and gene expression influenced by simulated microgravity in human lymphoblastoid cells

Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based “simulated” microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations

Busy as a bee.

Gift of Dr. Mary Jane Esplen.Piano vocal [instrumentation]C major [key]Moderate swing [tempo]Popular song [form/genre]Bees, flowers ; photograph: Lou Breese [illustration]Publisher's advertisement on back cover [note