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. 2024 May;31(5-6):335-344.
doi: 10.1038/s41434-023-00430-0. Epub 2023 Nov 27.

Engineered compact pan-neuronal promoter from Alphaherpesvirus LAP2 enhances target gene expression in the mouse brain and reduces tropism in the liver

Carola J Maturana  1
Affiliations

Engineered compact pan-neuronal promoter from Alphaherpesvirus LAP2 enhances target gene expression in the mouse brain and reduces tropism in the liver

Carola J Maturana. Gene Ther. 2024 May.

Abstract

Small promoters capable of driving potent neuron-restricted gene expression are required to support successful brain circuitry and clinical gene therapy studies. However, converting large promoters into functional MiniPromoters, which can be used in vectors with limited capacity, remains challenging. In this study, we describe the generation of a novel version of alphaherpesvirus latency-associated promoter 2 (LAP2), which facilitates precise transgene expression exclusively in the neurons of the mouse brain while minimizing undesired targeting in peripheral tissues. Additionally, we aimed to create a compact neural promoter to facilitate packaging of larger transgenes. Our results revealed that MiniLAP2 (278 bp) drives potent transgene expression in all neurons in the mouse brain, with little to no expression in glial cells. In contrast to the native promoter, MiniLAP2 reduced tropism in the spinal cord and liver. No expression was detected in the kidney or skeletal muscle. In summary, we developed a minimal pan-neuronal promoter that drives specific and robust transgene expression in the mouse brain when delivered intravenously via AAV-PHP.eB vector. The use of this novel MiniPromoter may broaden the range of deliverable therapeutics and improve their safety and efficacy by minimizing the potential for off-target effects.

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Conflict of interest statement

The author declares no competing interests.

