RESUMEN
Magnetic resonance imaging-guided focused ultrasound combined with microbubbles injected in the bloodstream (MRIgFUS) temporarily increases the permeability of the blood-brain barrier (BBB), which facilitates the entry of intravenously administered adeno-associated viruses (AAVs) from the blood to targeted brain areas. To date, the properties of the AAVs used for MRIgFUS delivery resulted in cell transduction limited to MRIgFUS-targeted sites. Considering future clinical applications, strategies are needed to deliver genes to multiple locations and large brain volumes while creating minimal BBB modulation. Here we combine MRIgFUS with a vector that has enhanced biodistribution following brain entry, AAV2-HBKO, to mediate broad gene delivery to targeted brain regions at levels with potential therapeutic relevance. Expression of a reporter gene was achieved in 13% and 21% of all neurons present in the striatum and thalamus, respectively, while targeting only 28% of the brain regions with MRIgFUS. Compared with AAV9, MRIgFUS-mediated delivery of AAV2-HBKO showed greater diffusion in the brain and a higher percentage of the neurons expressing the transgene. MRIgFUS AAV2-HBKO gene delivery to the brain has the potential to reach levels that are functionally and clinically relevant, and this even when using relatively low intravenous AAV dosages, compared with what is currently used in clinical trials.
RESUMEN
Focused ultrasound combined with intravenously injected microbubbles (FUS) is known to non-invasively, locally, and transiently increase the permeability of the blood-brain barrier (BBB). A promising approach for non-invasive gene delivery to the brain is to administer recombinant adeno-associated viruses (AAVs) intravenously and allow them to cross the BBB at precise FUS-targeted brain regions. FUS-AAV delivery has been achieved in animal models; however, the key elements influencing, guiding, and monitoring the success of FUS-AAV delivery to the brain remain largely unknown. We systematically compared the ability of AAV1, AAV2, AAV5, AAV8, AAV9, and AAVrg to enter four specific brain regions and transduce two main cell types: neurons and astrocytes. Our results demonstrate that the AAV serotype, the extent of FUS-induced BBB permeability, and the intrinsic properties of the targeted brain tissue influence the observed biodistribution, diffusion and transduction of AAV to cells of the cerebrovasculature and brain parenchyma. Non-invasive contrast-enhanced MR imaging was found to predict the efficacy of FUS-AAV delivery. Notably, we also found that AAVs with high biodistribution to peripheral organs result in low gene delivery to the brain when combined with FUS. Gene delivery by AAV1, AAV2, AAV5, AAV8 and AAV9 was highly and selectively localized to FUS-targeted brain areas. To obtain non-invasive gene delivery to multiple brain regions with one area of FUS-BBB modulation, we combined a modified AAV2 vector harboring enhanced retrograde transport properties (AAVrg) with FUS-mediated brain delivery. This allowed for gene delivery from the FUS-targeted site to multiple connected brain regions. This study demonstrates that MR imaging can be used as a non-invasive indication of AAV delivery to the brain, and that the properties of AAV serotypes influence the efficacy of gene delivery to the brain with FUS. AAVs that have minimal peripheral biodistribution are ideal candidates for enhanced, and perhaps exclusive with future serotypes, delivery to the brain with FUS. The characterization of parameters influencing FUS-AAV delivery to the brain are critical to the design of safe and efficient gene therapies, from preclinical studies to future clinical applications.
