Abstract
Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.
Keywords: genome editing, Campylobacter jejuni, long-term effect, retina
Jo et al. demonstrated that intravitreally administered adeno-associated virus encoding the Cas9 ortholog from Campylobacter jejuni targeted to Hif1a did not induce histologic and functional aberration in the retina. Despite the accumulation of on-target insertions and deletions (indels), there were no indels at the potential off-target sites.
Introduction
Genome engineering tools such as CRISPR-Cas9 systems open an avenue for correcting pathogenic mutations and controling gene expression.1, 2, 3 For example, in retinal diseases, subretinally or intravitreally administered Cas9 via adeno-associated viruses (AAVs), ribonucleoproteins, or plasmids can be used to cleave genetic sequences with mutations and suppress the expression of genes associated with pathological conditions.4, 5, 6, 7, 8, 9, 10 Besides correcting mutated gene segments, Cas9 can be applied therapeutically to disrupt disease-causing wild-type genes. Examples of such suppression include the depletion of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and VEGF receptor 2, which play roles in disease pathogenesis.4, 7, 8 In particular, the ability to target intracellular proteins (e.g., HIF-1α) and their coding genes (e.g., HIF1A), which are undruggable or untargetable by currently available therapeutic agents with high specificity, represents an additional advantage of CRISPR-Cas9-mediated therapeutic approaches.
Despite recent advances in genome engineering technologies, there are still concerns about in vivo genome editing regarding long-term or permanent genetic modification and unexpected off-target effects.1, 2 Attempts have been made to use non-viral administration tools for the transient expression of Cas9,1, 11 but viral vectors, including AAV, are still the most practical method to induce genome editing in vivo, especially in organs such as brain, liver, and eye, to which different serotypes of AAVs allow tissue-specific tropism.12, 13, 14 Although AAV or lentiviral vectors are utilized in many preclinical tests and clinical trials for gene therapy, it is essential to resolve concerns about the long-term effects of AAV encoding genome editing machinery, which results in prolonged expression of Cas9 in transfected cells.4
In our previous study, we showed that the efficient delivery of Cas9 derived from Campylobacter jejuni (CjCas9) with a single-guide RNA (sgRNA) in a single AAV vector resulted in the depletion of the Vegfa or Hif1a gene in murine retinal and retinal pigment epithelium (RPE) cells at 6 weeks post-injection.4 CjCas9-edited RPE reduced choroidal neovascularization, which is one of the major pathologic features of age-related macular degeneration (AMD).
In this study, we assessed the histologic integrity of retinal tissues and the electrical properties of retinal neurons of mice at 14 months after intravitreal injection of AAV encoding CjCas9 targeted to Hif1a, which effectively induced small insertions and deletions (indels) at the target site without detectable off-target effects. These long-term observations might provide support for in vivo genome editing of selected target genes associated with pathological conditions in various retinal diseases, including AMD, Leber congenital amaurosis, and retinitis pigmentosa.
Results
Long-Term Effects of CjCas9 on Histologic Integrity and Functions of Retinal Tissues at 14 Months
We previously demonstrated that intravitreally administered AAV encoding CjCas9 targeted to Vegfa and Hif1a (AAV-CjCas9: Vegfa and AAV-CjCas9: Hif1a, respectively) resulted in 20%–30% indels at target sites in RPE cells and effectively inhibited the formation of choroidal neovascular membranes compared to AAV encoding CjCas9 targeted to Rosa26 (AAV-CjCas9: Rosa26) or PBS.4 With the same procedure, mice were treated with intravitreal injections of AAV-CjCas9: Rosa26, AAV-CjCas9: Hif1a, and AAV-CjCas9: Vegfa at a concentration of 2 × 1010 viral genomes in 2 μL PBS. Interestingly, even at 14 months after the treatment, AAV-CjCas9: Rosa26 and AAV-CjCas9: Hif1a did not induce any definite changes in histologic integrity (Figure 1A), the number of apoptotic cells identified by terminal deoxynucleotidyl transferase dUTP nick end labeling (Figure 1B), or the area of opsin positivity (Figure 1C). In contrast, AAV-CjCas9: Vegfa reduced the thickness of the retinal tissues (Figure S1), which was also seen in mice with an RPE-specific Vegfa deletion.15 There was an evident expression of the hemagglutinin (HA) epitope, which is conjugated to the C terminus of CjCas9 in AAV-CjCas9-treated tissues (Figure 1C).
