AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes.


Journal

Gene therapy
ISSN: 1476-5462
Titre abrégé: Gene Ther
Pays: England
ID NLM: 9421525

Informations de publication

Date de publication:
Mar 2024
Historique:
received: 29 09 2023
accepted: 19 12 2023
revised: 12 12 2023
medline: 18 3 2024
pubmed: 11 1 2024
entrez: 10 1 2024
Statut: ppublish

Résumé

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

Identifiants

pubmed: 38200264
doi: 10.1038/s41434-023-00436-8
pii: 10.1038/s41434-023-00436-8
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

175-186

Subventions

Organisme : U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)
ID : EY019014
Organisme : U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)
ID : EY030163
Organisme : Robert Wood Johnson Foundation (RWJF)
ID : Harold Amos Faculty Development Award

Informations de copyright

© 2024. The Author(s), under exclusive licence to Springer Nature Limited.

Références

Wong KA, Benowitz LI. Retinal ganglion cell survival and axon regeneration after optic nerve injury: role of inflammation and other factors. Int J Mol Sci. 2022;23:10179.
pubmed: 36077577 pmcid: 9456227 doi: 10.3390/ijms231710179
Sanz-Morello B, Ahmadi H, Vohra R, Saruhanian S, Freude KK, Hamann S, et al. Oxidative stress in optic neuropathies. Antioxidants. 2021;10:1538.
pubmed: 34679672 pmcid: 8532958 doi: 10.3390/antiox10101538
Newman NJ, Yu-Wai-Man P, Biousse V, Carelli V. Understanding the molecular basis and pathogenesis of hereditary optic neuropathies: towards improved diagnosis and management. Lancet Neurol. 2023;22:172–88.
pubmed: 36155660 doi: 10.1016/S1474-4422(22)00174-0
Quigley HA. Glaucoma. Lancet. 2011;377:1367–77.
pubmed: 21453963 doi: 10.1016/S0140-6736(10)61423-7
Shu DY, Chaudhary S, Cho KS, Lennikov A, Miller WP, Thorn DC, et al. Role of oxidative stress in ocular diseases: a balancing act. Metabolites. 2023;13:187.
pubmed: 36837806 pmcid: 9960073 doi: 10.3390/metabo13020187
Fague L, Liu YA, Marsh-Armstrong N. The basic science of optic nerve regeneration. Ann Transl Med. 2021;9:1276.
pubmed: 34532413 pmcid: 8421956 doi: 10.21037/atm-20-5351
Levin LA, Patrick C, Choudry NB, Sharif NA, Goldberg JL. Neuroprotection in neurodegenerations of the brain and eye: lessons from the past and directions for the future. Front Neurol. 2022;13:964197.
pubmed: 36034312 pmcid: 9412944 doi: 10.3389/fneur.2022.964197
Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358–78.
pubmed: 30710128 pmcid: 6927556 doi: 10.1038/s41573-019-0012-9
Zinn E, Vandenberghe LH. Adeno-associated virus: fit to serve. Curr Opin Virol. 2014;8:90–7.
pubmed: 25128609 doi: 10.1016/j.coviro.2014.07.008
Cwerman-Thibault H, Augustin S, Ellouze S, Sahel JA, Corral-Debrinski M. Gene therapy for mitochondrial diseases: leber hereditary optic neuropathy as the first candidate for a clinical trial. C R Biol. 2014;337:193–206.
pubmed: 24702846 doi: 10.1016/j.crvi.2013.11.011
Martin KR, Quigley HA. Gene therapy for optic nerve disease. Eye. 2004;18:1049–55.
pubmed: 15534589 doi: 10.1038/sj.eye.6701579
Mak KY, Rajapaksha IG, Angus PW, Herath CB. The adeno-associated virus - a safe and promising vehicle for liverspecific gene therapy of inherited and non-inherited disorders. Curr Gene Ther. 2017;17:4–16.
pubmed: 28292253 doi: 10.2174/1566523217666170314141931
Bennett J. Taking stock of retinal gene therapy: looking back and moving forward. Mol Ther. 2017;25:1076–94.
pubmed: 28391961 pmcid: 5417792 doi: 10.1016/j.ymthe.2017.03.008
Gao J, Hussain RM, Weng CY. Voretigene neparvovec in retinal diseases: a review of the current clinical evidence. Clin Ophthalmol. 2020;14:3855–69.
pubmed: 33223822 pmcid: 7671481 doi: 10.2147/OPTH.S231804
Russell S, Bennett J, Wellman JA, Chung DC, Yu ZF, Tillman A, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–60.
pubmed: 28712537 pmcid: 5726391 doi: 10.1016/S0140-6736(17)31868-8
Buning H, Perabo L, Coutelle O, Quadt-Humme S, Hallek M. Recent developments in adeno-associated virus vector technology. J Gene Med. 2008;10:717–33.
