Challenges to Gene Editing Approaches in the Retina

Abstract

Retinal gene therapy has recently been at the cutting edge of clinical development in the diverse field of genetic therapies. The retina is an attractive target for genetic therapies such as gene editing due to the distinctive anatomical and immunological features of the eye, known as immune privilege, so that inherited retinal diseases (IRDs) have been studied in several clinical studies. Thus, rapid strides are being made toward developing targeted treatments for IRDs. Gene editing in the retina faces a group of heterogenous challenges, including editing efficiencies, off-target effects, the anatomy of the target organ, immune responses, inactivation, and identifying optimal application methods. As clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) based technologies are at the forefront of current gene editing advances, their specific editing efficiency challenges and potential off-target effects were assessed. The immune privilege of the eye reduces the likelihood of systemic immune responses following retinal gene therapy, but possible immune responses must not be discounted. Immune responses to gene editing in the retina may be humoral or cell mediated, with immunologically active cells, including microglia, implicated in facilitating possible immune responses to gene editing. Immunogenicity of gene therapeutics may also lead to the inactivation of edited cells, reducing potential therapeutic benefits. This review outlines the broad spectrum of potential challenges currently facing retinal gene editing, with the goal of facilitating further advances in the safety and efficacy of gene editing therapies.

Zusammenfassung

Im vielfältigen Gebiet der Gentherapien steht die retinale Gentherapie in der vordersten Reihe der klinischen Entwicklung und findet zunehmend Anwendung im klinischen Alltag. Die Netzhaut ist aufgrund der einzigartigen anatomischen und immunologischen Eigenschaften des Auges, bezeichnet als Immunprivileg, ein attraktives Anwendungsgebiet für Gentherapien, wie etwa Geneditierung. Daher werden insbesondere die hereditären Netzhauterkrankungen (engl: Inherited retinal Dystrophies [IRDs]) in mehreren klinischen Studien untersucht. Folglich wurden rasche Fortschritte bei der Entwicklung zielgerichteter Therapien für IRDs gemacht. Dennoch steht die retinale Geneditierung zahlreichen heterogenen Herausforderungen, wie etwa deren Effizienz, Off-Target-Effekten, der Anatomie des Zielorgans, Immunreaktionen, der Inaktivierung und Ermittlung optimaler Anwendungsmethoden, gegenüber. Da Gentherapien basierend auf der CRISPR/Cas-Technologie (Clustered regular interspaced palindromic Repeats/CRISPR-assoziierte Nuklease) zunehmend an Bedeutung gewinnen, wurden ihre spezifischen Herausforderungen in Bezug auf die Editing-Effizienz und mögliche Off-Target-Effekte beurteilt. Das Immunprivileg des Auges verringert die Wahrscheinlichkeit systemischer Immunreaktionen nach einer retinalen Gentherapie, dennoch dürfen mögliche Immunreaktionen nicht außer Acht gelassen werden. Diese Immunreaktionen, induziert durch Geneditierung, können humoraler sowie zellvermittelter Natur sein, wobei immunologisch aktive Zellen wie Mikroglia mögliche Immunreaktionen auf die Geneditierung begünstigen können. Eine Immunogenität der Gentherapeutika kann auch zur Inaktivierung der editierten Zellen führen, was eine Reduzierung des therapeutischen Nutzens zur Folge hätte. Dieses Review evaluiert das breite Spektrum der Herausforderungen, denen die Geneditierung retinaler Zellen derzeit gegenübersteht, mit dem Ziel, weitere Fortschritte bei der Sicherheit und Wirksamkeit von Therapien mit Geneditierung zu ermöglichen.