Figure 1.
Histologic Evaluation of Retinal Tissues at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice
(A) Representative H&E images of the murine retina at 14 months after intravitreal injection of PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a. (B) The number of apoptotic cells in retinal tissues observed at 400× magnification (n = 6). Error bars indicate SEM. (C) Representative immunofluorescence images of the murine retina at 14 months after intravitreal injection of PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a. GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segment; ONL, outer nuclear layer; OS, outer segment. Scale bars, 25 μm.
In line with these results in histologic integrity, AAV-CjCas9: Rosa26 and AAV-CjCas9: Hif1a did not affect the scotopic and photopic responses upon electroretinogram (ERG) analyses (Figures 2A–2F). These ERG results suggest that there were no definite long-term functional changes in retinal tissues after intravitreal administration of AAV-CjCas9: Rosa26 and AAV-CjCas9: Hif1a. In addition, there were no significant differences in body weights among groups treated with AAV-CjCas9 targeting different target genes (Figure S2).
Figure 2.
Electrical Properties of Retinal Neuronal Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice
Amplitudes of scotopic and photopic light responses of mice treated with PBS (No AAV), AAV-CjCas9: Rosa26, and AAV-CjCas9: Hif1a on ERG analyses (ns = 6–10). (A) Amplitudes of the scotopic b-wave at −24 dB. (B) Amplitudes of the scotopic a-wave at 0 dB. (C) Amplitudes of the scotopic b-wave at 0 dB. (D) Amplitudes of the photopic b-wave at 5 dB. (E) Amplitudes of the flicker response at 0 dB. (F) Amplitudes of the oscillatory potential at 5 dB. One-way ANOVA. NS, not significant. Error bars indicate SEM.
Long-Term Indel Rates and Off-Target Effects Induced by CjCas9 in the Mouse Retina
To assess the long-term genome editing effects of CjCas9, we investigated the presence of indels in the mouse retina at 14 months following AAV injection. At 6 weeks post-injection, Hif1a- and Vegfa-specific CjCas9 achieved indels with frequencies of 58 ± 5% and 20 ± 2% in the retina, respectively, and 31 ± 2% and 22 ± 3% in the RPE, respectively.4 At 14 months, CjCas9-induced indels were detected at the Rosa26, Hif1a, and Vegfa target sites with frequencies of 49 ± 14%, 79 ± 2%, and 49 ± 7% in retinal cells treated with AAV-CjCas9: Rosa26, AAV-CjCas9: Hif1a, and AAV-CjCas9: Vegfa, respectively (Figure 3A; Figure S3A). In RPE cells, indels were detected with frequencies of 28 ± 4%, 45 ± 7%, and 23 ± 5% at the Rosa26, Hif1a, and Vegfa target sites, respectively (Figure 3B; Figure S3B), indicating that indels were persistent and significantly increased in the mouse retina for a period of up to 14 months after the initial injection. Accordingly, AAV-CjCas9: Vegfa decreased the VEGF levels in the retinal and RPE cells at 14 months after the initial injection (Figures S4A and S4B). In contrast, there was no definite change in the VEGF levels in the normal retinal and RPE tissues by treatment with AAV-CjCas9: Hif1a (Figures S4C and S4D). It is also remarkable that the major mutation patterns at the Hif1a, Vegfa, or Rosa26 target sites were identical between 6 weeks and 14 months after post-intravitreal injection of AAV-CjCas9: Hif1a, Vegfa, or Rosa26 (Figure S5; Tables S1–S3).
Figure 3.
Long-Term Genome Editing Efficacy in Retinal and RPE Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice
Mutation frequencies at Hif1a and Rosa26 target sites in (A) retinal and (B) RPE cells determined using deep sequencing at 14 months after intravitreal injection of AAV-CjCas9. Error bars indicate SEM. n = 4 for AAV-uninjected control (mock); n = 7 and n = 3 for AAV-CjCas9: Hif1a and AAV-CjCas9: Rosa26, respectively. 6W, 6 weeks post-injection; 14Mo, 14 months post-injection. **p < 0.01, Student’s t test.
