Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: Comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors

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Abstract

Recombinant adeno-associated viral vectors (AAVs) are very promising gene transfer tools for the nervous system. We have compared the efficiency of gene expression of seven AAV serotypes in young adult rats following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. AAV serotypes 1–6 and 8 and a lentiviral vector (LV) were used, all encoding green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. AAV vectors were titer matched at 5 × 1011 genomic copies (GC)/ml and 1 μl was injected into the red nucleus. The proportion of transduced neurons in the red nucleus was determined at 1 and 4 weeks post-injection. AAV1 would be the vector of choice if the aim would be to overexpress a transgene at high level for a longer period of time. AAV5 and AAV8 would be the preferred serotype if onset of expression is should be somewhat delayed. The use of lentiviral vectors should be considered when transduction of both glial cells and neurons is required. Serotypes 3 and 4 did not transduce red nucleus neurons. AAV1, AAV6 and LV would be the vectors of choice if the aim of the experiment would be to rapidly express a transgene. The current data are important for the design of experiments that aim to study the effects of transgene products on the regenerative capacity of injured red nucleus neurons.

Introduction

Adeno-associated viral vectors (AAVs) have evolved into one of the most attractive gene delivery systems for various tissues amenable for gene therapy. AAV vectors are of special interest for transduction of the central nervous system because of their ability to efficiently transduce neurons while inducing minimal immune responses in the host brain (Kaplitt et al., 1994, Kaplitt et al., 2007, McCown et al., 1996, Klein et al., 2002, Klein et al., 2008, Peel and Klein, 2000, Ruitenberg et al., 2004, Tenenbaum et al., 2004, Cearley and Wolfe, 2006, Cearley and Wolfe, 2007, Taymans et al., 2007, Tuszynski, 2007, Cearley et al., 2008, Lawlor et al., 2009). To date, the most widely used AAV vector has been AAV serotype 2.

One strategy to change tropism of the AAV vector entails cross packaging of the AAV2 genome in capsids of other AAV serotypes (Grimm et al., 2003, Burger et al., 2004). Different AAV serotypes transduced muscle cells of mice with differential efficiency (Louboutin et al., 2005, Zincarelli et al., 2008). The efficiencies of transduction in skeletal muscle with AAV7 and AAV8 were similar to AAV1 and higher than that seen with AAV2 and AAV5 (Louboutin et al., 2005, Hasbrouck and High, 2008). Various AAV serotypes have also been shown to efficiently and differentially transduce neuronal cells in the brain. For instance, AAV1, AAV5 and AAV8 are able to transduce neuronal cells in the hippocampus, substantia nigra and striatum (Kaplitt et al., 1994, Klein et al., 2002, Klein et al., 2008, Peel and Klein, 2000, Burger et al., 2004, Broekman et al., 2006). Non-neuronal transduction was observed when other serotypes were used, such as rh24 (Lawlor et al., 2009), whereas, hu.32, hu.11, pi.2, hu.48R3, and rh.8 resulted in preferential green fluorescent protein (GFP) expression in astrocytes or oligodendrocytes in the rodent brain (Cearley et al., 2008).

So far, the tropism and transduction properties of AAV serotypes for brain structures that have projections through the spinal cord (e.g. the red nucleus or corticospinal tract) has not been systematically evaluated, even though long lasting transduction of the red nucleus using AAV2 (Ruitenberg et al., 2002) and the red nucleus and corticospinal neurons with AAV5 vectors (Foust et al., 2008) has been documented. The red nucleus (RN) is an important target for gene therapeutic proof of concept studies because this relatively small nucleus in the brainstem contains the cell bodies of the rubrospinal tract (RST) that runs through the lateral white matter of the spinal cord. The RST is a well-defined spinal nerve tract and injury to the RST results in functional deficits of the fore and hind limbs (Kobayashi et al., 1997, Hendriks et al., 2006). The relatively small size of the nucleus makes this structure an interesting candidate for delivery of potential therapeutic genes (Ruitenberg et al., 2002, Ruitenberg et al., 2004, Blits and Bunge, 2006, Kwon et al., 2007). With this in mind, we compared the tropism, transduction efficiency and temporal transgene expression profile of 7 cross-packaged AAV serotypes and lentiviral vectors (LVs) following a single stereotactic injection into the RN (see Fig. 1). The results will facilitate the design and selection of AAV vectors to target the RN and advance the application of viral vectors for spinal cord injury research.

Section snippets

Production of AAV particles

AAV vectors encoding enhanced green fluorescent protein were all generated using the plasmid pTRCGW (Hermens et al., 1999, Blits et al., 2004). This vector contains a transgene expression cassette that carries the human cytomegalovirus (CMV) promoter to drive transgene expression, the transgenic cDNA encoding the marker protein GFP and a downstream SV40 polyadenylation (polyA) signal. Downstream of this sequence, the vector contained the cis-acting woodchuck posttranscriptional regulatory

Transduction efficiencies of AAV serotypes and LV in the red nucleus

Transduction efficiency was determined at 1 week and 1 month post-injection (Fig. 1). GFP staining of coronal sections of the brain stem revealed different expression patterns for the different serotypes at both time points. One week post-injection, serotypes 1, 2 and 6, as well as LV, all directed transgene expression in the red nucleus. The AAV-transduced cells had a neuronal phenotype based on co-localisation of GFP and NeuN expression. In contrast, LV transduced non-neuronal cell types as

Discussion

This study compared the transduction efficiency, temporal expression profile and cellular tropism of seven AAV serotype vectors and LV after a single stereotactic injection of viral vector in the RN of adult rats. We report differential transduction efficiencies, onset of transgene expression and cellular tropism. These observations will facilitate the design and selection of AAV vectors for gene transfer to the rubrospinal nucleus, an important and widely used target structure for studies on

Acknowledgements

We thank Corrinna Burger for supplying AAV vectors for pilot experiments, Eric Timmermans for technical assistance isolating AAV and Jurgen Kleischmidt and James Wilson for generously providing plasmids for cross packaging. These studies were supported by the International Spinal Research Trust (STR 090), SenterNovem, Amsterdam Molecular Therapeutics and the Netherlands Institute for Neuroscience.

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