Focused Ultrasound Blood-Brain Barrier Opening (FUS-BBBO) can deliver adeno-associated viral vectors (AAVs) to treat genetic disorders of the brain. However, such disorders often affect large brain regions. Moreover, the applicability of FUS-BBBO in the treatment of brain-wide genetic disorders has not yet been evaluated. Herein, we evaluated the transduction efficiency and safety of opening up to 105 sites simultaneously. Increasing the number of targeted sites increased gene delivery efficiency at each site. We achieved transduction of up to 60% of brain cells with comparable efficiency in the majority of the brain regions. Furthermore, gene delivery with FUS-BBBO was safe even when all 105 sites were targeted simultaneously without negative effects on animal weight or neuronal loss. To evaluate the application of multi-site FUS-BBBO for gene therapy, we used it for gene editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system and found effective gene editing, but also a loss of neurons at the targeted sites. Overall, this study provides a brain-wide map of transduction efficiency, shows the synergistic effect of multi-site targeting on transduction efficiency, and is the first example of large brain volume gene editing after noninvasive gene delivery with FUS-BBBO.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Numerical data is available in the attached source data file. Images used in the generation of this data are available upon reasonable request.
Turner TJ, Zourray C, Schorge S, Lignani G. Recent advances in gene therapy for neurodevelopmental disorders with epilepsy. J Neurochem. 2021;157:229–62.
Simonato M, Bennett J, Boulis NM, Castro MG, Fink DJ, Goins WF, et al. Progress in gene therapy for neurological disorders. Nat Rev Neurol. 2013;9:277–91.
Joshi CR, Labhasetwar V, Ghorpade A. Destination brain: the past, present, and future of therapeutic gene delivery. J Neuroimmune Pharmacol. 2017;12:51–83.
Keeler GD, Kumar S, Palaschak B, Silverberg EL, Markusic DM, Jones NT, et al. Gene therapy-induced antigen-specific Tregs inhibit neuro-inflammation and reverse disease in a mouse model of multiple sclerosis. Mol Ther. 2018;26:173–83.
Spronck EA, Brouwers CC, Vallès A, de Haan M, Petry H, van Deventer SJ, et al. AAV5-miHTT gene therapy demonstrates sustained huntingtin lowering and functional improvement in Huntington disease mouse models. Mol Ther-Methods Clin Dev. 2019;13:334–43.
Flotte TR, Cataltepe O, Puri A, Batista AR, Moser R, McKenna-Yasek D, et al. AAV gene therapy for Tay-Sachs disease. Nat Med. 2022;28:251–9.
Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR. Gene therapy clinical trials worldwide to 2017: an update. J Gene Med. 2018;20:e3015.
Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet. 2011;12:341–55.
Snyder BR, Gray SJ, Quach ET, Huang JW, Leung CH, Samulski RJ, et al. Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery. Hum Gene Ther. 2011;22:1129–35.
Hayashi T, Akikawa R, Kawasaki K, Egawa J, Minamimoto T, Kobayashi K, et al. Macaques exhibit implicit gaze bias anticipating others’ false-belief-driven actions via medial prefrontal cortex. Cell Rep. 2020;30:4433–4444.e5.
Johnson TB, White KA, Brudvig JJ, Cain JT, Langin L, Pratt MA, et al. AAV9 gene therapy increases lifespan and treats pathological and behavioral abnormalities in a mouse model of CLN8-batten disease. Mol Ther. 2021;29:162–75.
Bartus RT, Baumann TL, Siffert J, Herzog CD, Alterman R, Boulis N, et al. Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients. Neurology. 2013;80:1698–701.
Szablowski JO, Lee-Gosselin A, Lue B, Malounda D, Shapiro MG. Acoustically targeted chemogenetics for the non-invasive control of neural circuits. Nat Biomed Eng. 2018;2:475–84.
Timbie KF, Mead BP, Price RJ. Drug and gene delivery across the blood-brain barrier with focused ultrasound. J Control Release. 2015;219:61–75.
Thévenot E, Jordão JF, O’Reilly MA, Markham K, Weng Y-Q, Foust KD, et al. Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound. Hum Gene Ther. 2012;23:1144–55.
Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR imaging–guided focal opening of the blood-brain barrier in rabbits. Radiology. 2001;220:640–6.
Samiotaki G, Acosta C, Wang S, Konofagou EE. Enhanced delivery and bioactivity of the neurturin neurotrophic factor through focused ultrasound—mediated blood—brain barrier opening in vivo. J Cereb Blood Flow Metab. 2015;35:611–22.
Upright NA, Baxter MG. Effect of chemogenetic actuator drugs on prefrontal cortex-dependent working memory in nonhuman primates. Neuropsychopharmacology. 2020;45:1793–8.
Rafii MS, Tuszynski MH, Thomas RG, Barba D, Brewer JB, Rissman RA, et al. Adeno-associated viral vector (serotype 2)–nerve growth factor for patients with alzheimer disease: a randomized clinical trial. JAMA Neurol. 2018;75:834–41.
Hynynen K. Ultrasound for drug and gene delivery to the brain. Adv Drug Deliv Rev. 2008;60:1209–17.
