Abstract
To promote the efficient and safe application of adeno-associated virus (AAV) vectors as a gene transfer tool in the central nervous system (CNS), transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH). No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×109 genomic copies of AAV would be fully cleared from blood after 2 days.
Highlights
Viral vector gene delivery is currently among the most widely used gene transfer tools for gene delivery in the central nervous system (CNS)
This study focused on transduction of the ventromedial nucleus of the hypothalamus (VMH) in the rat brain, which is involved in energy homeostasis [10], fear [11] and female reproductive behavior [12]
A vector containing AAV2 terminal repeats flanking a short hairpin RNA (shRNA) targeting the leptin receptor and an enhanced green fluorescent protein expression cassette was packaged with an AAV1, AAV5 or AAV8 capsid
Summary
Viral vector gene delivery is currently among the most widely used gene transfer tools for gene delivery in the central nervous system (CNS). The inverted terminal repeats (ITRs) flanking the two viral genes rep (replication) and cap (capsid) of the wild type virus are the only elements necessary for virus replication and encapsidation. To design a rAAV vector, rep and cap are replaced by a promoter followed by the gene of interest or short hairpin RNA (shRNA) and subsequently provided in trans from a plasmid without ITRs. For the determination of gene function in distinct areas of the brain it is of importance to optimize the AAV-mediated transfer and expression of genes or shRNAs. For the determination of gene function in distinct areas of the brain it is of importance to optimize the AAV-mediated transfer and expression of genes or shRNAs For this purpose AAV serotypes have been studied for their transduction efficiency in the brain. AAV2/1, AAV2/5 and AAV2/8 have been shown to effectively transduce rat hypothalamus [3], striatum [4,5], hippocampus [4,6,7], substantia nigra [4,6,8] and red nucleus [9]
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