Figures

Fig. 1
Fig. 1. Bioinformatic design of six MiniPromoters targeting neuron specific.
A Prediction of potential transcription factor binding sites (TFBSs) at the 5’ end of the native LAP2 sequence using TRANSFAC®. The identification of elements potentially regulating neuron expression will allow us to design a series of 5’ deletions (21, 46, 65, 91, 149, and 172 bp) immediately upstream of the TATA box (red box), resulting in six promoter variants (black box). The MiniPromoter variant sizes were 383, 358, 339, 313, 255, and 232 bp. Native mCherry expression in 293 T cells is shown at 3 days post transfection with B LAP2 full-length 404 bp, C MiniLAP2 variant 383 bp, D MiniLAP2 variant 358 bp, E MiniLAP2 variant 339 bp, F MiniLAP2 variant 313 bp, G MiniLAP2 variant 255 bp, H MiniLAP2 variant 232 bp plasmid. Scale bars, 100 µm. I Percentage of 293 T cells expressing mCherry following transfection with LAP2 or various MiniLAP2 variant plasmids at the 3-day time point. MLAP2, MiniLAP2 variant.
Fig. 2
Fig. 2. MiniLAP2 and LAP2 drive transgene expression in mouse brains with equivalent efficiencies.
A The LAP2 sequence was modified upstream (5’ end) of the transcriptional start signal (as indicated by the TATA box). The first 160 base pairs (bps) of LAP2 were removed (as shown in grey), although the CREB-binding motif was preserved. A modified CTCF motif (blue) is added immediately upstream of the CREB site. B LAP2 (404 bp) and MiniLAP2 (278 bp) promoters were individually packaged into ssAAV-PHP.eB vector to drive the transcription of mCherry fluorescent reporter. Packaged AAV vectors were administered by unilateral intravenous injection into the retro-orbital sinus at a dose of 5 × 1011 gc per mouse. Negative control mice were inoculated with buffer without AAV. The brain, spinal cord, liver, kidney, and quadriceps muscles were collected 30 days after inoculation. C Representative mCherry immunofluorescence images of sagittal brain sections documenting transgene expression driven by both LAP2 and MiniLAP2, with no expression observed in negative control mice. Nuclei were counterstained with DAPI (blue). D Representative confocal images show native mCherry immunofluorescence (red) from MiniLAP2 in hippocampus, cortex, and cerebellum. Scale bar, 1 mm.
Fig. 3
Fig. 3. MiniLAP2 drives widespread and potent transgene expression in all regions of the mouse brain.
Representative confocal images documenting anti-mCherry signals (green) in the brain tissue of mice 30 days after inoculation with PHP.eB-MiniLAP2, including A anterior olfactory nucleus, B Cx visual area, C pallidum, D superior colliculus of the midbrain (MB), E inferior colliculus of the MB, F HPC CA1, G HPC CA3, H hypothalamus, I hindbrain (HB) pontine reticular nuclei, J vestibular nuclei of the medulla oblongata, K Cx somatosensory, L HPC CA2, M HPC dentate gyrus, N striatum, and O cerebellum VI. Representative confocal images of PHP.eB-LAP2 mediated mCherry expression (green) in the P Cx somatosensory, Q HPC CA2, R HPC dentate gyrus, S striatum, and T cerebellum VI. All images are stacked confocal sections (scale bar, 100 µm). U Quantification of the fluorescence intensity of the mCherry signal driven by PHP.eB-MiniLAP2 and PHP.eB-LAP2 in the cortex (Cx), hippocampus (HPC), striatum (Str), and cerebellum (Cb). V Quantification of the percentage of mCherry-positive cells after inoculation with AAV-MiniLAP2 or AAV-LAP2 in the Cx, HPC, Str, and Cb. Data are represented as the mean ± SEM; n = 4 animals per group; three tissue sections were analyzed for each animal. Statistical significance was determined by Student’s t-test, with a P-value < 0.05 considered to be statistically significant. **P < 0.002; ns, not significant.
Fig. 4
Fig. 4. PHP.eB-MiniLAP2 transgene expression was predominantly detected in neurons.
A, B Representative confocal images of sagittal brain sections from mice 30 days after inoculation with PHP.eB-MiniLAP2, and immunostained with mCherry antibody (green). The staining patterns of (A1, B1) neurons (detected by staining with anti-NeuN, magenta), (A2, B2) astrocytes (detected by staining with anti-GFAP, magenta), (A3, B3) microglia (detected by staining with anti-Iba1, magenta), and (A4, B4) oligodendrocytes (detected by staining with anti-Olig2, magenta) in the cortex (Cx) and hippocampus (HPC). Arrows denote the merging of both channels (white). While NeuN, GFAP, and Iba1 signals can be detected both within the cell nucleus and cytoplasm, Olig2 signal is limited primarily to the nucleus (scale bar, 100 µm). C Percentage of transgene-expressing neurons, astrocytes, microglia, and oligodendrocytes in the Cx and HPC. Images are stacked confocal sections; n = 4 per group; three tissue sections were analyzed per animal.
Fig. 5
Fig. 5. MiniLAP2 drives transgene expression in GABAergic, glutamatergic, and dopaminergic neurons.
Representative confocal images of sagittal brain sections from mice immunostained with mCherry antibody (green) and A anti-GAD67 (magenta) to detect GABAergic neurons in the hippocampus CA1, B anti-vGLUT2 (magenta) to detect glutamatergic neurons in the cerebellum VIII, and C anti-TH (magenta) to detect dopaminergic neurons in the substantia nigra. Representative confocal image of a cross section of the lumbar spinal cord immunostained with mCherry antibody (green) and anti-NeuN (magenta) are shown for D, E AAV-MiniLAP2 and F, G AAV-LAP2. E, G A higher-magnification image of the dorsal horn reveals minimal transgene expression. Arrows denote the merging of both channels (white). Scale bars: 100 and 200 µm. F Percentage of cells expressing mCherry driven by negative control, AAV-LAP2 and AAV-MiniLAP2. Data are reported as mean ± SEM; n = 4 animals per group; three tissue sections were analyzed for each animal); ***P < 0.001; ns, not significant by one-way ANOVA with Tukey’s post-hoc test.
Fig. 6
Fig. 6. Systemic administration of PHP.eB-LAP2 and PHP.eB-MiniLAP2 leads to differential transgene expression in liver, kidney, and muscle tissues.
Representative images of mCherry-positive cells (brown) in A the kidney, B liver, and C skeletal muscle tissue from mice inoculated with (A1, B1, C1) negative control, (A2, B2, C2) PHP.eB-LAP2, or (A3, B3, C3) PHP.eB-MiniLAP2. (D1, E1) Representative images of mCherry immunostaining and (D2, E2) images corresponding to the morphometric analysis used to quantify mCherry immunostaining in liver tissue from mice inoculated with PHP.eB-LAP2 and PHP.eB-MiniLAP2. The inset squares show the fields shown at a higher magnification (on the right). Arrows indicate the immunostained cells. Scale bars, 200 µm (A–C), 1 mm, and 100 µm (D, E), as indicated. F Percentage of mCherry-positive nuclei per unit area (μm2) of liver tissue from mice inoculated with PHP.eB-LAP2 or PHP.eB-MiniLAP2; n = 4 animals per group, with two tissue sections analyzed per animal.
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