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

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.

These simulations are integrated into the treatment planning to automatically compensate the driving signal to ensure a constant acoustic pressure for all grid points.Simulated pressure change in the x-y plane (lateral-elevational) (A) and the x-z plane (lateral-axial) (B), respectively.

Figure S1 .
Figure S1.US-guided identification of FUS target areas: superimposition of US-guided images and anatomical annotations from the Allen Mouse Brain Atlas and Allen Reference Atlas -Mouse Brain (http://atlas.brain-map.org/,Mouse, P56, coronal) (A).Experimental therapy array setup for FUS treatment and array specifications (B).Abbreviations: RH: right hemisphere; LH: left hemisphere.

Figure S3 .
Figure S3.Representative planes of T2*-weighted MRI images post-FUS treatment at 600, 740 and 420 kPa, respectively, in the right hemisphere (RH) of treated subjects.For subjects treated at 420 and 600 kPa, changes in T2*-weighted MRI images due to insonation could not be detected at these FUS pressures.

Figure S4 .
Figure S4.Methodology for the creation of the passive acoustic maps (PAMs) based on passive cavitation detection (PCD).This methodology is further detailed in Supplementary FigureS5.A treatment plan is created and RF data are collected with the imaging array while the therapeutic array is transmitting.The backscattered echoes primarily originate from oscillating microbubbles.Processing in the Fourier domain allows selection of specific frequency bands (like the harmonics here) and yields a passive acoustic map.This map is processed and overlaid on the ultrasound image in real-time during the treatment.Localizing the maximum of the passive acoustic map shows the agreement with the targeted area.We then create an integrated map with all grid positions.

Figure S5 .
Figure S5.Steps for the angular spectrum passive acoustic mapping processing.First, RF data are collected with the imaging array while the therapeutic array is transmitting.The backscattered echoes primarily originate from oscillating microbubbles.The two-dimensional Fourier transform of the RF data is then utilized to perform the angular spectrum backpropagation.Processing in the Fourier domain allows selection of specific frequency bands (like the harmonics here) and yields a passive acoustic map.This map is processed and overlaid on the ultrasound image in real-time during the treatment.Localizing the maximum of the passive acoustic map shows the agreement with the targeted area.

Figure S6 .
Figure S6.Comparison of average levels of acoustic emissions in the harmonics and the broadband frequency range for different acoustic pressures during FUS treatment over 2 minutes.The bold line represents the average value and the interval represent the range (minimum-maximum).Each pressure group pools 3 animals each having 5 focus locations: 420 kPa PNP (A), 600 kPa PNP (B), 740 kPa PNP (C).

Figure S8 .
Figure S8.Protein and gene expression assessments from insonified and control untreated brain tissue.A. Protein expression assessment.Protein bands of PKM2 and β-actin control from mice brain at 3 weeks p.i. of AAV9.B. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assessment of expression: PKM2 mRNA fold increase in hemispheres of treated mice (n = 4) and no-FUS AAV9-injected control mice (n = 3).C. Gene expression of TSPO (translocator protein) from hemispheres of mice brain at 3 weeks p.i. pf AAV9.Brown-Forsythe and Welch ANOVA tests were performed for statistical analysis.ns: not significant (P> 0.05).

Figure S9 .
Figure S9.Evaluation of the acoustic pressure over space for calibration.Simulated acoustic pressure decreases when the beam is steered away from the array's geometric focus (indicated by the red dot).These simulations are integrated into the treatment planning to automatically compensate the driving signal to ensure a constant acoustic pressure for all grid points.Simulated pressure change in the x-y plane (lateral-elevational) (A) and the x-z plane (lateral-axial) (B), respectively.