We next investigated whether the long-term expression of CjCas9 and its sgRNA might lead to off-target effects. In our previous report, two or one in vitro cleavage sites were identified by Digenome-seq using the Hif1a- or the Vegfa-specific sgRNA, respectively.4 Hence, we performed targeted deep sequencing at these Digenome sequencing (Digenome-seq) captured potential off-target sites and found that CjCas9 did not cause any detectable off-target mutations at these sites (Figure 4A; Figure S6A). We further examined the genome-wide specificity of CjCas9 at potential off-target sites that differed from the on-target sites by up to 4 nt in the mouse genome, identified using the Cas-OFFinder algorithm (http://www.rgenome.net/cas-offinder/).16 CjCas9-edited retinal and RPE cells did not show any detectable off-target indels at 7 homologous sites for Hif1a and 14 homologous sites for Vegfa (Figure 4B; Figure S6B).
Figure 4.
Long-Term Genome Editing Specificity in Retinal and RPE Cells at 14 Months after Intravitreal Injection of AAV-CjCas9 in Mice
Indel frequencies at potential off-target sites of CjCas9 targeted to Hif1a identified by (A) Digenome-seq and (B) Cas-OFFinder. Genomic DNA isolated from retinal and RPE cells at 14 months after intravitreal injection of AAV-CjCas9 was subjected to targeted deep sequencing. Mismatched nucleotides are indicated in blue, and protospacer-adjacent motifs are indicated in orange. On, on-target site; OT, off-target sites. Error bars indicate SEM. n = 3 for AAV-uninjected control (-AAV); n = 7 for AAV-CjCas9: Hif1a.
Discussion
In this report, we describe the long-term effects of CjCas9 delivered via AAV to the mouse retina and RPE. Fourteen months after the successful targeting of Hif1a via intravitreal injection of AAV-CjCas9: Hif1a, we observed no definite histologic or functional abnormalities in the retinal tissues compared to age-matched controls. These data are in line with a previous report showing that there were no significant morphological, functional, or transcriptional abnormalities in RPE-specific Hif1a knockout mice.15
HIF-1α is a transcription factor that controls the expression of various angiogenesis-related genes under ischemic and/or hypoxic conditions.17 In particular, HIF-1α is upregulated in pathological states associated with various retinal vascular diseases, including AMD and diabetic retinopathy; a key effect is that HIF-1α promotes the expression of VEGF-A, one of the most potent players in pathological angiogenesis.15, 17, 18 Because there are several HIF-1α-independent pathways that also regulate VEGF-mediated physiological angiogenesis,19, 20 HIF-1α is not required for the maintenance of physiological vasculature.15 In contrast, VEGF is a trophic factor for the maintenance of normal retinal neuronal cells, RPE cells, and vascular endothelial cells.15, 21, 22 As expected, AAV-CjCas9: Vegfa resulted in severe histologic changes of retinal tissues, while AAV-CjCas9: Hif1a induced no change in histologic integrity and retinal functions. These data demonstrated that targeting Hif1a with AAV-CjCas9 could be a safe way to address pathological angiogenesis without introducing unwanted toxicities related to histologic integrity, apoptotic activity, and off-target effects.
In the treatment of retinal diseases such as Leber congenital amaurosis, AAV is a widely utilized platform for therapeutic gene delivery and has demonstrated long-term efficacy and safety.23, 24, 25 Intravitreally administered AAVs do not significantly affect the gene expression in other organs.26, 27, 28 Similarly, both AAV-CjCas9: Vegfa and Hif1a did not induce any change in body weights of treated mice, regardless of their local effects. Nevertheless, there have been concerns about increased off-target effects associated with prolonged Cas9 expression. In our study, indels were detected at the on-target sites with frequencies of 79 ± 2% and 45 ± 7% in Hif1a-edited retinal and RPE cells, respectively. The indel rate increased at the Vegfa and Hif1a target sites in the Cas9-edited retina from 6 weeks to 14 months after the initial injection. Apparently, continuous Cas9 expression led to the induction of additional double-strand breaks and indels in cells, considering that most retinal cells are differentiated and non-transformed. Despite the increase in indel frequencies at on-target sites, there were no detectable off-target effects up to 14 months post-injection, suggesting that, because of high CjCas9 specificity, long-term expression did not cause indels at potential off-target sites in vivo.
