Promoters and serotypes: targeting of adeno‐associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo

Abstract

The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

“…32,33 Of course, increased transduction and stronger transgene expression from gene therapy applications may not necessarily lead to better outcomes, and there is likely to be a fine balance between therapeutic and detrimental expression levels, especially for genes that encode secretable proteins. [34][35][36] This should also be taken into account when choosing promoters that may increase the specificity of transgene expression [37][38][39] and when designing vector systems that allow the temporal regulation of transgene expression. [40][41][42][43] Although comparable in terms of overall transduction efficiency, the tropism profile between rAAV2/2 and rAAV2/6 was considerably different.…”

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

“…Kugler and colleagues have shown that gene expression from AAV2 or AAV5 can be restricted to neurons in vitro by incorporating the hSYN or CBA promoter or restricted mainly to astrocytes by using the mCMV promoter [18,20,27]. The hSYN promoter also confers neuronal specificity in vivo [20,27].…”

“…Kugler and colleagues have shown that gene expression from AAV2 or AAV5 can be restricted to neurons in vitro by incorporating the hSYN or CBA promoter or restricted mainly to astrocytes by using the mCMV promoter [18,20,27]. The hSYN promoter also confers neuronal specificity in vivo [20,27]. Klein, Meyer and colleagues have demonstrated transduction of cultures of astrocytes, microglia and cortical neurons using AAV2 or AAV8 with the CBA promoter [12,17,18].…”

Laser-evoked synaptic transmission in cultured hippocampal neurons expressing channelrhodopsin-2 delivered by adeno-associated virus

“…Second, long-term expression, from months to years, is achievable. Due to high rate of infectivity, rAAV can be used to introduce multiple genes into the same neurons in pre-selected brain regions (Shevtsova et al, 2005) without epigenetic silencing (Zhu et al, 2007). This broadens the experimental possibilities so that other genes whose products act as biosensors for different signaling systems, such as for calcium (Miyawaki, 2003;Palmer and Tsien, 2006;Wallace et al, 2008) and neurotransmitter release (Miesenbock et al, 1998), could also be introduced into the same neuron using rAAV as the delivery method.…”