pubmed: 18452237 doi: 10.1002/jgm.1205
Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol. 2016;21:75–80.
pubmed: 27596608 pmcid: 5138125 doi: 10.1016/j.coviro.2016.08.003
O’Donnell J, Taylor KA, Chapman MS. Adeno-associated virus-2 and its primary cellular receptor–Cryo-EM structure of a heparin complex. Virology. 2009;385:434–43.
pubmed: 19144372 doi: 10.1016/j.virol.2008.11.037
Hadaczek P, Mirek H, Bringas J, Cunningham J, Bankiewicz K. Basic fibroblast growth factor enhances transduction, distribution, and axonal transport of adeno-associated virus type 2 vector in rat brain. Hum Gene Ther. 2004;15:469–79.
pubmed: 15144577 doi: 10.1089/10430340460745793
Kashiwakura Y, Tamayose K, Iwabuchi K, Hirai Y, Shimada T, Matsumoto K, et al. Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection. J Virol. 2005;79:609–14.
pubmed: 15596854 pmcid: 538679 doi: 10.1128/JVI.79.1.609-614.2005
Asokan A, Hamra JB, Govindasamy L, Agbandje-McKenna M, Samulski RJ. Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry. J Virol. 2006;80:8961–9.
pubmed: 16940508 pmcid: 1563945 doi: 10.1128/JVI.00843-06
Kurzeder C, Koppold B, Sauer G, Pabst S, Kreienberg R, Deissler H. CD9 promotes adeno-associated virus type 2 infection of mammary carcinoma cells with low cell surface expression of heparan sulphate proteoglycans. Int J Mol Med. 2007;19:325–33.
pubmed: 17203208
Van Vliet KM, Blouin V, Brument N, Agbandje-McKenna M, Snyder RO. The role of the adeno-associated virus capsid in gene transfer. Methods Mol Biol. 2008;437:51–91.
pubmed: 18369962 pmcid: 7120696 doi: 10.1007/978-1-59745-210-6_2
Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol. 2002;76:791–801.
pubmed: 11752169 pmcid: 136844 doi: 10.1128/JVI.76.2.791-801.2002
Ramachandran PS, Lee V, Wei Z, Song JY, Casal G, Cronin T, et al. Evaluation of dose and safety of AAV7m8 and AAV8BP2 in the non-human primate retina. Hum Gene Ther. 2017;28:154–67.
pubmed: 27750461 pmcid: 5312498 doi: 10.1089/hum.2016.111
Ross AG, Chaqour B, McDougald DS, Dine KE, Duong TT, Shindler RE, et al. Selective upregulation of sirt1 expression in retinal ganglion cells by AAV-mediated gene delivery increases neuronal cell survival and alleviates axon demyelination associated with optic neuritis. Biomolecules. 2022;12:830.
pubmed: 35740955 pmcid: 9221096 doi: 10.3390/biom12060830
Yue J, Khan RS, Duong TT, Dine KE, Cui QN, O’Neill N, et al. Cell-specific expression of human sirt1 by gene therapy reduces retinal ganglion cell loss induced by elevated intraocular pressure. Neurotherapeutics. 2023;20:896–907.
pubmed: 36941497 doi: 10.1007/s13311-023-01364-6
McDougald DS, Dine KE, Zezulin AU, Bennett J, Shindler KS. SIRT1 and NRF2 gene transfer mediate distinct neuroprotective effects upon retinal ganglion cell survival and function in experimental optic neuritis. Invest Ophthalmol Vis Sci. 2018;59:1212–20.
pubmed: 29494741 pmcid: 5839257 doi: 10.1167/iovs.17-22972
Alam F, Syed H, Amjad S, Baig M, Khan TA, Rehman R. Interplay between oxidative stress, SIRT1, reproductive and metabolic functions. Curr Res Physiol. 2021;4:119–24.
pubmed: 34746831 pmcid: 8562188 doi: 10.1016/j.crphys.2021.03.002
Singh V, Ubaid S. Role of silent information regulator 1 (SIRT1) in regulating oxidative stress and inflammation. Inflammation. 2020;43:1589–98.
pubmed: 32410071 doi: 10.1007/s10753-020-01242-9
Li L, Zhi D, Cheng R, Li J, Luo C, Li H. The neuroprotective role of SIRT1/PGC-1alpha signaling in limb postconditioning in cerebral ischemia/reperfusion injury. Neurosci Lett. 2021;749:135736.
pubmed: 33600904 doi: 10.1016/j.neulet.2021.135736
Nita M, Grzybowski A. Interplay between reactive oxygen species and autophagy in the course of age-related macular degeneration. EXCLI J. 2020;19:1353–71.
pubmed: 33192217 pmcid: 7658465
Samarin J, Wessel J, Cicha I, Kroening S, Warnecke C, Goppelt-Struebe M. FoxO proteins mediate hypoxic induction of connective tissue growth factor in endothelial cells. J Biol Chem. 2010;285:4328–36.