The lifespan of C57BL/6 mice is estimated to be less than 30 months (878 ± 10 days for male mice and 794 ± 6 days for female mice).29 Thus, our observation period (14 months) corresponds to half of the expected lifespan of the study animals. Our results show that intravitreally administered AAV-CjCas9 to a carefully selected target such as Hif1a effectively induced and maintained indels in retinal tissues for more than 1 year. Furthermore, this treatment did not affect retinal histologic integrity or functions and, importantly, did not aggravate off-target effects over this extended period of time. Taken together, our results provide evidence of long-term safety of CjCas9 expression in the eye, in support of in vivo genome editing for therapeutic treatments of various retinal diseases.
Materials and Methods
Animals
Eight-week-old male C57BL/6 mice were purchased from Central Laboratory Animal and maintained under a 12-hr:12-hr dark:light cycle. All animal experiments were performed under the guidelines of the Association for Research in Vision and Ophthalmology statement for the use of animals in ophthalmic and vision research and approved by the institutional animal care and use committees of both Seoul National University and Seoul National University Hospital.
Construction of CjCas9 and sgRNA Plasmids
A human codon-optimized CjCas9 coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientific) and cloned into the p3s plasmid. The trans-activating CRISPR RNA sequence and the precursor CRISPR RNA sequence were fused with a TGAA linker to form an sgRNA sequence. sgRNAs were transcribed under the control of the U6 promoter.
AAV Vectors Encoding CjCas9 and sgRNAs
AAV inverted-terminal-repeat-based vector plasmids carrying an sgRNA sequence and the CjCas9 gene with a nuclear localization sequence and an HA tag at the C terminus were constructed. For retinal delivery, AAV vectors encoding CjCas9 under the control of the short form of elongation factor 1α promoter; EGFP linked to the C terminus of CjCas9 with the self-cleaving T2A peptide; and a U6 promoter-driven sgRNA specific to the Rosa26, Vegfa, or Hif1a gene were constructed.
Production and Characterization of AAV Vectors
To produce AAV vectors, they were pseudotyped in AAV9 capsids. HEK293T cells were transfected with pAAV-ITR-CjCas9-sgRNA, pAAVED2/9, and a helper plasmid. HEK293T cells were cultured in DMEM with 2% fetal bovine serum (FBS). Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation with PEIpro (Polyplus-transfection) and triple transfection with plasmids at a molar ratio of 1:1:1 in HEK293T cells. After 72 hr of incubation, cells were lysed and particles were purified by iodixanol (Sigma-Aldrich) step-gradient ultracentrifugation. The number of vector genomes was determined by qPCR.
Intravitreal Injection of AAV
After mice were deeply anesthetized, AAV9-CjCas9 (2 × 1010 viral genomes in 2 μL PBS) was injected into the vitreous cavity of the retina using a customized Nanofil syringe with a 33G blunt needle (World Precision Instruments) under an operating microscope (Leica).
Histologic Evaluation
At 14 months after AAV-mediated delivery of CjCas9, paraffin blocks were prepared from enucleated eyes. Thin sections were prepared for H&E staining and TUNEL (Sigma) assays. TUNEL-positive cells were evaluated in 10 randomly selected fields in each slide at 400× magnification.
Immunofluorescence
At 14 months after AAV-mediated delivery of CjCas9, paraffin blocks were prepared from enucleated eyes. Thin sections were immunostained with anti-HA antibody (1:1,000; catalog no. 3F10, Roche) and anti-opsin antibody (1:1,000; catalog no. AB5405, Millipore), followed by treatment with Alexa Fluor 488 or 594 IgG (1:500; Thermo Fisher). Nuclear staining was performed using DAPI (Sigma). Then, the slides were observed under a fluorescence microscope (Leica).
Isolation of Retina and RPE Sheets
Enucleated eyes were incubated with 0.1% hyaluronidase (Sigma-Aldrich) for 45 min at 37°C, after removal of the lens, and then transferred in PBS for 30 min on ice. Next, the neural retina was completely removed from enucleated eyes, and only the RPE-choroid-scleral complex was incubated with Trypsin-EDTA solution for 45 min at 37°C. RPE cell sheets were isolated by shaking the eyecup using microforceps, and only monolayer RPE sheets were collected using a glass capillary on the microscope.
