In vivo biodistribution analysis of transmission competent and defective RNA virus-based episomal vector

Figures

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  Figure 1. In vivo imaging of REVec. (a) In vivo luciferase activity of Lewis rats at 3, 6, 9, and 12 weeks after IN administration of 104 FFU REVec-Luciferase. Dorsal and lateral images are shown at indicated time points. Total flux rate at each time point (photons/sec) is shown below. (n = 3). (b) In vivo luciferase activity of Lewis rats after IC administration of 104 FFU REVec-Luciferase. Luciferase activity was measured as above at 2, 4, 6, 8, 10, and 12 weeks post-administration. Dorsal, ventral, and lateral images are shown at indicated time points. Total flux rate at each time point (photons/sec) is shown below (n = 3).

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  Figure 2. Tissue tropism of REVec and ΔG-REVec. (a) Body weight of Lewis rats administered IC with 104 FFU REVec-GFP and ΔG-REVec-GFP (n = 3). (b) Animals inoculated with REVec-GFP or ΔG-REVec-GFP were sacrificed at 12 weeks after injection and the level of transgene expression was examined in indicated tissues by Western blot analysis for GFP, β-Actin, and α-Tubulin. Data are representative of triplicate experiment. Full-length immunoblots are presented in Supplementary Fig. S1. (c) Vector copy number per μg of total RNA in various tissues were determined by RT-qPCR. (n = 3).

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  Figure 3. Transgene expression in different brain regions and cell types. (a) Immunohistochemical analysis of anterior to posterior coronal brain sections (20 μm) at 2 and 12 weeks post-injection with 104 FFU REVec-GFP or ΔG-REVec-GFP. Higher exposure images of GFP (GFP High) are shown for mock and ΔG-REVec-GFP. Boxes 1 and 2 indicate high magnification images of selected brain regions showing magnified detail of GFP expression. (b) Immunohistochemistry of brain sections at 12 weeks after REVec injection co-labeled with GFP (green) and antibodies to microglia (Iba-1), astrocyte (GFAP), and neuron (NeuN). Cell specific markers are labeled in red and counterstained with DAPI. Scale bar, 50 μm.

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  Figure 4. Histology analysis of brain at 12 weeks post-administration. (a) H&E staining of mock, REVec-GFP or ΔG-REVec-GFP brain sections (10 μm). (b) Immunohistochemistry of coronal brain sections (20 μm) stained with CD8 antibody (red) and counterstained with DAPI. CD8 T cells in brain parenchyma and meninges are shown. Scale bar, 50 μm.

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  Figure 5. Quantification of infectious vector titer in brain and CSF. (a) Infectious vector titer in brain was quantified by focus-forming assay by inoculating Vero cells with serial dilutions of brain homogenates (n = 3). (b) Infectious titer of REVec in CSF was determined as above (n = 3). Representative images of Vero cells inoculated with brain homogenates or CSF and immunostained with BoDV-P antibody is shown to the right. Scale bar, 100 μm.

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  Figure 6. Vector shedding analysis. Vector copy number in feces and blood were determined by RT-qPCR at 2 and 12 weeks post-administration with (a) REVec-GFP or (b) ΔG-REVec-GFP (n = 3).

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  Figure 7. Detection of anti-REVec antibodies in serum. (a) Immunofluorescence analysis of Vero cells or Vero cells persistently infected with REVec-GFP using serum from mock, REVec, or ΔG-REVec inoculated animals. (b) Immunoblot analysis using serum from mock, REVec, or ΔG-REVec inoculated animals showing immunoreactivity of serum against each of the viral proteins and transgene. Western blot analysis using antibodies specific to the proteins of interest are shown to the right. Full-length immunoblots are presented in Supplementary Fig. S2. (c) REVec neutralization assay. The 100 FFU of REVec-GFP was pre-incubated with serial dilutions of serum obtained from mock treated, REVec, or ΔG-REVec injected animals, and inoculated on Vero cells. At 3 days post-inoculation, number of infected cells were quantified. Neutralization activity of serum obtained from REVec or ΔG-REVec were compared to control serum at each dilution. Data are presented as % neutralization compared to the control serum (n = 3).