Comparative transduction efficiency of AAV vector serotypes 1-6 in the substantia nigra and striatum of the primate brain

Abstract

Vectors derived from adeno-associated virus (AAV) are promising candidates for neural cell transduction in vivo because they are nonpathogenic and achieve long-term transduction in the central nervous system. AAV serotype 2 (AAV2) is the most widely used AAV vector in clinical trials based largely on its ability to transduce neural cells in the rodent and primate brain. Prior work in rodents suggests that other serotypes might be more efficient; however, a systematic evaluation of vector transduction efficiency has not yet been performed in the primate brain. In this study, AAV viral vectors of serotypes 1-6 with an enhanced green-fluorescent protein (GFP) reporter gene were generated at comparable titers, and injected in equal amounts into the brains of Chlorocebus sabaeus. Vector injections were placed in the substantia nigra (SN) and the caudate nucleus (CD). One month after injection, immunohistochemistry for GFP was performed and the total number of GFP+ cells was calculated using unbiased stereology. AAV5 was the most efficient vector, not only transducing significantly more cells than any other serotype, but also transducing both NeuN+ and glial-fibrillary-acidic protein positive (GFAP+) cells. These results suggest that AAV5 is a more effective vector than AAV2 at delivering potentially therapeutic transgenes to the nigrostriatal system of the primate brain.

Figure 1

Bright-field microscopy of immunohistochemistry for GFP on transduced substantia nigra tissue. Tissue that has been transduced with vectors of each serotype (by column) is shown in progressive magnifications (by row) with boxes in low power images showing the area magnified in subsequent rows. All vectors were able to transduce neural cells to produce the gene product GFP. Bars are 1 mm in the top row, 100 µm in the middle row, and 10 µm in the bottom row. GFP, green-fluorescent protein.

Figure 2

Bright-field microscopy of immunohistochemistry for GFP on transduced caudate tissue. Tissue that has been transduced with vectors of each serotype (by column) is shown in progressive magnifications (by row) with boxes in low power images showing the area magnified in subsequent rows. All vectors were able to transduce neural cells to produce the gene product GFP. Bars are 1 mm in the top row, 100 µm in the middle row, and 10 µm in the bottom row. GFP, green-fluorescent protein.

Figure 3

Quantification of vector transduction. (a) The numbers of transduced cells expressing GFP, determined by unbiased stereology, are shown for each vector serotype in the CD and the SN. The number (n) of monkeys studied was as follows: for AAV1, n = 4 CD, n = 4 SN; AAV2, n = 2 CD, n = 6 SN; AAV3, n = 5 CD, n = 5 SN; AAV4, n = 3 CD, n = 3 SN; AAV5, n = 5 CD, n = 6 SN; AAV6, n = 4 CD, n = 3 SN. AAV5 transduced significantly (*) more cells than any other vector type in both CD and SN. (b) The volume of tissue (mm³) containing transduced cells expressing GFP is shown by serotype in CD and SN. AAV1 and AAV5 transduced significantly (*) more tissue volume than other vectors studied. AAV, adeno-associated virus; CD, caudate nucleus; GFP, green-fluorescent protein; SN, substantia nigra.

Figure 4

Confocal microscopy of fluorescence immunohistochemistry for neural phenotypes in tissue transduced by AAV1, AAV2, and AAV5. Tissue was labeled with the antibody to GFP (green), the neuronal marker NeuN (red), and the glial marker GFAP (blue). Merged images are shown in the left column (and again at higher power in the far right column), followed by separated images, and show several yellow cells at yellow arrows indicating NeuN⁺ cells (red) that are also GFP⁺ (green). Blue arrows point to GFAP⁺ cells (blue) that are not GFP⁺, and red arrows indicate NeuN⁺ (red) cells that are not GFP⁺. In the case of AAV5, there are also aqua arrows that indicate GFAP⁺ (blue) cells that are also GFP⁺ (green). Bar = 100 µm. AAV, adeno-associated virus; GFP, green-fluorescent protein; GFAP, glial-fibrillary-acidic protein.