Key words

retina - genetics - molecular biology

Schlüsselwörter

Netzhaut - Genetik - Molekularbiologie

Publication History

Received: 29 September 2021

Accepted: 31 January 2022

Article published online:
22 March 2022

• References

• 1 Ledford H. CRISPR treatment inserted directly into the body for first time. Nature 2020; 579: 185
• 2 Ziccardi L, Cordeddu V, Gaddini L. et al. Gene therapy in retinal dystrophies. Int J Mol Sci 2019; 20: 5722
• 3 Maeder ML, Gersbach CA. Genome-editing Technologies for Gene and Cell Therapy. Mol Ther 2016; 24: 430-446
• 4 Bordet T, Behar-Cohen F. Ocular gene therapies in clinical practice: viral vectors and nonviral alternatives. Drug Discov Today 2019; 24: 1685-1693
• 5 [Anonymous] FDA approves hereditary blindness gene therapy. Nat Biotechnol 2018; 36: 6
• 6 Daiger SP, Sullivan LS, Bowne SJ. Genes and mutations causing retinitis pigmentosa. Clin Genet 2013; 84: 132-141
• 7 Abouzeid H, Li Y, Dharmaraj S. et al. Monogenic inheritance of Leber congenital amaurosis: causation by a novel mutation in CRB1. Invest Ophthalmol Vis Sci 2004; 45: 4730
• 8 Raj RK, Dhoble P, Anjanamurthy R. et al. Genetic characterization of Stargardt clinical phenotype in South Indian patients using sanger and targeted sequencing. Eye Vis (Lond) 2020; 7: 3
• 9 Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013; 31: 397-405
• 10 Jinek M, Chylinski K, Fonfara I. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337: 816-821
• 11 The Royal Swedish Academy of Sciences. Press release: The Nobel Prize in Chemistry 2020. Accessed October 24, 2020 at: https://www.nobelprize.org/prizes/chemistry/2020/press-release/
• 12 Redman M, King A, Watson C. et al. What is CRISPR/Cas9?. Arch Dis Child Educ Pract Ed 2016; 101: 213-215
• 13 Gabriel R, Lombardo A, Arens A. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol 2011; 29: 816-823
• 14 Pattanayak V, Ramirez CL, Joung JK. et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 2011; 8: 765-770
• 15 Gaj T, Liu J, Anderson KE. et al. Protein delivery using Cys2-His2 zinc-finger domains. ACS Chem Biol 2014; 9: 1662-1667
• 16 Gaj T, Guo J, Kato Y. et al. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 2012; 9: 805-807
• 17 Guilinger JP, Pattanayak V, Reyon D. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat Methods 2014; 11: 429-435
• 18 Cradick TJ, Antico CJ, Fine EJ. et al. On-Target Cleavage and Off-Target Activity of TALEN ‘Nickases’. Mol Ther 2013; 21 (Suppl. 01) S125
• 19 Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9. J Clin Invest 2014; 124: 4154
• 20 Cho SW, Kim S, Kim JM. et al. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013; 31: 230-232
• 21 Fu Y, Foden JA, Khayter C. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013; 31: 822-826
• 22 Bruegmann T, Deecke K, Fladung M. Evaluating the efficiency of gRNAs in CRISPR/Cas9 mediated genome editing in poplars. Int J Mol Sci 2019; 20: 3623
• 23 Matson AW, Hosny N, Swanson ZA. et al. Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system. PLoS One 2019; 14: e0226107
• 24 Ryan DE, Taussig D, Steinfeld I. et al. Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res 2018; 46: 792-803
• 25 Maruyama T, Dougan SK, Truttmann MC. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 2015; 33: 538-542
• 26 Yourik P, Fuchs RT, Mabuchi M. et al. Staphylococcus aureus Cas9 is a multiple-turnover enzyme. RNA 2019; 25: 35-44
• 27 Sternberg SH, Redding S, Jinek M. et al. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 2014; 507: 62-67
• 28 Ding X, Seebeck T, Feng Y. et al. Improving CRISPR-Cas9 Genome Editing Efficiency by Fusion with Chromatin-Modulating Peptides. CRISPR J 2019; 2: 51-63
• 29 Park J, Yoon J, Kwon D. et al. Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins. Sci Rep 2021; 11: 16199
• 30 Čermák T, Curtin SJ, Gil-Humanes J. et al. A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants. Plant Cell 2017; 29: 1196-1217