Genomic DNA Extraction and Mutation Analysis
The collected retina and RPE sheets were incubated with lysis buffer, and genomic DNA was extracted according to the manufacturer’s protocol (NucleoSpin Tissue, Macherey-Nagel). Genomic DNA was analyzed by targeted deep sequencing. On-target or off-target loci were amplified using 100 ng genomic DNA for targeted deep sequencing. Deep sequencing libraries were generated by PCR with the following primers: mouse Rosa26, 5′-CGGGAGTCTTCTGGGCAGGCTTAA-3′ (forward), 5′-CCGAGGCGGATCACAAGCAA-3′ (reverse); mouse Hif1a, 5′-GTCCCCATATATGAAGAGCAC-3′ (forward), 5′-CAATATCTGACTGAAAATCACCT-3′ (reverse); mouse Vegfa, 5′-CCCTTGGGATCTTGCATC-3′ (forward), 5′-TACTACGGAGCGAGAAGAG-3′ (reverse). TruSeq HT Dual Index primers were used to label each sample. Pooled libraries were subjected to paired-end sequencing using MiniSeq (Illumina). Indel frequencies were calculated as described previously.30
ERG
Mice were maintained in a dark-adapted state for over 16 hr. After deep anesthesia, pupils were dilated with an eye drop containing phenylephrine hydrochloride (5 mg/mL) and tropicamide (5 mg/mL). Full-field ERG was performed using the Universal Testing and Analysis System, Electrophysiologic 2000 (UTAS E-2000; LKC Technologies). The responses were recorded at a gain of 2 k, utilizing a notch filter at 60 Hz, and were bandpass filtered between 0.1 and 1500 Hz. In the light-adapted photopic state, with a 30-cd/m2 background light to desensitize the rods and isolate cones, photopic cone responses were recorded in response to a single flash of 0 dB. The amplitudes of the a-wave were measured from the baseline to the lowest negative-going voltage, whereas peak b-wave amplitudes were estimated from the trough of the a-wave to the highest peak of the positive b-wave.
Statistics
No statistical methods were used to predetermine sample size for in vitro or in vivo experiments. All group results are expressed as mean ± SEM, if not stated otherwise. Comparisons between groups were made using the two-tailed Student’s t test or one-way ANOVA and Tukey post hoc tests for multiple groups. Statistical analysis was performed in GraphPad Prism 5.
Accession Numbers
The accession number for the deep sequencing data reported in this paper is NCBI: PRJNA435661. Details of the primers used in this study are available on request.
Author Contributions
D.H.J. and T.K. drafted the manuscript. D.H.J. performed experiments on the histologic evaluation of retinal tissues. T.K. performed experiments on the design of the AAV encoding CjCas9 and sgRNA, followed by genome analysis of on-target and off-target mutations. C.S.C. performed animal experiments. Jin Hyoung Kim, J.-S.K., and Jeong Hun Kim edited the manuscript. J.-S.K. and Jeong Hun Kim designed the study and revised the manuscript.
Conflicts of Interest
J.-S.K. is a co-founder and shareholder of ToolGen.
Acknowledgments
We thank Dr. Sung Wook Park for his assistance in animal experiments. This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF), funded by the Korean government, MSIP (NRF-2015M3A9E6028949 and 2017M3A9B4062654 to J.H.K.); the Development of Platform Technology for Innovative Medical Measurements Program from the Korea Research Institute of Standards and Science (KRISS-2018-GP2018-0018 to J.H.K.); the Creative Materials Discovery Program through the NRF, funded by the Ministry of Science and ICT (2018M3D1A1058826 to J.H.K.); the Basic Science Research Program through the NRF, funded by the Ministry of Education (2017R1A6A3A04004741 to D.H.J.); and the Institute for Basic Science (IBS-R021-D1 to J.-S.K.).
Footnotes
Supplemental Information includes six figures and three tables and can be found with this article online at https://doi.org/10.1016/j.ymthe.2018.10.009.
Contributor Information
Jin-Soo Kim, Email: jskim01@snu.ac.kr.
Jeong Hun Kim, Email: steph25@snu.ac.kr.