Felix M-S, Borloz E, Metwally K, Dauba A, Larrat B, Matagne V, et al. Ultrasound-mediated blood-brain barrier opening improves whole brain gene delivery in mice. Pharmaceutics. 2021;13:1245.
Downs ME, Buch A, Sierra C, Karakatsani ME, Chen S, Konofagou EE, et al. Long-term safety of repeated blood-brain barrier opening via focused ultrasound with microbubbles in non-human primates performing a cognitive task. PloS One. 2015;10:e0125911.
Staahl BT, Benekareddy M, Coulon-Bainier C, Banfal AA, Floor SN, Sabo JK, et al. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol. 2017;35:431–4.
Maggio I, Zittersteijn HA, Wang Q, Liu J, Janssen JM, Ojeda IT, et al. Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components. Gene Therapy. 2020;27:209–25.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.
Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33:102–6.
Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat Methods. 2016;13:868–74.
Park H, Oh J, Shim G, Cho B, Chang Y, Kim S, et al. In vivo neuronal gene editing via CRISPR–Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer’s disease. Nat Neurosci. 2019;22:524–8.
Zhou H, Liu J, Zhou C, Gao N, Rao Z, Li H, et al. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR–dCas9-activator transgenic mice. Nat Neurosci. 2018;21:440–6.
Hana S, Peterson M, McLaughlin H, Marshall E, Fabian AJ, McKissick O, et al. Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice. Gene Ther. 2021;28:646–58.
Yang Q, Zhou Y, Chen J, Huang N, Wang Z, Cheng Y. Gene therapy for drug-resistant glioblastoma via lipid-polymer hybrid nanoparticles combined with focused ultrasound. Int J Nanomed. 2021;16:185.
Yan S, Tu Z, Li S, Li X-J. Use of CRISPR/Cas9 to model brain diseases. Prog Neuro-Psychopharmacol Biol Psychiatry. 2018;81:488–92.
Wu W-H, Tsai Y-T, Justus S, Lee T-T, Zhang L, Lin C-S, et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol Ther. 2016;24:1388–94.
Kofoed RH, Dibia CL, Noseworthy K, Xhima K, Vacaresse N, Hynynen K, et al. Efficacy of gene delivery to the brain using AAV and ultrasound depends on serotypes and brain areas. J Control Release. 2022;351:667–80.
Badea A, Ali-Sharief AA, Johnson GA. Morphometric analysis of the C57BL/6J mouse brain. Neuroimage. 2007;37:683–93.
Wang S, Samiotaki G, Olumolade O, Feshitan JA, Konofagou EE. Microbubble type and distribution dependence of focused ultrasound-induced blood–brain barrier opening. Ultrasound Med Biol. 2014;40:130–7.
McDannold N, Vykhodtseva N, Hynynen K. Effects of acoustic parameters and ultrasound contrast agent dose on focused-ultrasound induced blood-brain barrier disruption. Ultrasound Med Biol. 2008;34:930–7.
Gray SJ, Foti SB, Schwartz JW, Bachaboina L, Taylor-Blake B, Coleman J, et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum Gene Ther. 2011;22:1143–53.
Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–40.
Li H, Heath JE, Trippett JS, Shapiro MG, Szablowski JO. Engineering viral vectors for acoustically targeted gene delivery. bioRxiv 2021: https://doi.org/10.1101/2021.07.26.453904.
Bing C, Ladouceur-Wodzak M, Wanner CR, Shelton JM, Richardson JA, Chopra R. Trans-cranial opening of the blood-brain barrier in targeted regions using astereotaxic brain atlas and focused ultrasound energy. J Ther Ultrasound. 2014;2:1–11.
Gorick CM, Breza VR, Nowak KM, Cheng VW, Fisher DG, Debski AC et al. Applications of focused ultrasound-mediated blood-brain barrier opening. Adv Drug Deliv Rev. 2022;191:114583.
Rich MC, Sherwood J, Bartley AF, Whitsitt QA, Lee M, Willoughby W, et al. Focused ultrasound blood brain barrier opening mediated delivery of MRI-visible albumin nanoclusters to the rat brain for localized drug delivery with temporal control. J Controlled Release. 2020;324:172–80.
Fletcher SMP, Choi M, Ogrodnik N, O’Reilly MA. A porcine model of transvertebral ultrasound and microbubble-mediated blood-spinal cord barrier opening. Theranostics. 2020;10:7758.
Cho EE, Drazic J, Ganguly M, Stefanovic B, Hynynen K. Two-photon fluorescence microscopy study of cerebrovascular dynamics in ultrasound-induced blood—brain barrier opening. J Cereb Blood Flow Metab. 2011;31:1852–62.
Goldim MPDS, Della Giustina A, Petronilho F. Using evans blue dye to determine blood‐brain barrier integrity in rodents. Curr Protoc Immunol. 2019;126:e83.
Downs ME, Buch A, Karakatsani ME, Konofagou EE, Ferrera VP. Blood-brain barrier opening in behaving non-human primates via focused ultrasound with systemically administered microbubbles. Sci Rep. 2015;5:1–13.