• 31 Spark Therapeutics. Three-year follow-up phase 3 data provide additional information on efficacy, durability and safety of investigational LUXTURNA^™ (voretigene neparvovec) in patients with biallelic RPE65-mediated inherited retinal disease. Accessed September 20, 2020 at: https://sparktx.com/press_releases/three-year-follow-up-phase-3-data-provide-additional-information-on-efficacy-durability-and-safety-of-investigational-luxturna-voretigene-neparvovec-in-patients-with-biallelic-rpe65-mediat/
• 32 Dong B, Nakai H, Xiao W. Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 2010; 18: 87-92
• 33 Odom GL, Gregorevic P, Allen JM. et al. Gene therapy of mdx mice with large truncated dystrophins generated by recombination using rAAV6. Mol Ther 2011; 19: 36-45
• 34 Sondergaard PC, Griffin DA, Pozsgai ER. et al. AAV. Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models. Ann Clin Transl Neurol 2015; 2: 256-270
• 35 Trapani I, Colella P, Sommella A. et al. Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med 2014; 6: 194-211
• 36 Lopes VS, Boye SE, Louie CM. et al. Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus. Gene Ther 2013; 20: 824-833
• 37 Bucher K, Rodríguez-Bocanegra E, Dauletbekov D. et al. Immune responses to retinal gene therapy using adeno-associated viral vectors – Implications for treatment success and safety. Prog Retin Eye Res 2021; 83: 100915
• 38 Dull T, Zufferey R, Kelly M. et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463-8471
• 39 Schlimgen R, Howard J, Wooley D. et al. Risks Associated With Lentiviral Vector Exposures and Prevention Strategies. J Occup Environ Med 2016; 58: 1159
• 40 Koirala A, Conley SM, Naash MI. A review of therapeutic prospects of non-viral gene therapy in the retinal pigment epithelium. Biomaterials 2013; 34: 7158-7167
• 41 Senzer N, Nemunaitis J, Nemunaitis D. et al. Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol Ther 2013; 21: 1096-1103
• 42 Koirala A, Makkia RS, Cooper MJ. et al. Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells. Biomaterials 2011; 32: 9483-9493
• 43 Han Z, Conley SM, Makkia RS. et al. DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice. J Clin Invest 2012; 122: 3221-3226
• 44 Medawar PB. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29: 58-69
• 45 Perez VL, Caspi RR. Immune mechanisms in inflammatory and degenerative eye disease. Trends Immunol 2015; 36: 354
• 46 Streilein JW. Ocular immune privilege: the eye takes a dim but practical view of immunity and inflammation. J Leukoc Biol 2003; 74: 179-185
• 47 Cunha-Vaz J, Bernardes R, Lobo C. Blood-retinal barrier. Eur J Ophthalmol 2011; 21 (Suppl. 06) 3-9
• 48 Streilein JW. Regulation of ocular immune responses. Eye (Lond) 1997; 11: 171-175
• 49 Cousins SW, McCabe MM, Danielpour D. et al. Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Invest Ophthalmol Vis Sci 1991; 32: 2201-2211
• 50 Stein-Streilein J. Immune regulation and the eye. Trends Immunol 2008; 29: 548-554
• 51 Taylor AW, Streilein JW, Cousins SW. Identification of alpha-melanocyte stimulating hormone as a potential immunosuppressive factor in aqueous humor. Curr Eye Res 1992; 11: 1199-1206
• 52 Griffith TS, Brunner T, Fletcher SM. et al. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995; 270: 1189-1192
• 53 Taylor AW. Ocular immunosuppressive microenvironment. Chem Immunol Allergy 2007; 92: 71-85
• 54 Zhou R, Caspi RR. Ocular immune privilege. F1000 Biol Rep 2010; 2: 3
• 55 ClinicalTrials.gov. https://sparktx.com/press_releases/three-year-follow-up-phase-3-data-provide-additional-information-on-efficacy-durability-and-safety-of-investigational-luxturna-voretigene-neparvovec-in-patients-with-biallelic-rpe65-mediat/
• 56 Simó R, Villarroel M, Corraliza L. et al. The retinal pigment epithelium: something more than a constituent of the blood-retinal barrier–implications for the pathogenesis of diabetic retinopathy. J Biomed Biotechnol 2010; 2010: 190724
• 57 Klaassen I, Van Noorden CJF, Schlingemann RO. Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog Retin Eye Res 2013; 34: 19-48
• 58 Naylor A, Hopkins A, Hudson N. et al. Tight Junctions of the Outer Blood Retina Barrier. Int J Mol Sci 2019; 21: 211