pubmed: 20018872 doi: 10.1074/jbc.M109.049650
Chaffiol A, Caplette R, Jaillard C, Brazhnikova E, Desrosiers M, Dubus E, et al. A new promoter allows optogenetic vision restoration with enhanced sensitivity in macaque retina. Mol Ther. 2017;25:2546–60.
pubmed: 28807567 pmcid: 5675708 doi: 10.1016/j.ymthe.2017.07.011
Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O, et al. Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther. 2008;16:458–65.
pubmed: 18209734 doi: 10.1038/sj.mt.6300389
Ross AG, McDougald DS, Khan RS, Duong TT, Dine KE, Aravand P, et al. Rescue of retinal ganglion cells in optic nerve injury using cell-selective AAV mediated delivery of SIRT1. Gene Ther. 2021;28:256–64.
pubmed: 33589779 pmcid: 8149296 doi: 10.1038/s41434-021-00219-z
Zuo L, Khan RS, Lee V, Dine K, Wu W, Shindler KS. SIRT1 promotes RGC survival and delays loss of function following optic nerve crush. Invest Ophthalmol Vis Sci. 2013;54:5097–102.
pubmed: 23821198 pmcid: 3726244 doi: 10.1167/iovs.13-12157
Cronin T, Vandenberghe LH, Hantz P, Juttner J, Reimann A, Kacso AE, et al. Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter. EMBO Mol Med. 2014;6:1175–90.
pubmed: 25092770 pmcid: 4197864 doi: 10.15252/emmm.201404077
Xu L, Daly T, Gao C, Flotte TR, Song S, Byrne BJ, et al. CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Hum Gene Ther. 2001;12:563–73.
pubmed: 11268288 doi: 10.1089/104303401300042500
Boye SE, Alexander JJ, Witherspoon CD, Boye SL, Peterson JJ, Clark ME, et al. Highly efficient delivery of adeno-associated viral vectors to the primate retina. Hum Gene Ther. 2016;27:580–97.
pubmed: 27439313 pmcid: 4991591 doi: 10.1089/hum.2016.085
Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther. 1999;10:2295–305.
pubmed: 10515449 doi: 10.1089/10430349950016942
Popa I, Harris ME, Donello JE, Hope TJ. CRM1-dependent function of a cis-acting RNA export element. Mol Cell Biol. 2002;22:2057–67.
pubmed: 11884594 pmcid: 133666 doi: 10.1128/MCB.22.7.2057-2067.2002
Graveley BR, Hertel KJ, Maniatis T. The role of U2AF35 and U2AF65 in enhancer-dependent splicing. RNA. 2001;7:806–18.
pubmed: 11421359 pmcid: 1370132 doi: 10.1017/S1355838201010317
Xu DH, Wang XY, Jia YL, Wang TY, Tian ZW, Feng X, et al. SV40 intron, a potent strong intron element that effectively increases transgene expression in transfected Chinese hamster ovary cells. J Cell Mol Med. 2018;22:2231–9.
pubmed: 29441681 pmcid: 5867124 doi: 10.1111/jcmm.13504
Rotondaro L, Mele A, Rovera G. Efficiency of different viral promoters in directing gene expression in mammalian cells: effect of 3’-untranslated sequences. Gene. 1996;168:195–8.
pubmed: 8654943 doi: 10.1016/0378-1119(95)00767-9
Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene. 2001;272:149–56.
pubmed: 11470520 doi: 10.1016/S0378-1119(01)00550-9
Hermonat PL, Quirk JG, Bishop BM, Han L. The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors. FEBS Lett. 1997;407:78–84.
pubmed: 9141485 doi: 10.1016/S0014-5793(97)00311-6
Tenenbaum L, Chtarto A, Lehtonen E, Velu T, Brotchi J, Levivier M. Recombinant AAV-mediated gene delivery to the central nervous system. J Gene Med. 2004;6:S212–22.
pubmed: 14978764 doi: 10.1002/jgm.506
Prosch S, Stein J, Staak K, Liebenthal C, Volk HD, Kruger DH. Inactivation of the very strong HCMV immediate early promoter by DNA CpG methylation in vitro. Biol Chem Hoppe Seyler. 1996;377:195–201.
pubmed: 8722321 doi: 10.1515/bchm3.1996.377.3.195
Chintalapudi SR, Morales-Tirado VM, Williams RW, Jablonski MM. Multipronged approach to identify and validate a novel upstream regulator of Sncg in mouse retinal ganglion cells. FEBS J. 2016;283:678–93.
pubmed: 26663874 doi: 10.1111/febs.13620
Claes M, Moons L. Retinal ganglion cells: global number, density and vulnerability to glaucomatous injury in common laboratory mice. Cells. 2022;11:2689.
pubmed: 36078097 pmcid: 9454702 doi: 10.3390/cells11172689
Rodriguez AR, de Sevilla Muller LP, Brecha NC. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina. J Comp Neurol. 2014;522:1411–43.
