Differential transduction following basal ganglia administration of distinct pseudotyped AAV capsid serotypes in nonhuman primates

Abstract

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson's disease (PD). Six intact young adult cynomolgus monkeys received a single 10 microl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP+ cells within the striatum revealed that AAV2/5-GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP+ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure.

Figure 1

GFP expression in the caudate nucleus after AAV2/1, AAV2/5, or AAV2/8 injection. Low- and high-power photomicrographs through the caudate nucleus of GFP⁺ delivered via (a,b) AAV2/1, (c,d) AAV2/5, and (e,f) AAV2/8 vectors demonstrating the robust transduction of striatal cells for each serotype. Note the intense neuronal and fiber network for all pseudotypes, although the fiber network was slightly diminished with AAV2/5. Bar in e and f represents the magnifications of 1 mm and 100 µm, and applies to a,c,e and b,d,f, respectively. AAV, adeno-associated virus; GFP, green fluorescent protein.

Figure 2

GFP expression in the putamen after AAV2/1, AAV2/5, or AAV2/8 injection. Low- and high-power photomicrographs through the putamen of GFP⁺ delivered via (a,b) AAV2/1, (c,d) AAV2/5, and (e,f) AAV2/8 vectors demonstrating the robust transduction of striatal cells for each pseudotype. Bar in e and f represents the magnifications of 2 mm and 100 µm, and applies to a,c,e and b,d,f, respectively. AAV, adeno-associated virus; GFP, green fluorescent protein.

Figure 3

Quantification of GFP⁺ cells and their distribution volume around the injection sites in caudate and putamen. (a) Stereological estimates of the number of GFP⁺ stained cells 3 months following intrastriatal AAV2/1-GFP, AAV2/5-GFP, and AAV2/8-GFP. One-way analysis of variance F(2, 21) = 3.604, P = 0.047, followed by Bonferroni post hoc test. *P = 0.047. (b) Quantification of GFP⁺ stained cell volume in the striatum 3 months following intrastriatal AAV1-GFP, AAV5-GFP, and AAV-8 GFP administration. *P < 0.05. AAV, adeno-associated virus; GFP, green fluorescent protein.

Figure 4

GFP expression in nigra after intranigral AAV2/1 or AAV2/8 injections. Robust expression of GFP⁺ in the substantia nigra 3 months following (a,b) AAV1-GFP and (c,d) AAV8-GFP. Bar in c and d represents the magnifications of 1 mm and 50 µm. AAV, adeno-associated virus; GFP, green fluorescent protein.

Figure 5

Transduction of neuronal cells by AAV pseudotypes. Confocal images (a,d,g) shows GFP⁺, cells (green color), extensively colocalized with (b,e,h) NeuN (red color) in the striatum as demonstrated by (c,f,i) the merged image (the yellow cells). Bar in (i) represents 100 µm for all panels. AAV, adeno-associated virus; GFP, green fluorescence protein.

Figure 6

Transduction of glial cells by AAV pseudotypes. Confocal images (a,d,g) shows GFP⁺ cells (green color), which rarely colocalized with (b,e,h) GFAP (red color) in the striatum as demonstrated by (c,f,i) the merged image (only a few yellow cells). Bar in (i) represents 100 µm for all panels. AAV, adeno-associated virus; GFP, green fluorescence protein.

Figure 7

Quantification of GFP-NeuN and GFP-GFAP colocalization. Quantification of colocalization of GFP⁺ delivered via each serotype with (a) NeuN and (b) GFAP. AAV, adeno-associated virus; GFP, green fluorescent protein.

Figure 8

Immune response following injections of AAV serotypes in striatum. (a) Low-power photomicrographs illustrating GFP⁺ staining derived from AAV2/1 (caudate) and AAV2/8 (putamen). (b) A modest inflammatory response seen using LN3 staining on an adjacent section. (c,d) High-power photomicrograph of GFP and LN3 staining shows limited immune response with abundant number of GFP⁺ cells. e is a confocal image that demonstrates virally expressed GFP. Note the absence of GFP within the needle tract (*). (f) A dense, but limited plexus, of GFAP positivity astrocytes were observed in the needle tract (*). (g) Note the absence of GFP/GFAP colocalization in the merged image indicating that activated astroglia are not transduced by the viral vectors. Bar in (a,b) represents 2 mm, whereas (c–g) represents 100 µm. AAV, adeno-associated virus; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein.