title: Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation.
abstract: More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×10(5) microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation-induced gene therapies.
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel.
authors.forename: Galina
authors.initials: G
authors.lastname: Shapiro
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Wong
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel.
authors.forename: Maxim
authors.initials: M
authors.lastname: Bez
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Tam
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Even
authors.affiliation: Department of Surgery, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Sheyn
authors.affiliation: Department of Surgery, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Shiran
authors.initials: S
authors.lastname: Ben-David
authors.affiliation: Department of Surgery, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Wafa
authors.initials: W
authors.lastname: Tawackoli
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel; Department of Surgery, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Gadi
authors.initials: G
authors.lastname: Pelled
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Katherine W
authors.initials: KW
authors.lastname: Ferrara
authors.affiliation: Skeletal Biotech Laboratory, The Hebrew University-Hadassah Faculty of Dental Medicine, Ein Kerem, Jerusalem 91120, Israel; Department of Surgery, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048, USA; Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Electronic address: Dan.Gazit@csmc.edu.
authors.forename: Dan
authors.initials: D
authors.lastname: Gazit
mesh_terms: D000818:Animals; D004247:DNA / Q000008:administration & dosage; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D045423:Microbubbles; D018482:Muscle, Skeletal / Q000378:metabolism; D010957:Plasmids; D016062:Porosity; D000069453:Ultrasonic Waves
chemical_list: D004247:DNA; D008156:Luciferases
keywords: “Gene therapy”; “Microbubbles”; “Sonoporation”; “Ultrasound”