• 59 Wang W, Kini A, Wang Y. et al. Metabolic Deregulation of the Blood-Outer Retinal Barrier in Retinitis Pigmentosa. Cell Rep 2019; 28: 1323
• 60 Detrick B, Hooks JJ. Immune regulation in the retina. Immunol Res 2010; 47: 153-161
• 61 Singh PK, Guest JM, Kanwar M. et al. Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes. JCI Insight 2017; 2: e92340
• 62 Wörnle M, Merkle M, Wolf A. et al. Inhibition of TLR3-mediated proinflammatory effects by Alkylphosphocholines in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2011; 52: 6536-6544
• 63 Ashander LM, Lie S, Ma Y. et al. Neutrophil Activities in Human Ocular Toxoplasmosis: An In Vitro Study With Human Cells. Invest Ophthalmol Vis Sci 2019; 60: 4652-4660
• 64 Giese MJ, Rayner SA, Fardin B. et al. Mitigation of neutrophil infiltration in a rat model of early Staphylococcus aureus endophthalmitis. Invest Ophthalmol Vis Sci 2003; 44: 3077-3082
• 65 Chen X, Han R, Hao P. et al. Nepetin inhibits IL-1β induced inflammation via NF-κB and MAPKs signaling pathways in ARPE-19 cells. Biomed Pharmacother 2018; 101: 87-93
• 66 Holtkamp GM, Kijlstra A, Peek R. et al. Retinal pigment epithelium-immune system interactions: cytokine production and cytokine-induced changes. Prog Retin Eye Res 2001; 20: 29-48
• 67 Kauppinen A, Paterno JJ, Blasiak J. et al. Inflammation and its role in age-related macular degeneration. Cell Mol Life Sci 2016; 73: 1765-1786
• 68 von Lintig J, Kiser PD, Golczak M. et al. The biochemical and structural basis for trans-to-cis isomerization of retinoids in the chemistry of vision. Trends Biochem Sci 2010; 35: 400-410
• 69 Vecino E, Rodriguez FD, Ruzafa N. et al. Glia-neuron interactions in the mammalian retina. Prog Retin Eye Res 2016; 51: 1-40
• 70 Rashid K, Akhtar-Schaefer I, Langmann T. Microglia in Retinal Degeneration. Front Immunol 2019; 10: 1975
• 71 Chen M, Xu H. Parainflammation, chronic inflammation, and age-related macular degeneration. J Leukoc Biol 2015; 98: 713-725
• 72 Langmann T. Microglia activation in retinal degeneration. J Leukoc Biol 2007; 81: 1345-1351
• 73 Kohno H, Chen Y, Kevany BM. et al. Photoreceptor proteins initiate microglial activation via Toll-like receptor 4 in retinal degeneration mediated by all-trans-retinal. J Biol Chem 2013; 288: 15326-15341
• 74 Zhao L, Zabel MK, Wang X. et al. Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration. EMBO Mol Med 2015; 7: 1179-1197
• 75 Gupta N, Brown KE, Milam AH. Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res 2003; 76: 463-471
• 76 Alves CH, Wijnholds J. Microglial Cell Dysfunction in CRB1-Associated Retinopathies. Adv Exp Med Biol 2019; 1185: 159-163
• 77 Karlstetter M, Scholz R, Rutar M. et al. Retinal microglia: just bystander or target for therapy?. Prog Retin Eye Res 2015; 45: 30-57
• 78 Wang N, Lai C, Liu C. et al. Origin of fundus hyperautofluorescent spots and their role in retinal degeneration in a mouse model of Goldmann-Favre syndrome. Dis Model Mech 2013; 6: 1113-1122
• 79 Marshall E. Gene therapy death prompts review of adenovirus vector. Science 1999; 286: 2244-2245
• 80 Hösel M, Broxtermann M, Janicki H. et al. Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors. Hepatology 2012; 55: 287-297
• 81 Zhu J, Huang X, Yang Y. The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J Clin Invest 2009; 119: 2388-2398
• 82 Reichel FF, Dauletbekov DL, Klein R. et al. AAV8 Can Induce Innate and Adaptive Immune Response in the Primate Eye. Mol Ther 2017; 25: 2648-2660
• 83 Mingozzi F, Meulenberg JJ, Hui DJ. et al. AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells. Blood 2009; 114: 2077-2086
• 84 Mendell JR, Campbell K, Rodino-Klapac L. et al. Dystrophin immunity in Duchenneʼs muscular dystrophy. N Engl J Med 2010; 363: 1429-1437
• 85 Mendell JR, Rodino-Klapac LR, Rosales-Quintero X. et al. Limb-girdle muscular dystrophy type 2D gene therapy restores α-sarcoglycan and associated proteins. Ann Neurol 2009; 66: 290-297
• 86 Mendell JR, Rodino-Klapac LR, Rosales XQ. et al. Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D. Ann Neurol 2010; 68: 629-638
