Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain

With a CMV-EGFP cassette flanked by AAV2 ITRs and packaged in AAV1, AAV2, AAV5, AAV6, AAV8 or AAV9 capsids, 80 nl injections (9.6×10^11 viral genomes/ml) into the mouse brain give GFP expression 21 days later that ranks serotypes by brain region. The order is AAV5>AAV9>AAV8>AAV1>AAV6>AAV2 in striatum, AAV9>AAV5>AAV8>AAV1>AAV6>AAV2 in hippocampus and AAV9>AAV8>AAV6>AAV5>AAV1>AAV2 in auditory cortex. AAV2 gives the lowest expression in all three regions, while AAV5, AAV8 and AAV9 give good expression throughout.

AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9 capsids carrying a CMV-EGFP cassette with AAV2 ITRs each drive GFP expression in neurons (NeuN-positive), GABA-positive inhibitory neurons, astrocytes (GFAP-positive), microglia (Iba1-positive) and oligodendrocytes (Olig2-positive) of mouse striatum, hippocampus and auditory cortex. GFP levels in a given cell type nonetheless differ markedly between serotypes.

AAV8 capsids carrying a CMV-EGFP cassette produce higher GFP levels in hippocampal astrocytes than any of AAV1, AAV2, AAV5, AAV6 or AAV9 capsids with the same cassette, including AAV5. Cumulative single-cell fluorescence distributions for GFAP-positive astrocytes from AAV8-injected mice are shifted systematically toward higher values than those from AAV5-injected mice.

AAV9 capsids carrying a CMV-EGFP cassette produce higher GFP levels in NeuN-positive neurons of mouse auditory cortex than AAV1 capsids with the same cassette. AAV9 also gives the highest overall GFP expression in the auditory cortex among AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9.

Injecting AAV1, AAV2, AAV5, AAV6, AAV8 or AAV9 vectors into mouse striatum, hippocampus or auditory cortex does not produce a chronic microglial reaction comparable to acute inflammation. Iba1-positive microglia counts 21 days after injection of any of these serotypes are about three-fold lower than 48 hours after striatal injection of 10 µg lipopolysaccharide, and show only small differences without a systematic increase in the injected hemisphere relative to the contralateral hemisphere. Only this single time point was examined.

AAV5 injected into the mouse dentate gyrus is retrogradely transported along axons to neurons in the ipsilateral lateral entorhinal cortex, and to a lesser extent the contralateral lateral entorhinal cortex, which then express the CMV-EGFP reporter (all 9 injected mice). When AAV5-GFP is co-injected with fluorescent cholera toxin subunit B (CTB), GFP is almost exclusively found in CTB-labeled entorhinal cells, and the GFP-positive cells concentrate in layer 2 of the ventral lateral entorhinal cortex, a subset of the CTB-labeled population.

Among injections of AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9 into mouse striatum, hippocampus and auditory cortex, only AAV5 injected into the dentate gyrus shows retrograde axonal transport, judged by clustered GFP-positive cell bodies more than 500 µm from the injection site in coronal sections up to 1 mm away along the anterior-posterior axis. The transport is highly selective: AAV5-injected mice show no distally labeled cell bodies in other regions with dense projections, such as the reciprocal callosal projections between auditory cortices.

Entorhinal cortex neurons labeled by retrograde AAV5-GFP transport after dentate gyrus injection are excitatory. Nearly all GFP-positive and CTB-positive entorhinal cells co-stain for the neuronal marker NeuN (CTB: 918 cells, GFP: 264 cells, 4 sections from one mouse), and none of 1033 cells counted in a GAD2-IRES-Cre; Ai9 (tdTomato) mouse co-express GFP and tdTomato in GAD2-expressing interneurons.

A self-complementary AAV8 genome (scAAV) expressing H2B-GFP from the human synapsin 1 promoter starts producing detectable fluorescent nuclei in mouse striatal neurons by 3 days after injection (80 nl, 8.1×10^7 genome copies). A single-stranded AAV8 (ssAAV) with the same cassette shows GFP-positive nuclei only at 8 to 21 days, when 2 to 9% of cells in the analyzed region exceed the GFP-expressing threshold. The fraction of expressing cells under scAAV8 rises further at 8 and 21 days.

At all time points, the median fluorescence of transduced cells is higher after striatal injection of self-complementary AAV8 than of single-stranded AAV8 carrying the same human synapsin 1 promoter H2B-GFP cassette. With scAAV8 the number of GFP-expressing cells increases only slightly from 8 to 21 days while the GFP level keeps rising. An H2B-mCherry version of the ssAAV8 and scAAV8 constructs gives similar results, with mCherry visible at 3 days for scAAV8 and at 8 days for ssAAV8.

Deleting the terminal resolution site (trs) of the right AAV2 ITR in a vector with a human synapsin 1 promoter-driven H2B-EGFP or H2B-mCherry cassette yields a self-complementary AAV genome. Packaged in AAV8 capsids, the vector DNA runs at about 4.8 kb on a denaturing alkaline agarose gel, double the 2422 bp (mCherry) and 2431 bp (GFP) bands of the single-stranded versions.

In mouse striatum, pressure injection of 80 nl of AAV5 (CMV-GFP, 9.6×10^11 viral genomes/ml) through a finely tapered glass capillary transduces only the targeted area, with no transduction in surrounding tissue within 100 to 750 µm of the pipette tip, and does not saturate GFP expression. Larger injection volumes increase GFP expression further but also cause extensive transduction outside the striatum.