Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala

Abstract

BackgroundIn recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype’s ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (αCaMKII).ResultsWe determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5′s decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined.ConclusionsFor laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.

Comparison of the efficacy of five adeno-associated virus vectors for transducing dorsal raphé nucleus cells in the mouse

“…These results agree with reports that AAV2/9 or AAV2/rh.10 are superior to AAV 2/2 or AAV 2/1 for transducing striatum (15). AAV2/9 and AAV2/rh.10 have also been found to transduce amygdala and substantia nigra more efficiently than AAV2/5 (7, 12). However, in these studies, AAV2/1 exhibited similar transduction efficiency to that of AAV2/9, reinforcing the likelihood that pseudotype efficacy depends on cell- or region-specific factors.…”

“…The titer, volume, and promoter for the fluorescent tag were the same among all viral vectors, allowing the extent of fluorescent marker expression to be used to determine the transduction efficiency of the different AVV serotypes in the DRN (7). To determine the time point with maximal viral transduction, mice were sacrificed at 15 and 30 days post-injection to collect the brains for immunofluorescence imaging.…”

Increasing the GluN2A/GluN2B Ratio in Neurons of the Mouse Basal and Lateral Amygdala Inhibits the Modification of an Existing Fear Memory Trace

“…Mice were killed by perfusion as described previously (Holehonnur et al, 2014). Briefly, the mice were anesthetized with an overdose of chloral hydrate (250 mg/kg) and then perfused with ϳ100 ml of PBS (1ϫ phosphate buffer, 150 mM NaCl) and then with 10% buffered formalin (Fisher Scientific).…”

“…Anisomycin (Sigma-Aldrich) was solubilized fresh before every behavior experiment at a concentration of 125 g/l in 1 M HCl and adjusted to pH 7.4 using 1 M NaOH (Nader et al, 2000). On the day of reactivation, mice were bilaterally infused with 0.3 l of anisomycin at a rate of 0.2 l/min using a 28 gauge infuser that projected 1 mm below the guide cannula, as described previously (Holehonnur et al, 2014). After infusion, the infusers were left in for an additional minute to allow the drug to diffuse away from the cannula, after which time they were withdrawn, the dummy cannula were inserted into the guide cannula, and the mice were returned to their home cages.…”

Vascular-targeted recombinant adeno-associated viral vectors for the treatment of rare diseases

“…Although other AAV serotypes like AAV9 are known to yield >10 fold higher titers than AAV2, the amounts of recombinant AAV-BR1 that are needed for gene therapy approaches can easily be produced in few large scale preparations. 23 The tropism of our selected vector AAV-BR1 for BBB-associated endothelial cells is unique and well suited for the treatment of neurovascular diseases, which was proven by using AAV-BR1 to normalize many of the severe neurological impairments in a mouse model of incontinentia pigmenti. Still, there are several open questions that are worth to be investigated further.…”