• 87 Nathwani AC, Tuddenham EGD, Rangarajan S. et al. Adenovirus-associated virus vector–mediated gene transfer in hemophilia B. N Engl J Med 2011; 365: 2357-2365
• 88 Manno CS, Arruda VR, Pierce GF. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342-347
• 89 Machitani M, Sakurai F, Wakabayashi K. et al. Inhibition of CRISPR/Cas9-Mediated Genome Engineering by a Type I Interferon-Induced Reduction in Guide RNA Expression. Biol Pharm Bull 2017; 40: 272-277
• 90 Chen Z, Zhong D, Li G. The role of microglia in viral encephalitis: a review. J Neuroinflammation 2019; 16: 76
• 91 Hooks JJ, Nagineni CN, Hooper LC. et al. IFN-β provides immuno-protection in the retina by inhibiting ICAM-1 and CXCL9 in retinal pigment epithelial cells. J Immunol 2008; 180: 3789-3796
• 92 Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther 2004; 11 (Suppl. 01) S10-S17
• 93 Xiong W, Wu DM, Xue Y. et al. AAV cis-regulatory sequences are correlated with ocular toxicity. Proc Natl Acad Sci U S A 2019; 116: 5785-5794
• 94 Ronzitti G, Gross DA, Mingozzi F. Human Immune Responses to Adeno-Associated Virus (AAV) Vectors. Front Immunol 2020; 11: 670
• 95 Vandenberghe LH, Bell P, Maguire AM. et al. Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey. Sci Transl Med 2011; 3: 88ra54
• 96 Li H, Lasaro MO, Jia B. et al. Capsid-specific T-cell responses to natural infections with adeno-associated viruses in humans differ from those of nonhuman primates. Mol Ther 2011; 19: 2021-2030
• 97 Mingozzi F, Maus MV, Hui DJ. et al. CD8+ T-cell responses to adeno-associated virus capsid in humans. Nat Med 2007; 13: 419-422
• 98 Banerjee A, Lu Y, Do K. et al. Validation of Induced Microglia-Like Cells (iMG Cells) for Future Studies of Brain Diseases. Front Cell Neurosci 2021; 15: 85
• 99 Wienert B, Shin J, Zelin E. et al. In vitro–transcribed guide RNAs trigger an innate immune response via the RIG-I pathway. PLoS Biol 2018; 16: e2005840
• 100 Schmidt A, Rothenfusser S, Hopfner KP. Sensing of viral nucleic acids by RIG-I: from translocation to translation. Eur J Cell Biol 2012; 91: 78-85
• 101 Schustak J, Twarog M, Wu X. et al. Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type i Interferon Response in Human RPE. J Immunol Res 2021; 2021: 9975628
• 102 Kang R, Zhu S, Zeh H. et al. The STING-STAT6 pathway drives Cas9-induced host response in human monocytes. Biochem Biophys Res Commun 2018; 506: 278-283
• 103 Jeffries AM, Marriott I. Human microglia and astrocytes express cGAS-STING viral sensing components. Neurosci Lett 2017; 658: 53-56
• 104 Cromer MK, Vaidyanathan S, Ryan DE. et al. Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells. Mol Ther 2018; 26: 2431-2442
• 105 Chew WL, Tabebordbar M, Cheng JKW. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat Methods 2016; 13: 868-874
• 106 Charlesworth CT, Deshpande PS, Dever DP. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med 2019; 25: 249-254
• 107 Wagner DL, Amini L, Wendering DJ. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med 2019; 25: 242-248
• 108 Moreno AM, Palmer N, Alemán F. et al. Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy. Nat Biomed Eng 2019; 3: 806-816
• 109 Mehta A, Merkel OM. Immunogenicity of Cas9 Protein. J Pharm Sci 2020; 109: 62-67
• 110 Kotterman MA, Yin L, Strazzeri JM. et al. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates. Gene Ther 2015; 22: 116
• 111 Haapaniemi E, Botla S, Persson J. et al. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 2018; 24: 927-930
• 112 Nelson CE, Wu Y, Gemberling MP. et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat Med 2019; 25: 427-432
• 113 Stephens CJ, Lauron EJ, Kashentseva E. et al. Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. J Control Release 2019; 298: 128-141
• 114 Jacobson SG, Cideciyan AV, Roman AJ. et al. Improvement and decline in vision with gene therapy in childhood blindness. N Engl J Med 2015; 372: 1920-1926
• 115 Bainbridge JWB, Mehat MS, Sundaram V. et al. Long-term effect of gene therapy on Leberʼs congenital amaurosis. N Engl J Med 2015; 372: 1887-1897