PET imaging of focused-ultrasound enhanced delivery of AAVs into the murine brain

Abstract

Rationale: Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. Methods: In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for in vivo PET quantification, whereas results for ex vivo assays are reported as percent injected dose per gram (%ID/g). Results: In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed in vivo by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[¹⁸F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Conclusion: Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies.

Keywords: Adeno-associated virus; Blood-brain barrier; Focused ultrasound; Gene therapy; Positron emission tomography.

Figure 1

Focused ultrasound treatment and BBB opening study by optical imaging. A. Illustration of FUS target area in the right hemisphere of the mouse brain. The beam follows a dorsal trajectory. B. Pre-treatment ultrasound-guided FUS target area (upper-left panel), post-FUS map localizing acoustic emissions from MBs for the entire treatment (upper-right panel), real-time passive acoustic monitoring (PAM) (lower-left panel) and power spectral density (PSD) plot localizing acoustic emissions from MBs for the entire treatment (lower-right panel) during FUS treatment. C. MRI 30 minutes post-FUS treatment (600 kPa) employing gadoteridol as contrast agent (left image), T2*-weighted MRI 30 minutes post-FUS treatment (600 kPa) (right image). D. Fluorescence microscopy (DAPI/Evans Blue) images of brain slices (100 μm) showing the FUS-treated (right) and contralateral (left) hemispheres. E. Brain images after FUS treatment followed by Evans Blue dye injection. Abbreviations: LH: left hemisphere. RH: right hemisphere.

Figure 2

Optimization of FUS treatment parameters and ex vivo/in vivo AAV accumulation study. A. Radiolabeling scheme of AAV9 by modification of lysine residues in the surface of the capsid with a peptide-multichelator via “click chemistry”. B. Schematic illustration of radiolabeled-AAV brain delivery and accumulation study. ⁶⁴Cu-AAV9 is systemically injected in C57BL/6 mice (n_CTL = 3, n₄₂₀ = 4, n₆₀₀ = 3, n₇₄₀ = 5) and capsid delivery and accumulation were assessed by PET/CT imaging (in vivo) and biodistribution (Bio-D)/autoradiography analysis (ex vivo). C. Representative coronal, sagittal and axial PET/CT images at 21 h post injection (p.i.) of ⁶⁴Cu-AAV9 of FUS-treated (420 kPa, 600 kPa, 740 kPa) and no-FUS AAV9-injected control (CTL) mice. D. Fold increase in ⁶⁴Cu-AAV9 accumulation within the entire FUS-treated brain hemisphere (RH). E. Ex vivo autoradiography at 22 h p.i. of ⁶⁴Cu-AAV9 at different FUS pressures and without FUS treatment (CTL). Red arrowheads highlight the insonified region in the central slice. F. Intensity and fold increase as assessed from autoradiography. G. Power spectra as a function of the treatment time for the different groups of pressure and all observations in all animals (420, 600, 740 kPa) Abbreviations: FUS: Focused ultrasound. HPC: hippocampus. LH: left hemisphere. RH: right hemisphere. IV: intravenous injection. MBs: microbubbles, CTL: Control. F. Ordinary one-way ANOVA and G. Two-way ANOVA each with multiple comparison tests. p-values are presented in the figures.

