A Viral Vector Engineered for Improved Noninvasive Gene Delivery to the Brain

Gene delivery to the brain with adeno-associated virus (AAV) has been a mainstay of in vivo neuroscience research and has significant therapeutic potential. Focused ultrasound (FUS) can be used to open the blood-brain barrier (FUS-BBBO) to enable noninvasive delivery of AAVs into the brain from the blood. However, the delivery of large molecules, e.g. AAVs, requires FUS pressures close to the safety margins and intravenous injection of AAVs leads to off-target peripheral tissue transduction. Here, we engineer a new AAV with improved brain transduction efficiency and tissue specificity after FUS-BBBO. To obtain a new AAV strain optimized for FUS-BBBO we modified a viral vector engineering method called Cre-recombination-based AAV targeted evolution (CREATE). In CREATE, a library of AAVs with mutated capsid proteins is injected intravenously into mice. When a particular AAV clone transduces a cell expressing Cre, its viral genome is modified and becomes detectable by a Cre-dependent PCR. We modified CREATE to engineer a FUS-BBBO-optimized AAV, which we called AAV.FUS. We first generated a viral vector library with a capsid modified by insertion of a randomized 7 amino-acid sequence between residues 588 and 589 of the AAV9 capsid. We then used FUS-BBBO to deliver the AAV library to one hemisphere of hSyn1-Cre mice, extracted the tissue, and identified 5 clones specifically enriched in the FUS-targeted regions. We observed improvement in transduction of all candidates compared to AAV9 (20-130% improvement). At the same time, each serotype transduced the liver less effectively (range: 13 – 643% reduction compared to AAV9). The top AAV.FUS candidate showed a 12.1-fold improvement in overall brain-tissue specificity after FUS-BBBO, 56% improvement in neuronal tropism, and regional transduction efficiency with hippocampus showing 4.7-fold better delivery than AAV9 (32-fold improvement in liver/hippocampus transduction ratio). Overall, FUS-BBBO gene delivery was improved by AAV engineering.