title: Ultrasound directs a transposase system for durable hepatic gene delivery in mice.
abstract: Our aim was to evaluate the delivery of transposase-based vectors by ultrasound targeted microbubble destruction (UTMD) in mice. DNA vectors were attached to cationic lipid microbubbles (1-3 μm in diameter), injected intravenously and delivered to the liver by destruction of the carrier bubbles with ultrasound in burst mode at 1.0 MHz, 20-μs pulse duration, 10-Hz pulse repetition frequency and ∼1.3-MPa acoustic peak negative pressure. We evaluated the expression and genomic integration of conventional (pcDNA3) and piggyBac transposase-based (pmGENIE) reporter vectors. In vivo, we observed UTMD-mediated liver-specific expression of pmGENIE for an average of 24 d, compared with 4 d with pcDNA3. Reporter expression was located predominately near blood vessels initially, whereas expression after 3 d was more evenly distributed through the parenchyma of the liver. We confirmed random genomic integration for pmGENIE in vitro; however, integration events for pmGENIE in vivo were targeted to specific areas of chromosome 14. Our results suggest that a combination of UTMD and non-viral DNA transposase vectors can mediate weeks of hepatic-specific gene transfer in vivo, and analyses performed by non-restrictive linear amplification-mediated (nrLAM) polymerase chain reaction, cloning and sequencing identify an unexpected tropism for integration within a specific sequence on chromosome 14 in mice. UTMD delivery of transgenes may be useful for the treatment of hepatic gene deficiency disorders.
authors.affiliation: Department of Cellular and Molecular Biology; John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Cynthia D
authors.initials: CD
authors.lastname: Anderson
authors.affiliation: Department of Anatomy, Biochemistry, and Physiology, John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Johann
authors.initials: J
authors.lastname: Urschitz
authors.affiliation: Department of Medicine, John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Mark
authors.initials: M
authors.lastname: Khemmani
authors.affiliation: Department of Anatomy, Biochemistry, and Physiology, John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Jesse B
authors.initials: JB
authors.lastname: Owens
authors.affiliation: Department of Anatomy, Biochemistry, and Physiology, John A. Burns School of Medicine, Honolulu, HI 96813.|Manoa Biosciences.
authors.forename: Stefan
authors.initials: S
authors.lastname: Moisyadi
authors.affiliation: Department of Medicine, John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Ralph V
authors.initials: RV
authors.lastname: Shohet
authors.affiliation: Department of Medicine, John A. Burns School of Medicine, Honolulu, HI 96813.
authors.forename: Chad B
authors.initials: CB
authors.lastname: Walton
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics; D003692:Delayed-Action Preparations / Q000008:administration & dosage / Q000737:chemistry / Q000528:radiation effects*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D057809:HEK293 Cells; D019220:High-Energy Shock Waves; D006801:Humans; D008099:Liver / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011829:Radiation Dosage; D013010:Sonication / Q000379:methods*; D014162:Transfection / Q000379:methods*; D019895:Transposases / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D003692:Delayed-Action Preparations; D019895:Transposases
keywords: Hepatic gene therapy; Microbubbles; Site-specific chromosomal integration; Transposase; Ultrasound
references.citation: Mol Ther. 2007 Jan;15(1):139-45
references.pmid: 17164785
references.citation: J Vis Exp. 2011 Jun 12;(52):
references.pmid: 21694693
references.citation: J Ultrasound Med. 2000 Feb;19(2):143-8, 154-68
references.pmid: 10680619
references.citation: J Immunother. 2009 Oct;32(8):837-44
references.pmid: 19752750
references.citation: Circ Res. 2009 Mar 13;104(5):563-5
references.pmid: 19286610
references.citation: Mol Ther. 2010 Apr;18(4):707-14
references.pmid: 20104210
references.citation: Circ Res. 2012 Mar 2;110(5):777-93
references.pmid: 22383712
references.citation: Mol Ther. 2009 Dec;17(12):2115-20
references.pmid: 19809403
references.citation: Proc Natl Acad Sci U S A. 2006 Oct 10;103(41):15008-13
references.pmid: 17005721
references.citation: Nat Rev Genet. 2011 May;12(5):316-28
references.pmid: 21468099
references.citation: Nature. 2002 Dec 5;420(6915):520-62
references.pmid: 12466850
references.citation: Mol Ther. 2001 Jun;3(6):947-57
references.pmid: 11407909
references.citation: Proc Natl Acad Sci U S A. 2010 May 4;107(18):8117-22
references.pmid: 20404201
references.citation: Hum Gene Ther. 2005 Jan;16(1):126-31
references.pmid: 15703495
references.citation: J Gene Med. 2007 Oct;9(10):833-42
references.pmid: 17721874
references.citation: Nucleic Acids Res. 2007;35(12):e87
references.pmid: 17576687
references.citation: Circulation. 2003 Aug 26;108(8):1022-6
references.pmid: 12912823
references.citation: Mol Ther. 2003 Sep;8(3):495-500
references.pmid: 12946323
references.citation: Nucleic Acids Res. 2011 Dec;39(22):e148
references.pmid: 21948799
references.citation: Gene Ther. 2008 Dec;15(24):1593-605
references.pmid: 18633447
references.citation: Curr Gene Ther. 2004 Mar;4(1):89-113
references.pmid: 15032617
references.citation: Circulation. 2009 Sep 15;120(11 Suppl):S230-7
references.pmid: 19752373
references.citation: Mol Ther. 2008 Mar;16(3):548-56
references.pmid: 18253155
references.citation: Haemophilia. 2004 Jul;10(4):309-18
references.pmid: 15230943
references.citation: Hum Gene Ther. 2012 Mar;23(3):311-20
references.pmid: 21992617
references.citation: Nat Protoc. 2010 Aug;5(8):1379-95
references.pmid: 20671722
references.citation: BMC Biotechnol. 2011 Mar 30;11:28
references.pmid: 21447194
references.citation: Hum Gene Ther. 2005 Jul;16(7):893-905
references.pmid: 16000070
references.citation: N Engl J Med. 2011 Dec 22;365(25):2357-65
references.pmid: 22149959
references.citation: J Am Coll Cardiol. 2005 Feb 1;45(3):329-35
references.pmid: 15680708
references.citation: Mol Ther. 2010 Jun;18(6):1200-9
references.pmid: 20372108
references.citation: J Gene Med. 2004 Apr;6(4):395-404
references.pmid: 15079814
references.citation: Nat Genet. 2009 Jun;41(6):753-61
references.pmid: 19412179
references.citation: Genetica. 1996 Jul;98(1):33-41
references.pmid: 8765680
references.citation: Gene Ther. 2008 Aug;15(16):1147-55
references.pmid: 18385766
references.citation: Nat Rev Drug Discov. 2004 Jun;3(6):527-32
references.pmid: 15173842