Supplemental Information
References
- 1.Yin H., Kauffman K.J., Anderson D.G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 2017;16:387–399. doi: 10.1038/nrd.2016.280. [DOI] [PubMed] [Google Scholar]
- 2.Cornu T.I., Mussolino C., Cathomen T. Refining strategies to translate genome editing to the clinic. Nat. Med. 2017;23:415–423. doi: 10.1038/nm.4313. [DOI] [PubMed] [Google Scholar]
- 3.Kim J.S. Genome editing comes of age. Nat. Protoc. 2016;11:1573–1578. doi: 10.1038/nprot.2016.104. [DOI] [PubMed] [Google Scholar]
- 4.Kim E., Koo T., Park S.W., Kim D., Kim K., Cho H.Y., Song D.W., Lee K.J., Jung M.H., Kim S. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 2017;8:14500. doi: 10.1038/ncomms14500. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Suzuki K., Tsunekawa Y., Hernandez-Benitez R., Wu J., Zhu J., Kim E.J., Hatanaka F., Yamamoto M., Araoka T., Li Z. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature. 2016;540:144–149. doi: 10.1038/nature20565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ruan G.X., Barry E., Yu D., Lukason M., Cheng S.H., Scaria A. CRISPR/Cas9-mediated genome editing as a therapeutic approach for Leber congenital amaurosis 10. Mol. Ther. 2017;25:331–341. doi: 10.1016/j.ymthe.2016.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Huang X., Zhou G., Wu W., Duan Y., Ma G., Song J., Xiao R., Vandenberghe L., Zhang F., D’Amore P.A., Lei H. Genome editing abrogates angiogenesis in vivo. Nat. Commun. 2017;8:112. doi: 10.1038/s41467-017-00140-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kim K., Park S.W., Kim J.H., Lee S.H., Kim D., Koo T., Kim K.E., Kim J.H., Kim J.S. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration. Genome Res. 2017;27:419–426. doi: 10.1101/gr.219089.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bakondi B., Lv W., Lu B., Jones M.K., Tsai Y., Kim K.J., Levy R., Akhtar A.A., Breunig J.J., Svendsen C.N., Wang S. In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol. Ther. 2016;24:556–563. doi: 10.1038/mt.2015.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Latella M.C., Di Salvo M.T., Cocchiarella F., Benati D., Grisendi G., Comitato A., Marigo V., Recchia A. In vivo editing of the human mutant rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina. Mol. Ther. Nucleic Acids. 2016;5:e389. doi: 10.1038/mtna.2016.92. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Gao X., Tao Y., Lamas V., Huang M., Yeh W.H., Pan B., Hu Y.J., Hu J.H., Thompson D.B., Shu Y. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature. 2018;553:217–221. doi: 10.1038/nature25164. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Nishiyama J., Mikuni T., Yasuda R. Virus-mediated genome editing via homology-directed repair in mitotic and postmitotic cells in mammalian brain. Neuron. 2017;96:755–768.e5. doi: 10.1016/j.neuron.2017.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Zincarelli C., Soltys S., Rengo G., Rabinowitz J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 2008;16:1073–1080. doi: 10.1038/mt.2008.76. [DOI] [PubMed] [Google Scholar]
- 14.Hung S.S., Li F., Wang J.H., King A.E., Bui B.V., Liu G.S., Hewitt A.W. Methods for in vivo CRISPR/Cas editing of the adult murine retina. Methods Mol. Biol. 2018;1715:113–133. doi: 10.1007/978-1-4939-7522-8_9. [DOI] [PubMed] [Google Scholar]
- 15.Kurihara T., Westenskow P.D., Bravo S., Aguilar E., Friedlander M. Targeted deletion of Vegfa in adult mice induces vision loss. J. Clin. Invest. 2012;122:4213–4217. doi: 10.1172/JCI65157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Bae S., Park J., Kim J.S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014;30:1473–1475. doi: 10.1093/bioinformatics/btu048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Semenza G.L. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J. Appl. Physiol. (1985) 2000;88:1474–1480. doi: 10.1152/jappl.2000.88.4.1474. [DOI] [PubMed] [Google Scholar]