pubmed: 24318667 pmcid: 3959221 doi: 10.1002/cne.23521
Wang Q, Zhuang P, Huang H, Li L, Liu L, Webber HC, et al. Mouse gamma-synuclein promoter-mediated gene expression and editing in mammalian retinal ganglion cells. J Neurosci. 2020;40:3896–914.
pubmed: 32300046 pmcid: 7219295 doi: 10.1523/JNEUROSCI.0102-20.2020
Ge J, Jin L, Tang X, Gao D, An Q, Ping W. Optimization of eGFP expression using a modified baculovirus expression system. J Biotechnol. 2014;173:41–6.
pubmed: 24445173 doi: 10.1016/j.jbiotec.2014.01.003
Martin KR, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta D, et al. Gene therapy with brain-derived neurotrophic factor as a protection: retinal ganglion cells in a rat glaucoma model. Invest Ophthalmol Vis Sci. 2003;44:4357–65.
pubmed: 14507880 doi: 10.1167/iovs.02-1332
Powell SK, Rivera-Soto R, Gray SJ. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy. Discov Med. 2015;19:49–57.
pubmed: 25636961 pmcid: 4505817
Wang L, Wang Z, Zhang F, Zhu R, Bi J, Wu J, et al. Enhancing transgene expression from recombinant AAV8 vectors in different tissues using woodchuck hepatitis virus post-transcriptional regulatory element. Int J Med Sci. 2016;13:286–91.
pubmed: 27076785 pmcid: 4829541 doi: 10.7150/ijms.14152
Gruh I, Wunderlich S, Winkler M, Schwanke K, Heinke J, Blomer U, et al. Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors. J Gene Med. 2008;10:21–32.
pubmed: 18022932 doi: 10.1002/jgm.1122
Ghazal P, Lubon H, Hennighausen L. Multiple sequence-specific transcription factors modulate cytomegalovirus enhancer activity in vitro. Mol Cell Biol. 1988;8:1809–11.
pubmed: 2837656 pmcid: 363343
Le Hir H, Nott A, Moore MJ. How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci. 2003;28:215–20.
pubmed: 12713906 doi: 10.1016/S0968-0004(03)00052-5
Huang MT, Gorman CM. Intervening sequences increase efficiency of RNA 3’ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 1990;18:937–47.
pubmed: 1690394 pmcid: 330348 doi: 10.1093/nar/18.4.937
Fonseca-Kelly Z, Nassrallah M, Uribe J, Khan RS, Dine K, Dutt M, et al. Resveratrol neuroprotection in a chronic mouse model of multiple sclerosis. Front Neurol. 2012;3:84.
pubmed: 22654783 pmcid: 3359579 doi: 10.3389/fneur.2012.00084
Shindler KS, Ventura E, Rex TS, Elliott P, Rostami A. SIRT1 activation confers neuroprotection in experimental optic neuritis. Invest Ophthalmol Vis Sci. 2007;48:3602–9.
pubmed: 17652729 doi: 10.1167/iovs.07-0131

Auteurs

Brahim Chaqour (B)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Thu T Duong (TT)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
GlaxoSmithKline, Collegeville, PA, 19426, USA.

Jipeng Yue (J)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Tehui Liu (T)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
Spark Therapeutics, Philadelphia, PA, 19104, USA.

David Camacho (D)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Kimberly E Dine (KE)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Julian Esteve-Rudd (J)

Gyroscope Therapeutics Limited, a Novartis Company, London, N7 9AS, UK.

Scott Ellis (S)

Gyroscope Therapeutics Limited, a Novartis Company, London, N7 9AS, UK.

Jean Bennett (J)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Kenneth S Shindler (KS)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA. kenneth.shindler@pennmedicine.upenn.edu.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. kenneth.shindler@pennmedicine.upenn.edu.
Department of Neurology, University of Pennsylvania, Philadelphia, PA, 19104, USA. kenneth.shindler@pennmedicine.upenn.edu.

Ahmara G Ross (AG)

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA, 19104, USA. ahmara.ross@pennmedicine.upenn.edu.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. ahmara.ross@pennmedicine.upenn.edu.
Department of Neurology, University of Pennsylvania, Philadelphia, PA, 19104, USA. ahmara.ross@pennmedicine.upenn.edu.

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