The engineered AAV2-HBKO promotes non-invasive gene delivery to large brain regions beyond ultrasound targeted sites

Focused ultrasound increases gene delivery to deep brain structure following the administration of a recombinant adeno-associated virus in the cerebrospinal fluid

“…AAV2-HBKO encoded green fluorescent protein (GFP) under control of the ubiquitous promoter CAG. AAV2-HBKO was administered at a dose of 2.16×10 12 GC/kg irrespective of the administration route, which is within the range previously used for FUS-MB-mediated AAV delivery to the brain following intravenous injection (1.67×10 12 GC/kg – 1.67×10 13 GC/kg)(16). Four weeks following AAV delivery and bilateral FUS-MB targeting to the striatum, the animals were sacrificed and tissues were harvested for subsequent analyses.…”

“…The level of AAV in the blood 21 min post intravenous administration was approximately twice the level of AAV 153 min post ICM injection (Figure 3D). Previous studies in mice have demonstrated that doubling the intravenous dosages of AAV2-HBKO do not significantly affect the percentage of transgene-positive cells obtained in the brain following FUS-MB delivery(16). Because of the significant clearance of AAV from the cerebrospinal fluid into the blood after 153 min, distribution route #2 (Figure 1C) is likely to be the primary delivery mechanism responsible for GFP protein and mRNA expression seen in animals injected ICM with AAV 120 min prior to FUS-MB (Figure 2K+L).…”

“…C Possible distribution route #2 is based on the fact that ICM-injected AAV will eventually get cleared from the CSF into the blood. Then, the AAV could enter the brain from the blood at FUS-targeted sites, similarly to when the AAV is injected intravenously (16).…”

“…This feature of AAV5 may be useful to determine the significance of distribution route #2 in FUS-MB delivery of ICM-injected AAV to the brain since we expect this distribution route to work poorly for AAV5. Opposed to AAV5, AAV2-HBKO shows decreased uptake in peripheral organs compared to AAV9, which could also explain the success of FUS-MB delivery of this serotype to the brain after ICM administration if the primary route is distribution route #2 (16,24). AAVs have been optimized separately for increased brain transduction following ICM administration and FUS-MB-mediated delivery (25,26).…”

“…DNA was extracted from organs using QIAamp DNA FFPE Tissue Kit (Qiagen, cat no 56404) and from blood samples using DNeasy Blood and Tissue Kit (Qiagen, cat no 69504) according to manufacturer's protocol except for the final elution step being done with a low TE buffer instead of the elution buffer provided in the kit. Digital droplet PCR analysis was conducted as previously described in detail (16). GFP primers were 50-ACT ACA ACA GCC ACA ACG TCT ATA TCA-30 and reverse primer 50-GGCGGATCTTGAAGTTCACC-30 (Invitrogen) and the probe was 50-6-FAM-CCG ACA AGC-ZENAGA AGA ACG GCA TCA-Iowa Black FQ-30 (Integrated DNA Technologies, Coralville, IA, USA).…”