- 18.Ferrara N., Gerber H.P., LeCouter J. The biology of VEGF and its receptors. Nat. Med. 2003;9:669–676. doi: 10.1038/nm0603-669. [DOI] [PubMed] [Google Scholar]
- 19.Arany Z., Foo S.Y., Ma Y., Ruas J.L., Bommi-Reddy A., Girnun G., Cooper M., Laznik D., Chinsomboon J., Rangwala S.M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha. Nature. 2008;451:1008–1012. doi: 10.1038/nature06613. [DOI] [PubMed] [Google Scholar]
- 20.Schmidt D., Textor B., Pein O.T., Licht A.H., Andrecht S., Sator-Schmitt M., Fusenig N.E., Angel P., Schorpp-Kistner M. Critical role for NF-kappaB-induced JunB in VEGF regulation and tumor angiogenesis. EMBO J. 2007;26:710–719. doi: 10.1038/sj.emboj.7601539. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Heo J.W., Kim J.H., Cho C.S., Jun H.O., Kim D.H., Yu Y.S., Kim J.H. Inhibitory activity of bevacizumab to differentiation of retinoblastoma cells. PLoS ONE. 2012;7:e33456. doi: 10.1371/journal.pone.0033456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Saint-Geniez M., Maharaj A.S., Walshe T.E., Tucker B.A., Sekiyama E., Kurihara T., Darland D.C., Young M.J., D’Amore P.A. Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors. PLoS ONE. 2008;3:e3554. doi: 10.1371/journal.pone.0003554. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Bainbridge J.W., Smith A.J., Barker S.S., Robbie S., Henderson R., Balaggan K., Viswanathan A., Holder G.E., Stockman A., Tyler N. Effect of gene therapy on visual function in Leber’s congenital amaurosis. N. Engl. J. Med. 2008;358:2231–2239. doi: 10.1056/NEJMoa0802268. [DOI] [PubMed] [Google Scholar]
- 24.Maguire A.M., Simonelli F., Pierce E.A., Pugh E.N., Jr., Mingozzi F., Bennicelli J., Banfi S., Marshall K.A., Testa F., Surace E.M. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. N. Engl. J. Med. 2008;358:2240–2248. doi: 10.1056/NEJMoa0802315. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Le Meur G., Lebranchu P., Billaud F., Adjali O., Schmitt S., Bézieau S., Péréon Y., Valabregue R., Ivan C., Darmon C. Safety and long-term efficacy of AAV4 gene therapy in patients with RPE65 Leber congenital amaurosis. Mol. Ther. 2018;26:256–268. doi: 10.1016/j.ymthe.2017.09.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Sugano E., Tabata K., Takahashi M., Nishiyama F., Shimizu H., Sato M., Tamai M., Tomita H. Local and systemic responses following intravitreous injection of AAV2-encoded modified Volvox channelrhodopsin-1 in a genetically blind rat model. Gene Ther. 2016;23:158–166. doi: 10.1038/gt.2015.99. [DOI] [PubMed] [Google Scholar]
- 27.Ye G.J., Budzynski E., Sonnentag P., Miller P.E., Sharma A.K., Ver Hoeve J.N., Howard K., Knop D.R., Neuringer M., McGill T. Safety and biodistribution evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus vector expressing retinoschisin. Hum. Gene Ther. Clin. Dev. 2015;26:165–176. doi: 10.1089/humc.2015.076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Cukras C., Wiley H.E., Jeffrey B.G., Sen H.N., Turriff A., Zeng Y., Vijayasarathy C., Marangoni D., Ziccardi L., Kjellstrom S. Retinal AAV8-RS1 gene therapy for X-linked retinoschisis: initial findings from a phase I/IIa trial by intravitreal delivery. Mol. Ther. 2018;26:2282–2294. doi: 10.1016/j.ymthe.2018.05.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Kunstyr I., Leuenberger H.G. Gerontological data of C57BL/6J mice. I. Sex differences in survival curves. J. Gerontol. 1975;30:157–162. doi: 10.1093/geronj/30.2.157. [DOI] [PubMed] [Google Scholar]
- 30.Kim D., Bae S., Park J., Kim E., Kim S., Yu H.R., Hwang J., Kim J.I., Kim J.S. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods. 2015;12:237–243. doi: 10.1038/nmeth.3284. 1, 243. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.