Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound

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Surgical infusion of gene therapy vectors has provided opportunities for biological manipulation of specific brain circuits in both animal models and human patients. Transient focal opening of the blood-brain barrier (BBB) by MR-guided focused ultrasound (MRgFUS) raises the possibility of noninvasive CNS gene therapy to target precise brain regions. However, variable efficiency and short follow-up of studies to date, along with recent suggestions of the potential for immune reactions following MRgFUS BBB disruption, all raise questions regarding the viability of this approach for clinical translation. The objective of the current study was to evaluate the efficiency, safety, and long-term stability of MRgFUS-mediated noninvasive gene therapy in the mammalian brain.


Focused ultrasound under the control of MRI, in combination with microbubbles consisting of albumin-coated gas microspheres, was applied to rat striatum, followed by intravenous infusion of an adeno-associated virus serotype 1/2 (AAV1/2) vector expressing green fluorescent protein (GFP) as a marker. Following recovery, animals were followed from several hours up to 15 months. Immunostaining for GFP quantified transduction efficiency and stability of expression. Quantification of neuronal markers was used to determine histological safety over time, while inflammatory markers were examined for evidence of immune responses.


Transitory disruption of the BBB by MRgFUS resulted in efficient delivery of the AAV1/2 vector to the targeted rodent striatum, with 50%–75% of striatal neurons transduced on average. GFP transgene expression appeared to be stable over extended periods of time, from 2 weeks to 6 months, with evidence of ongoing stable expression as long as 16 months in a smaller cohort of animals. No evidence of substantial toxicity, tissue injury, or neuronal loss was observed. While transient inflammation from BBB disruption alone was noted for the first few days, consistent with prior observations, no evidence of brain inflammation was observed from 2 weeks to 6 months following MRgFUS BBB opening, despite delivery of a virus and expression of a foreign protein in target neurons.


This study demonstrates that transitory BBB disruption using MRgFUS can be a safe and efficient method for site-specific delivery of viral vectors to the brain, raising the potential for noninvasive focal human gene therapy for neurological disorders.

ABBREVIATIONS AAV = adeno-associated virus; AAV1/2 = AAV serotype 1/2; BBB = blood-brain barrier; BSA = bovine serum albumin; DAB = 3,3′-diaminobenzidine; DAPI = 4′,6-diamino-2-phenylindole; Gd-DTPA = gadopentetate dimeglumine; GFP = green fluorescent protein; MRgFUS = MR-guided focused ultrasound; PBS = phosphate-buffered saline; rAAV = recombinant AAV; RF = radiofrequency; TBST = Tris-buffered saline with Triton.

Article Information

Correspondence Michael G. Kaplitt: Weill Cornell Medical College, New York, NY.

INCLUDE WHEN CITING Published online April 27, 2018; DOI: 10.3171/2017.8.JNS17790.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.



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    MRgFUS facilitates AAV-mediated gene delivery to the brain. A: Gd-DTPA–enhanced T1-weighted images collected after sonication showed disruption of the BBB and Gd-DTPA extravasation in brain parenchyma (dashed line). B: The brain tissue was harvested 3 weeks after sonication and the colocalization of GFP and NeuN was visualized using immunostaining. Bar = 50 μm. C: DAB visualization of GFP transduction from serial sections centered on the targeted area (serial sections through the center of the targeted points).

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    MRgFUS facilitates stable, long-term GFP transduction. A: High-magnification immunostaining for GFP and NeuN reveals a dominantly neuronal population of GFP-transduced cells in the striatum. Bar = 50 μm. B: Bar graph showing that quantification of striatal GFP transduction is stable over time. GFP-positive neurons are expressed as a percentage of the total number of striatal neurons per 20 hpf per animal (see Methods). C: Bar graph demonstrating that the MRgFUS-mediated GFP transduction is restricted mainly to neurons.

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    Detection of striatal MRgFUS-facilitated AAV-mediated GFP transduction 16 months after sonication. Gd-DTPA–enhanced T1-weighed images collected after sonication showed disruption of the BBB and Gd-DTPA extravasation in brain parenchyma (dashed line). Histological analysis of the brain harvested 16 months after sonication showed GFP transduction mostly in neurons. Bar = 50 μm.

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    MRgFUS-facilitated AAV-mediated gene delivery in peripheral organs is present in the short term but not the long term. Analysis of high-power immunofluorescent images of tissue collected from animals with MRgFUS with AAV1/2.GFP, and animals with AAV1/2.GFP stereotactically administered in the striatum shows no long-term GFP transgene expression in the liver, heart, and lungs. Bar = 100 μm. FUS = MRgFUS.

