title: Evaluation of lateral spread of transgene expression following subretinal AAV-mediated gene delivery in dogs.
abstract: Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these AAV serotypes.
authors.affiliation: Department of Small Animal Clinical Sciences, Michigan State University, East Lansing, MI, USA.
authors.forename: Ashlee R
authors.initials: AR
authors.lastname: Bruewer
authors.forename: Freya M
authors.initials: FM
authors.lastname: Mowat
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Bartoe
authors.forename: Sanford L
authors.initials: SL
authors.lastname: Boye
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: Simon M
authors.initials: SM
authors.lastname: Petersen-Jones
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D004285:Dogs; D004596:Electroretinography; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D056965:Injections, Intraocular; D008297:Male; D008856:Microscopy, Fluorescence; D009900:Optic Nerve / Q000648:ultrastructure / Q000821:virology*; D011401:Promoter Regions, Genetic; D012160:Retina / Q000648:ultrastructure / Q000821:virology*; D012163:Retinal Detachment / Q000503:physiopathology / Q000821:virology*; D014779:Virus Replication
chemical_list: D000199:Actins; D036022:Capsid Proteins; D049452:Green Fluorescent Proteins
references.citation: Invest Ophthalmol Vis Sci. 2009 Feb;50(2):801-13
references.pmid: 18775863
references.citation: Mol Ther. 2008 May;16(5):916-23
references.pmid: 18388922
references.citation: Mol Ther. 2009 Mar;17(3):463-71
references.pmid: 19066593
references.citation: N Engl J Med. 2008 May 22;358(21):2231-9
references.pmid: 18441371
references.citation: Hum Mol Genet. 2002 Aug 1;11(16):1823-33
references.pmid: 12140185
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: Gene Ther. 2003 Dec;10(26):2112-8
references.pmid: 14625565
references.citation: Methods Enzymol. 2000;316:743-61
references.pmid: 10800712
references.citation: Mol Ther. 2011 Feb;19(2):293-301
references.pmid: 21045809
references.citation: Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7827-32
references.pmid: 18511559
references.citation: Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1669-81
references.pmid: 16565408
references.citation: Invest Ophthalmol Vis Sci. 2007 May;48(5):1959-67
references.pmid: 17460247
references.citation: Proc Natl Acad Sci U S A. 1993 May 1;90(9):3968-72
references.pmid: 8387203
references.citation: Vision Res. 2009 Nov;49(22):2636-52
references.pmid: 19375447
references.citation: Cell Transplant. 2001;10(3):317-27
references.pmid: 11437077
references.citation: Curr Gene Ther. 2005 Jun;5(3):339-48
references.pmid: 15975011
references.citation: Gene Ther. 2008 Mar;15(6):463-7
references.pmid: 18004402
references.citation: Mol Ther. 2012 Nov;20(11):2019-30
references.pmid: 22828504
references.citation: Arch Ophthalmol. 2012 Jan;130(1):9-24
references.pmid: 21911650
references.citation: Mol Vis. 2009 Sep 11;15:1835-42
references.pmid: 19756181
references.citation: Vet Ophthalmol. 2009 May-Jun;12(3):192-204
references.pmid: 19392879
references.citation: Mol Ther. 2008 Oct;16(10):1648-56
references.pmid: 18682697
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6328-33
references.pmid: 11972042
references.citation: Hum Mol Genet. 2010 Jul 1;19(13):2581-93
references.pmid: 20378608
references.citation: Nat Genet. 2001 May;28(1):92-5
references.pmid: 11326284
references.citation: Am J Ophthalmol. 2007 Dec;144(6):791-811
references.pmid: 17964524
references.citation: Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):2132-7
references.pmid: 22308428
references.citation: Gene Ther. 2008 Jun;15(11):849-57
references.pmid: 18418417
references.citation: Adv Exp Med Biol. 2008;613:21-35
references.pmid: 18188925