Transduction patterns in the CNS following various routes of AAV-5-mediated gene delivery.

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo.

The efficient control of gene expression in vivo from lentiviral vectors remains technically challenging. To analyze inducible gene expression in a human setting, we generated 'human immune system' (HIS) mice by transplanting newborn BALB/c Rag2(-/-)IL-2Rgamma(c)(-/-) immunodeficient mice with human hematopoietic stem cells transduced with a doxycycline-inducible lentiviral vector. We compared several methods of doxycycline delivery to mice, and could accurately measure doxycycline in vivo using a new sensitive detection assay. Two different lentiviral vector designs with constitutive (TRECMV-V14) or autoregulatory (TREAuto-V14) expression of an optimized reverse tetracycline transactivator were used to transduce human hematopoietic stem cells. After transplantation into immunodeficient mice, we analyzed the expression of the green fluorescent protein (GFP) reporter gene in the human hematopoiesis-derived cells that develop and accumulate in the generated HIS mice. We show efficient inducible GFP expression in adult HIS mice containing TREAuto-V14-transduced human cells, whereas GFP expression is poor with the TRECMV-V14 vector. Multiple cycles of doxycycline exposure in the TREAuto-V14 group result in repeated cycles of GFP expression with no loss of intensity. These findings are of major interest for gene therapy and basic research settings that require inducible gene expression.