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    No evidence of long-term inflammatory response induced by unilateral striatal MRgFUS-mediated gene transfer to the brain. Both Iba1 (microglia marker) and GFAP (astrocytic marker) returned to baseline levels by week 2, suggesting that local inflammatory response is transitory. Bar = 50 μm.



Abbott NJPatabendige AADolman DEYusof SRBegley DJ: Structure and function of the blood-brain barrier. Neurobiol Dis 37:13252010


Alonso AReinz ELeuchs BKleinschmidt JFatar MGeers B: Focal delivery of AAV2/1-transgenes into the rat brain by localized ultrasound-induced BBB opening. Mol Ther Nucleic Acids 2:e732013


Bell PGao GHaskins MEWang LSleeper MWang H: Evaluation of adeno-associated viral vectors for liver-directed gene transfer in dogs. Hum Gene Ther 22:9859972011


Breous ESomanathan SBell PWilson JM: Inflammation promotes the loss of adeno-associated virus-mediated transgene expression in mouse liver. Gastroenterology 141:348357357.e1–357.e32011


Carty NLee DDickey CCeballos-Diaz CJansen-West KGolde TE: Convection-enhanced delivery and systemic mannitol increase gene product distribution of AAV vectors 5, 8, and 9 and increase gene product in the adult mouse brain. J Neurosci Methods 194:1441532010


Elias WJHuss DVoss TLoomba JKhaled MZadicario E: A pilot study of focused ultrasound thalamotomy for essential tremor. N Engl J Med 369:6406482013


Elias WJLipsman NOndo WGGhanouni PKim YGLee W: A randomized trial of focused ultrasound thalamotomy for essential tremor. N Engl J Med 375:7307392016


Feigin AKaplitt MGTang CLin TMattis PDhawan V: Modulation of metabolic brain networks after subthalamic gene therapy for Parkinson’s disease. Proc Natl Acad Sci U S A 104:19559195642007


Fiandaca MSVarenika VEberling JMcKnight TBringas JPivirotto P: Real-time MR imaging of adeno-associated viral vector delivery to the primate brain. Neuroimage 47 (Suppl 2):T27T352009


Foley CPRubin DGSantillan ASondhi DDyke JPCrystal RG: Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption. J Control Release 196:71782014


Fu HKang LJennings JSMoy SSPerez ADirosario J: Significantly increased lifespan and improved behavioral performances by rAAV gene delivery in adult mucopolysaccharidosis IIIB mice. Gene Ther 14:106510772007


Fu HMuenzer JSamulski RJBreese GSifford JZeng X: Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol Ther 8:9119172003


Hsu PHWei KCHuang CYWen CJYen TCLiu CL: Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound. PLoS One 8:e576822013


Huang QDeng JWang FChen SLiu YWang Z: Targeted gene delivery to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Exp Neurol 233:3503562012


Hynynen K: Focused ultrasound for blood-brain disruption and delivery of therapeutic molecules into the brain. Expert Opin Drug Deliv 4:27352007


Hynynen KMcDannold NVykhodtseva NJolesz FA: Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits. Radiology 220:6406462001


Jeanmonod DWerner BMorel AMichels LZadicario ESchiff G: Transcranial magnetic resonance imaging-guided focused ultrasound: noninvasive central lateral thalamotomy for chronic neuropathic pain. Neurosurg Focus 32(1):E12012


Jordão JFThévenot EMarkham-Coultes KScarcelli TWeng YQXhima K: Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial focused ultrasound. Exp Neurol 248:16292013


Kaplitt MGFeigin ATang CFitzsimons HLMattis PLawlor PA: Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 369:209721052007


Kaplitt MGLeone PSamulski RJXiao XPfaff DWO’Malley KL: Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat Genet 8:1481541994


Kovacs ZIKim SJikaria NQureshi FMilo BLewis BK: Disrupting the blood-brain barrier by focused ultrasound induces sterile inflammation. Proc Natl Acad Sci U S A 114:E75E842017


LeWitt PARezai ARLeehey MAOjemann SGFlaherty AWEskandar EN: AAV2-GAD gene therapy for advanced Parkinson’s disease: a double-blind, sham-surgery controlled, randomised trial. Lancet Neurol 10:3093192011


Manno CSPierce GFArruda VRGlader BRagni MRasko JJ: Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12:3423472006 (Erratum in Nat Med 12:592 2006)


Marks WJ JrBartus RTSiffert JDavis CSLozano ABoulis N: Gene delivery of AAV2-neurturin for Parkinson’s disease: a double-blind, randomised, controlled trial. Lancet Neurol 9:116411722010


Marks WJ JrOstrem JLVerhagen LStarr PALarson PSBakay RA: Safety and tolerability of intraputaminal delivery of CERE-120 (adeno-associated virus serotype 2-neurturin) to patients with idiopathic Parkinson’s disease: an open-label, phase I trial. Lancet Neurol 7:4004082008


