title: Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases.
abstract: Mutations in the retinal pigment epithelium (RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are non-toxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy.
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, BMSB 781, Oklahoma City, OK 73104, USA.
authors.forename: Adarsha
authors.initials: A
authors.lastname: Koirala
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Conley
authors.forename: Rasha
authors.initials: R
authors.lastname: Makkia
authors.forename: Zhao
authors.initials: Z
authors.lastname: Liu
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Cooper
authors.forename: Janet R
authors.initials: JR
authors.lastname: Sparrow
authors.forename: Muna I
authors.initials: MI
authors.lastname: Naash
mesh_terms: D000818:Animals; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000627:therapeutic use; D017353:Gene Deletion; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology*; D019745:cis-trans-Isomerases / Q000235:genetics*; D000099178:Retinoid Isomerohydrolase
chemical_list: D004247:DNA; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
keywords: DNA nanoparticle; Non-viral gene therapy; RPE65; Retinal pigment epithelium; S/MAR
references.citation: J Gene Med. 2009 Jun;11(6):486-97
references.pmid: 19340848
references.citation: Graefes Arch Clin Exp Ophthalmol. 2012 Aug;250(8):1117-28
references.pmid: 22644094
references.citation: Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1473-9
references.pmid: 15790918
references.citation: Hum Gene Ther. 2008 Oct;19(10):979-90
references.pmid: 18774912
references.citation: FASEB J. 2010 Apr;24(4):1178-91
references.pmid: 19952284
references.citation: J Clin Invest. 2012 Sep;122(9):3221-6
references.pmid: 22886305
references.citation: Nanomedicine (Lond). 2012 Apr;7(4):521-39
references.pmid: 22356602
references.citation: Mol Imaging. 2011 Oct;10(5):327-39
references.pmid: 21521549
references.citation: N Engl J Med. 2008 May 22;358(21):2240-8
references.pmid: 18441370
references.citation: Mol Ther. 2006 Mar;13(3):565-72
references.pmid: 16223604
references.citation: J Biol Chem. 1997 Apr 25;272(17):11463-70
references.pmid: 9111059
references.citation: J Neurosci. 2011 Jul 13;31(28):10403-11
references.pmid: 21753017
references.citation: Am J Ophthalmol. 2007 Dec;144(6):791-811
references.pmid: 17964524
references.citation: Mol Ther. 2006 Nov;14(5):613-26
references.pmid: 16784894
references.citation: Mol Cell Biol. 2002 Apr;22(8):2598-606
references.pmid: 11909954
references.citation: Hum Gene Ther. 2009 Sep;20(9):999-1004
references.pmid: 19583479
references.citation: Chromosome Res. 2003;11(5):413-21
references.pmid: 12971718
references.citation: J Biol Chem. 2002 May 24;277(21):19173-82
references.pmid: 11897783
references.citation: Nat Genet. 1998 Dec;20(4):344-51
references.pmid: 9843205
references.citation: Gene Ther. 2006 Jul;13(13):1048-51
references.pmid: 16525478
references.citation: Invest Ophthalmol Vis Sci. 2006 Mar;47(3):1177-84
references.pmid: 16505056
references.citation: Biochemistry. 2000 Sep 19;39(37):11370-80
references.pmid: 10985782
references.citation: Mol Cell Biol. 2005 Mar;25(6):2260-72
references.pmid: 15743822
references.citation: J Biol Chem. 2003 Aug 29;278(35):32578-86
references.pmid: 12807905
references.citation: PLoS Med. 2006 Oct;3(10):e347
references.pmid: 17032058
references.citation: Curr Biol. 2009 Oct 13;19(19):1665-9
references.pmid: 19781940
references.citation: Mol Ther. 2009 Apr;17(4):641-50
references.pmid: 19223866
references.citation: PLoS Med. 2005 Nov;2(11):e333
references.pmid: 16250670
references.citation: FASEB J. 2006 Oct;20(12):2036-49
references.pmid: 17012256
references.citation: Chromosome Res. 2003;11(5):435-45
references.pmid: 12971720
references.citation: PLoS One. 2012;7(12):e52189
references.pmid: 23272225
references.citation: Hum Gene Ther. 2004 Dec;15(12):1255-69
references.pmid: 15684701
references.citation: Prog Retin Eye Res. 2010 Sep;29(5):376-97
references.pmid: 20452457
references.citation: Mol Ther. 2012 Jan;20(1):63-72
references.pmid: 21952168
references.citation: Neuroscience. 2011 Oct 27;194:220-6
references.pmid: 21839809
references.citation: Methods Enzymol. 2000;316:313-24
references.pmid: 10800683
references.citation: Gene Ther. 2011 Jan;18(1):82-7
references.pmid: 20739959
references.citation: EMBO Rep. 2002 Apr;3(4):349-54
references.pmid: 11897664
references.citation: Mol Ther. 2007 Jul;15(7):1348-55
references.pmid: 17457320
references.citation: Arch Ophthalmol. 2012 Jan;130(1):9-24
references.pmid: 21911650
references.citation: PLoS One. 2006 Dec 20;1:e38
references.pmid: 17183666
references.citation: J Mol Biol. 1997 Nov 28;274(2):181-96
references.pmid: 9398526
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 30;102(35):12413-8
references.pmid: 16116091
references.citation: Biomaterials. 2011 Dec;32(35):9483-93
references.pmid: 21885113
references.citation: Mol Ther. 2003 Dec;8(6):936-47
references.pmid: 14664796
references.citation: Genet Vaccines Ther. 2004 Apr 27;2:3
references.pmid: 15109394
references.citation: Hum Mol Genet. 2013 Apr 15;22(8):1632-42
references.pmid: 23335596
references.citation: Mol Ther. 2003 Dec;8(6):948-56
references.pmid: 14664797
references.citation: PLoS One. 2009;4(4):e5290
references.pmid: 19390689
references.citation: Mol Vis. 2005 Feb 28;11:152-62
references.pmid: 15765048
references.citation: Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3088-93
references.pmid: 9501220
references.citation: Gene Ther. 2010 Jul;17(7):815-26
references.pmid: 20237510
references.citation: PLoS One. 2009 Oct 12;4(10):e7410
references.pmid: 19823583