Figure 3

⁶⁴Cu-AAV9 accumulation study through in vivo quantitative PET/CT imaging and ex vivo biodistribution analysis. A. USg-FUS target and post-FUS cavitation localization area from PAM, maximum intensity projection (MIP) PET/CT image at 21 h post ⁶⁴Cu-AAV9 systemic injection in C57BL/6 mice. B. Representative coronal, axial and sagittal plane PET/CT images of an example mouse at 21 h post injection (p.i.) of ⁶⁴Cu-AAV9 after brain lateral FUS treatment (600 kPa). Radiotagged AAV capsid accumulation has been highlighted with a dotted green circle for clarity. C. Image-based blood subtracted radioactivity (%ID/cc) in the brain region of interest (ROI, 10-11 mm³) of FUS-treated AAV9-injected (n = 5) and no-FUS AAV9-injected (n = 3) C57BL/6 mice at 21 h p.i. D. Image-based blood subtracted peak radioactivity (%ID/cc) in the brain ROI (10-11 mm³) of FUS-treated AAV9-injected (n = 5) and no-FUS AAV9-injected (n = 3) C57BL/6 mice at 21 h p.i. E. Biodistribution of ⁶⁴Cu-AAV9 (%ID/g) in left and right (FUS) hemispheres of FUS-treated AAV9-injected (n = 5) and no-FUS AAV9-injected (n = 3) C57BL/6 mice at 22 h p.i. F. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assessment of ssDNA (PKM2) in brain hemispheres of FUS-treated AAV9-injected (n= 4) and no-FUS AAV9-injected (CTL, n= 3). G. Correlation between ssDNA genome copies (PKM2) delivered and radiolabeled capsid uptake (%ID/cc). Abbreviations: %ID/g: Percent injected dose per gram of tissue. %ID/cc: Percent injected dose per cubic centimeter. C-L: contra-lateral. FUS: focused ultrasound. LH: left hemisphere. RH: right hemisphere. MBs: microbubbles. IV: intravenous injection. PAM: passive acoustic mapping. Brown-Forsythe and Welch ANOVA with multiple comparison correction was performed for the statistical analysis between groups and a paired t test compared the mean values between the FUS-treated and contralateral hemisphere in the same group. p-values are indicated in the panels.

Figure 4

Schematic illustration of PET reporter gene for gene expression quantification with a PET probe. 1) AAV9 capsid internalizes into the cell after systemic injection in C57BL/6 mice. 2) Capsid uncages and gene is delivered into the cell. 3) Gene transcription and mRNA synthesis. 4) mRNA translation and protein synthesis (PKM2). 5) PET reporter probe ([¹⁸F]DASA-10) internalizes into the cell. 6) PET reporter probe reversibly binds to gene-encoded protein for in vivo quantification.

Figure 5

[¹⁸F]DASA-10 accumulation study by in vivo quantitative PET/CT imaging and ex vivo biodistribution analysis 3 weeks after AAV9 injection followed by FUS at 600 kPa. A. Schematic illustration of workflow, USg-FUS and post-FUS brain cavitation localization area from PAM for mice treated at 600 kPa, maximum intensity projection (MIP) PET/CT image after [¹⁸F]DASA-10 systemic injection in C57BL/6 mice. B. Representative coronal, axial and sagittal plane PET/CT images of an example mouse acquired immediately post systemic injection of [¹⁸F]DASA-10. Areas of [¹⁸F]DASA-10 retention have been highlighted with a dotted green circle for clarity. C. Image-based blood subtracted radioactivity (%ID/cc) in regions of interest (ROIs) of left and right brain hemispheres of FUS-treated AAV9-injected, no-FUS AAV9-injected and no-FUS no-AAV9-injected groups after injection of [¹⁸F]DASA-10 (n = 3 per group). D. Image-based blood subtracted peak radioactivity (%ID/cc) in brain ROI (10-11 mm³) in FUS-treated AAV9-injected, no-FUS AAV9-injected and no-FUS no-AAV9-injected groups after injection of [¹⁸F]DASA-10 (n = 3 per group). E. Biodistribution post injection (p.i.) of [¹⁸F]DASA-10 (%ID/g) in left and right hemispheres of FUS-treated AAV9-injected, no-FUS AAV9-injected and no-FUS no-AAV9-injected groups (n = 3 per group). Abbreviations: %ID/g: Percent injected dose per gram of tissue. %ID/cc: Percent injected dose per cubic centimeter. LH: left hemisphere. RH: right hemisphere. FUS: focused ultrasound, MBs: microbubbles. IV: intravenous injection. Brown-Forsythe and Welch ANOVA with multiple comparison correction was performed between groups and a paired t test compared the mean values between the FUS-treated and contralateral hemisphere in same group. p-values are indicated in the panels.