Huh H, Park TY, Seo H, Han M, Jung B, Choi HJ, et al. A local difference in blood–brain barrier permeability in the caudate putamen and thalamus of a rat brain induced by focused ultrasound. Sci Rep. 2020;10:1–11.
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21.
Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci. 1988;85:5166–70.
Lao Y-H, Ji R, Zhou JK, Snow KJ, Kwon N, Saville E, et al. Focused ultrasound–mediated brain genome editing. Proc Natl Acad Sci. 2023;120:e2302910120.
Tsai S-J. Therapeutic potential of transcranial focused ultrasound for Rett syndrome. Med Sci Monitor Int Med J Exp Clin Res. 2016;22:4026.
Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B, Masellis M, et al. Blood–brain barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat Commun. 2018;9:1–8.
Rusch BD, Abercrombie HC, Oakes TR, Schaefer SM, Davidson RJ. Hippocampal morphometry in depressed patients and control subjects: relations to anxiety symptoms. Biol Psychiatry. 2001;50:960–4.
Chatzikonstantinou A. Epilepsy and the hippocampus. Hippocampus Clin Neurosci. 2014;34:121–42.
Isnard J, Guénot M, Ostrowsky K, Sindou M, Mauguière F. The role of the insular cortex in temporal lobe epilepsy. Ann Neurol: Off J Am Neurol Assoc Child Neurol Soc. 2000;48:614–23.
Upright NA, Baxter MG. Effects of nicotinic antagonists on working memory performance in young rhesus monkeys. Neurobiol Learning Memory. 2021;184:107505.
Deverman BE, Pravdo PL, Simpson BP, Kumar SR, Chan KY, Banerjee A, et al. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain. Nat Biotechnol. 2016;34:204–9.
Chan KY, Jang MJ, Yoo BB, Greenbaum A, Ravi N, Wu W-L, et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat Neurosci. 2017;20:1172–9.
Ajmone-Cat MA, Onori A, Toselli C, Stronati E, Morlando M, Bozzoni I, et al. Increased FUS levels in astrocytes leads to astrocyte and microglia activation and neuronal death. Sci Rep. 2019;9:1–15.
Tsai H-C, Tsai C-H, Chen W-S, Inserra C, Wei K-C, Liu H-L. Safety evaluation of frequent application of microbubble-enhanced focused ultrasound blood-brain-barrier opening. Sci Rep. 2018;8:1–13.
Jordão JF, Thévenot E, Markham-Coultes K, Scarcelli T, Weng Y-Q, Xhima K, et al. Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound. Exp Neurol. 2013;248:16–29.
McMahon D, Oakden W, Hynynen K. Investigating the effects of dexamethasone on blood-brain barrier permeability and inflammatory response following focused ultrasound and microbubble exposure. Theranostics. 2020;10:1604.
Gilkes JA, Bloom MD, Heldermon CD. Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10. Gene Ther. 2016;23:263–71.
McTeague LM, Rosenberg BM, Lopez JW, Carreon DM, Huemer J, Jiang Y, et al. Identification of common neural circuit disruptions in emotional processing across psychiatric disorders. Am J Psychiatry. 2020;177:411–21.
Urban DJ, Roth BL. DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility. Ann Rev Pharmacol Toxicol. 2015;55:399–417.
Adachi M, Keefer EW, Jones FS. A segment of the Mecp2 promoter is sufficient to drive expression in neurons. Hum Mol Genet. 2005;14:3709–22.
Dou Y, Lin Y, Wang TY, Wang XY, Jia YL, Zhao CP. The CAG promoter maintains high‐level transgene expression in HEK293 cells. FEBS Open Biol. 2021;11:95–104.
Yang S, Li S, Li X-J. Shortening the half-life of Cas9 maintains its gene editing ability and reduces neuronal toxicity. Cell Rep. 2018;25:2653–2659.e3.
Wang S, Olumolade OO, Sun T, Samiotaki G, Konofagou EE. Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus. Gene Ther. 2015;22:104–10.
Pouliopoulos AN, Murillo MF, Noel RL, Batts AJ, Ji R, Kwon N, et al. Non-invasive optogenetics with ultrasound-mediated gene delivery and red-light excitation. Brain Stimulation. 2022;15:927–41.
The authors thank Dr. Mingshan Xue (Baylor College of Medicine) for helpful discussions, and Gang Bao’s laboratory (Rice University) for providing the usage of the ultracentrifuge. This research was supported by the Dunn Foundation John S. Dunn Foundation award for collaborative research and by The Welch Foundation grant (C-2048-20200401). Related work was funded by Harold Y and G. Leila Mathers Foundation.
The work was funded by John S. Dunn Foundation award for collaborative research and The Welch Foundation. Related work was supported by and Harold Y and G. Leila Mathers Foundation.
Animal experiments were conducted in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee of Rice University.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Nouraein, S., Lee, S., Saenz, V.A. et al. Acoustically targeted noninvasive gene therapy in large brain volumes. Gene Ther (2023). https://doi.org/10.1038/s41434-023-00421-1