Mastakov MYBaer KXu RFitzsimons HDuring MJ: Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol Ther 3:2252322001


McCaffrey GDavis TP: Physiology and pathophysiology of the blood-brain barrier: P-glycoprotein and occludin trafficking as therapeutic targets to optimize central nervous system drug delivery. J Investig Med 60:113111402012


McCarty DMDiRosario JGulaid KMuenzer JFu H: Mannitol-facilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice. Gene Ther 16:134013522009


McDannold NVykhodtseva NRaymond SJolesz FAHynynen K: MRI-guided targeted blood-brain barrier disruption with focused ultrasound: histological findings in rabbits. Ultrasound Med Biol 31:152715372005


Mingozzi FHigh KA: Immune responses to AAV in clinical trials. Curr Gene Ther 11:3213302011


Morgenstern PFMarongiu RMusatov SAKaplitt MG: Adeno-associated viral gene delivery in neurodegenerative disease. Methods Mol Biol 793:4434552011


Nance ETimbie KMiller GWSong JLouttit CKlibanov AL: Non-invasive delivery of stealth, brain-penetrating nanoparticles across the blood-brain barrier using MRI-guided focused ultrasound. J Control Release 189:1231322014


Neuwelt EABalaban EDiehl JHill SFrenkel E: Successful treatment of primary central nervous system lymphomas with chemotherapy after osmotic blood-brain barrier opening. Neurosurgery 12:6626711983


Neuwelt EABarnett PAMcCormick CIFrenkel EPMinna JD: Osmotic blood-brain barrier modification: monoclonal antibody, albumin, and methotrexate delivery to cerebrospinal fluid and brain. Neurosurgery 17:4194231985


Neuwelt EADahlborg SA: Chemotherapy administered in conjunction with osmotic blood-brain barrier modification in patients with brain metastases. J Neurooncol 4:1952071987


Neuwelt EADiehl JTVu LHHill SAMichael AJFrenkel EP: Monitoring of methotrexate delivery in patients with malignant brain tumors after osmotic blood-brain barrier disruption. Ann Intern Med 94:4494541981


Neuwelt EAGoldman DLDahlborg SACrossen JRamsey FRoman-Goldstein S: Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function. J Clin Oncol 9:158015901991


Neuwelt EAHill SAFrenkel EP: Osmotic blood-brain barrier modification and combination chemotherapy: concurrent tumor regression in areas of barrier opening and progression in brain regions distant to barrier opening. Neurosurgery 15:3623661984


Neuwelt EAMaravilla KRFrenkel EPRapaport SIHill SABarnett PA: Osmotic blood-brain barrier disruption. Computerized tomographic monitoring of chemotherapeutic agent delivery. J Clin Invest 64:6846881979


Rafii MSBaumann TLBakay RAOstrove JMSiffert JFleisher AS: A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer’s disease. Alzheimers Dement 10:5715812014


Salegio EASamaranch LKells APForsayeth JBankiewicz K: Guided delivery of adeno-associated viral vectors into the primate brain. Adv Drug Deliv Rev 64:5986042012


Schuster DJDykstra JARiedl MSKitto KFBelur LRMcIvor RS: Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse. Front Neuroanat 8:422014


Thévenot EJordão JFO’Reilly MAMarkham KWeng YQFoust KD: Targeted delivery of self-complementary adeno-associated virus serotype 9 to the brain, using magnetic resonance imaging-guided focused ultrasound. Hum Gene Ther 23:114411552012


Treat LHMcDannold NVykhodtseva NZhang YTam KHynynen K: Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRI-guided focused ultrasound. Int J Cancer 121:9019072007


Treat LHMcDannold NZhang YVykhodtseva NHynynen K: Improved anti-tumor effect of liposomal doxorubicin after targeted blood-brain barrier disruption by MRI-guided focused ultrasound in rat glioma. Ultrasound Med Biol 38:171617252012


Tuszynski MHThal LPay MSalmon DPU HSBakay R: A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nat Med 11:5515552005


Tuszynski MHYang JHBarba DU HSBakay RAPay MM: Nerve growth factor gene therapy: activation of neuronal responses in Alzheimer disease. JAMA Neurol 72:113911472015


Wang LCao OSwalm BDobrzynski EMingozzi FHerzog RW: Major role of local immune responses in antibody formation to factor IX in AAV gene transfer. Gene Ther 12:145314642005


Wang SKugelman TBuch AHerman MHan YKarakatsani ME: Non-invasive, focused ultrasound-facilitated gene delivery for optogenetics. Sci Rep 7:399552017


Wang SOlumolade OOSun TSamiotaki GKonofagou EE: Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus. Gene Ther 22:1041102015




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