Figure 6

Assessment of ⁶⁴Cu-AAV9 accumulation and PKM2 transduction within the same cohort. A. Schematic illustration of FUS-assisted ⁶⁴Cu-AAV9:EF1A-PKM2 delivery for assessment of uptake and transgene expression, 21 h and 3 weeks after systemic capsid administration, respectively, within the same C57BL/6 mouse cohort. B. Representative PET/CT images for ⁶⁴Cu-AAV9 accumulation and PKM2 expression with [¹⁸F]DASA-10. Areas of ⁶⁴Cu-AAV9 accumulation and [¹⁸F]DASA-10 retention have been highlighted with a dotted green circle for clarity. C. Image-based blood subtracted radioactivity (%ID/cc) in the brain ROI (10-11 mm³) of FUS-treated AAV9-injected (n = 6) and no-FUS AAV9-injected (n = 3) C57BL/6 mice at 21 h post injection (p.i.) of ⁶⁴Cu-AAV9. D. Image-based blood subtracted peak radioactivity (%ID/cc) in brain ROI (10-11 mm³) of FUS-treated AAV9-injected (n = 6) and no-FUS AAV9-injected (n = 3) C57BL/6 mice at 21 h p.i. of ⁶⁴Cu-AAV9. E. Image-based blood subtracted radioactivity (%ID/cc) in brain ROI (10-11 mm³) in FUS-treated AAV9-injected (n = 6) and no-FUS AAV9-injected (n = 3) C57BL/6 mice immediately after injection of [¹⁸F]DASA-10. F. Image-based blood subtracted peak radioactivity (%ID/cc) in brain ROI (10-11 mm³) of FUS-treated AAV9-injected (n = 6) and no-FUS AAV9-injected (n = 3) C57BL/6 mice after injection of [¹⁸F]DASA-10. G. Biodistribution (Bio-D) p.i. of [¹⁸F]DASA-10 (%ID/g) in the left and right hemispheres of FUS-treated AAV9-injected (n = 6) and no-FUS AAV9-injected (n = 3) C57BL/6 mice. H. Correlation between ⁶⁴Cu-AAV9 accumulation and [¹⁸F]DASA-10 retention in brains of FUS-treated AAV9-injected (n = 6) subjects. Abbreviations: %ID/g: Percent injected dose per gram of tissue. %ID/cc: Percent injected dose per cubic centimeter. C-L: contra-lateral. LH: left hemisphere. RH: right hemisphere. FUS: focused ultrasound, MBs: microbubbles. IV: intravenous injection. BW: body weight. Brown-Forsythe and Welch ANOVA with multiple comparison correction was performed between groups and a paired t test compared the mean values between the FUS-treated and contralateral hemisphere in the same group. p-values are indicated in the panels.

Figure 7

Assessment of transduction of PKM2 and mNeonGreen (mNG) reporter genes by optical imaging three weeks after capsid injection. A. Schematic illustration of FUS-assisted delivery of AAV capsids encapsulating PKM2 or mNG reporter genes for transduction assessment with optical imaging in C57BL6 mice. B. Schematic illustration of brain sectioning for optical imaging. C. Fluorescence microscopy images of brain slices (100 μm) of the FUS-treated and no-FUS AAV9-injected control (CTL) mice. Assessment of PKM2 transduction through anti-PKM2 protein staining (DAPI/FITC) or mNG fluorescence detection (DAPI/mNG). D. Magnified regions of slices of the left hemisphere of brains receiving FUS treatment in the right hemisphere. C-D. Red arrows indicate locally-enhanced gene expression. Abbreviations: LH: left hemisphere. RH: right hemisphere. FUS: focused ultrasound, MBs: microbubbles. IV: intravenous injection, CTL: control.