Focused ultrasound as a novel strategy for noninvasive gene delivery to retinal Müller glia

• Published in: Theranostics 2020-01, Vol.10 (7), p.2982-2999
• Main Authors: Touahri, Yacine, Dixit, Rajiv, Kofoed, Rikke Hahn, Miloska, Kristina, Park, EunJee, Raeisossadati, Reza, Markham-Coultes, Kelly, David, Luke Ajay, Rijal, Hibo, Zhao, Jiayi, Lynch, Madelaine, Hynynen, Kullervo, Aubert, Isabelle, Schuurmans, Carol
• Format: Article
• Language: English
• Subjects: Animals; Blood-Brain Barrier; Blood-Retinal Barrier; Cell cycle; Dependovirus - genetics; Ependymoglial Cells; Gene expression; Gene therapy; Gene Transfer Techniques; Genes, Synthetic; Genetic Vectors - administration & dosage; Genetic Vectors - pharmacokinetics; Glial Fibrillary Acidic Protein - administration & dosage; Glial Fibrillary Acidic Protein - genetics; Green Fluorescent Proteins - administration & dosage; Green Fluorescent Proteins - genetics; Intravitreal Injections; Kidney - chemistry; Liver - chemistry; Luminescent Proteins - administration & dosage; Luminescent Proteins - genetics; Magnetic Resonance Imaging; Mammals; Mice; Mice, Inbred C57BL; Microbubbles; Photoreceptors; Promoter Regions, Genetic; Rats; Red Fluorescent Protein; Research Paper; Retina; Retinal detachment; Sonication - adverse effects; Sonication - methods; Tissue Distribution; Transduction, Genetic; Ultrasonic imaging
• ISSN: 1838-7640; 1838-7640
• DOI: 10.7150/thno.42611

Müller glia are specialized retinal cells with stem cell properties in fish and frogs but not in mammals. Current efforts to develop gene therapies to activate mammalian Müller glia for retinal repair will require safe and effective delivery strategies for recombinant adeno-associated viruses (AAVs), vectors of choice for clinical translation. Intravitreal and subretinal injections are currently used for AAV gene delivery in the eye, but less invasive methods efficiently targeting Müller glia have yet to be developed. : As gene delivery strategies have been more extensively studied in the brain, to validate our vectors, we initially compared the glial tropism of AAV-PHP.eB, an AAV9 that crosses the blood-brain and blood-retinal barriers, for its ability to drive fluorescent protein expression in glial cells in both the brain and retina. We then tested the glial transduction of AAV2/8-GFAP-mCherry, a virus that does not cross blood-brain and blood-retinal barriers, for its effectiveness in transducing Müller glia in murine retinal explants . For assays we used larger rat eyes, performing invasive intravitreal injections, and non-invasive intravenous delivery using focused ultrasound (FUS) (pressure amplitude: 0.360 - 0.84 MPa) and microbubbles (Definity, 0.2 ml/kg). : We showed that AAV-PHP.eB carrying a ubiquitous promoter (CAG) and green fluorescent protein (GFP) reporter, readily crossed the blood-brain and blood-retinal barriers after intravenous delivery in mice. However, murine Müller glia did not express GFP, suggesting that they were not transduced by AAV-PHP.eB. We thus tested an AAV2/8 variant, which was selected based on its safety record in multiple clinical trials, adding a glial fibrillary acidic protein (GFAP) promoter and mCherry (red fluorescent protein) reporter. We confirmed the glial specificity of AAV2/8-GFAP-mCherry, showing effective expression of mCherry in astrocytes after intracranial injection in the mouse brain, and of Müller glia in murine retinal explants. For experiments we switched to rats because of their larger size, injecting AAV2/8-GFAP-mCherry intravitreally, an invasive procedure, demonstrating passage across the inner limiting membrane, leading to Müller glia transduction. We then tested an alternative non-invasive delivery approach targeting a different barrier - the inner blood-retinal-barrier, applying focused ultrasound (FUS) to the retina after intravenous injection of AAV2/8 and microbubbles in rats, using magnet