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. 2011 Sep;22(9):1143-53.
doi: 10.1089/hum.2010.245. Epub 2011 Jun 1.

Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors

Steven J Gray  1 , Stacey B Foti, Joel W Schwartz, Lavanya Bachaboina, Bonnie Taylor-Blake, Jennifer Coleman, Michael D Ehlers, Mark J Zylka, Thomas J McCown, R Jude Samulski
Affiliations

Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors

Steven J Gray et al. Hum Gene Ther. 2011 Sep.

Abstract

With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

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Figures

FIG. 1.
FIG. 1.
CMV, CBh, and CBA promoters demonstrate distinct kinetics in cultured rat hippocampal slices. Rat hippocampal slices were infected with 5×107 VG after 7 days in culture, and GFP expression was monitored for 5 days. The time course of GFP expression was examined for the CMV (A–E), CBh (F–J), and CBA (K–O) promoters at five time points. All images are mapped to the pseudo-color gray wedge depicted in (O). All images are set to equivalent scales with the scale bar in (O) set to 600 μm. All three promoters demonstrated observable fluorescence 24 hr after infection and steadily increased during the 5-day time course (P). The fluorescence intensity for each measurement was normalized to a fluorescent test strip recorded each day. The values are scaled to account for exposure time required for each image. Note: The exposure times were adjusted to ensure all images were acquired within the dynamic range of the detector, and the detector linearity was evaluate each day for the entire time course (P). Color images available online at www.liebertonline.com/hum
FIG. 2.
FIG. 2.
GFP expression remains constant independent of cell size, and the CMV, CBA, and CBh promoters express in a broad range of cells. Rat hippocampal slices were infected with 5×105 VG after 7 days in culture, and GFP expression was monitored after 5 days. (A) Cell size was measured by summating the area for each cell throughout the three-dimensional data set. Individual cells were outlined manually, and the total intensity was measured for each plane. A relative GFP concentration was determined by dividing integrated intensity values by the summated area values. The individual cells were subsequently binned according to summated area to depict the size of these cells. Because of the large range of cell sizes, the individual bins increase in size based on an exponential scale. The CBA, CMV, and CBh promoters all demonstrate a homogeneous GFP concentration independent of the size of the cell. (B–D) Images are z-stack maximal projections of native GFP fluorescence. Representative images of greater than 10 fields are shown. (B) CBA, (C) CBh, (D) CMV. Scale bar (100 μm) is shown in (B).
FIG. 3.
FIG. 3.
In vivo expression of CMV, CBA, and CBh promoters. Adult mice were injected with equal titers of scAAV9/GFP under the control of the CMV (A–C), CBA (D–F), or CBh (G–I) promoter, and then were killed 4 weeks postinjection to assess GFP-positive cells. (A), (D), and (G) show the hippocampus (bregma, −2.0). (B), (E), and (H) show the striatum (bregma, −2.0), with the insets showing an additional ×5 magnification. (C), (F), and (I) show the ventral horn of the cervical spinal cord. Scale bars: 200 μm. All images are representative of at least three mice.
FIG. 4.
FIG. 4.
Three- and 10-week expression profiles of the CBA, CMV, and CBh promoters in the spinal cord after intrathecal injection. Mice received a lumbar puncture injection of 1.25×109 VG of scAAV9/GFP, and then they were killed 3 or 10 weeks postinjection to assess GFP-positive cells. Rows A and B illustrate spinal cords subjected to anti-GFP immunohistochemistry. Rows C and D show representative confocal coimmunofluorescence (co-IF) images using anti-GFP (green), anti-substance P (blue), and anti-GS-IB4 (red). For each row, the left panel shows the CMV promoter, the middle panel shows the CBA promoter, and the right panel shows the CBh promoter. Row A, lumbar spinal cord at 3 weeks postinjection; row B, lumbar spinal cord at 10 weeks postinjection (CBA was not tested at this time point); row C, lumbar DRG at 3 weeks postinjection; row D, lumbar DRG at 10 weeks postinjection (CBA was not tested at this time point, and the 10-week CBh DRG image [bottom right] was not stained with anti-substance P). Scale bars: 200 μm. Images are representative of at least three mice for each variable.
FIG. 5.
FIG. 5.
In vivo expression of the MeP promoter at 4 weeks. Adult mice were injected intravenously with 2×1011 VG of scAAV9/MeP-GFP, and then killed 4 weeks postinjection to assess GFP-positive cells. (A–C) IHC using an anti-GFP antibody in the hippocampus (A), striatum (B), and spinal cord (C). The inset in (B) shows an additional ×5 magnification. Representative confocal images showing co-IF with the indicated antibodies in the hippocampus and spinal cord are provided as Supplemental Figure 1. Scale bars: 200 μm.
FIG. 6.
FIG. 6.
In vivo expression of the MeP promoter at 14 weeks in the CNS. Adult mice were injected intravenously with 2×1011 VG of scAAV9/MeP-GFP, and then killed 14 weeks postinjection to assess GFP-positive cells in the CNS. (A–C) IHC using an anti-GFP antibody in the hippocampus (A), striatum (B), and spinal cord (C). The inset in (B) shows an additional ×5 magnification. Rows D–F show alternate hippocampal (D), striatal (E), and spinal cord (F) sections prepared in parallel with no primary antibody. Rows G–I are representative confocal images showing co-IF with GFP and NeuN antibodies in the hippocampus, striatum, and spinal cord, respectively. Scale bars: 200 μm.
FIG. 7.
FIG. 7.
In vivo expression of the MeP promoter at 14 weeks in peripheral organs. Adult mice shown in Fig. 6 were also assessed for GFP expression in the liver (A and E), heart (B and F), kidney (C and G), and spleen (D and H). Panels A–D show IF using an anti-GFP antibody, and panels E–H show neighboring sections prepared in parallel with no primary antibody. Color images available online at www.liebertonline.com/hum
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