Wen-Hsuan Wu

Unexpected mutations after CRISPR-Cas9 editing in vivo

To the Editor: CRISPR–Cas9 editing shows promise for correcting disease-causing mutations. For example, in a recent study we used CRISPR-Cas9 for sight restoration in blind rd1 mice by correcting a mutation in the Pde6b gene1. However, concerns persist regarding secondary mutations in regions not targeted by the single guide RNA (sgRNA)2. Algorithms generate likely off-target sites for a given gRNA, but these algorithms may miss mutations. Whole-genome sequencing (WGS) has been used to assess the presence of small insertions and deletions (indels)3 but not to probe for single-nucleotide variants (SNVs) in a whole organism. We performed WGS on a CRISPR–Cas9-edited mouse to identify all off-target mutations and found an unexpectedly high number of SNVs compared with the widely accepted assumption that CRISPR causes mostly indels at regions homologous to the sgRNA. We tested four sgRNAs in cells then chose the sgRNA with the highest activity for in vivo targeting. DNA was isolated from two CRISPR-repaired mice (F03 and F05) and one uncorrected control1. CRISPR–Cas9-treated mice were sequenced at an average depth of 50×, and the control was sequenced at 30×. Variant calls were confirmed by at least 23× sequencing coverage (Supplementary Tables 1 and 2). Multiple variant-calling software pipelines identified indels and SNVs (Fig. 1 and Supplementary Methods). In the CRISPR-treated mice, targeted alleles were repaired1. Off-target mutations were identified as those present in the CRISPR-treated animals but absent in the uncorrected control. All pipelines showed that F03 harbored 164 indels and 1,736 SNVs (63 and 885 of these, respectively, associated with known genes). F05 harbored 128 indels and 1,696 SNVs (51 and 865 of these, respectively, associated with known genes) (Fig. 1). The same 117 indels and 1,397 SNVs were detected in both of the CRISPR-treated mice, which indicated nonrandom targeting. SNVs appeared to slightly favor transitions over transversions (Supplementary Fig. 1). The mutation rate detected in CRISPRtreated mice was substantially higher than that generated by spontaneous germline mutations (3 to 4 indels and 90 to 100 SNVs, de novo, per generation)4,5. As additional controls, each of the variants was compared with the FVB/NJ genome in the mouse dbSNP database (v138), and each of the SNVs was also compared with all 36 strains in the Mouse Genome Project (v3). None of the CRISPR-generated offtarget mutations were found in any of these strains, which further confirmed that these WGS-identified SNVs were the result of CRISPR–Cas9 off targeting. All pipelines identified 6 and 3 indels and 60 and 51 SNVs in F03 and F05 mice, respectively, in exonic regions only (Fig. 1); 5 indels and 24 SNVs caused nonsynonymous mutations in protein-coding sequences (Supplementary Tables 3 and 4). Of these, all five indels and one SNV (introducing a premature stop codon) were expected to be deleterious. Several mutated protein-coding genes were associated with a human and/ or mouse phenotype (Supplementary Tables 3 and 4). Of the 29 coding-sequence variants, 7 variants were mutated identically in both mice. 24 CRISPR-associated variants were selected, and all were confirmed by Sanger sequencing (Supplementary Fig. 2 and Supplementary Methods). Among the top-fifty sequences predicted for off targeting, none were mutated. Additionally, there was poor sequence homology between the sgRNA and sequences near the actual off-target coding and noncoding variants (Supplementary Fig. 3). Our results suggest current in silico modeling cannot predict bona fide off-target sites. Together, these results indicate that at least certain sgRNAs may target loci independently of their target in vivo. The unpredictable generation of these variants is of concern. The impact of the numerous mutations occurring in noncoding RNAs or other regulatory intragenic regions could be detrimental to key cellular processes (Supplementary Fig. 4 and Supplementary Table 5)6. Although our CRISPR-treated mice did not display obvious extraocular phenotypes, it is possible the mice may reveal phenotypes in time, when they are challenged or bred to homozygosity. The present study demonstrates WGS analysis of both indels and SNVs as the most thorough method for identifying off-target mutations and shows a significantly higher number of potentially deleterious CRISPR–Cas9-induced mutations than have been previously reported3. It is not clear whether improved sgRNA

Long-term safety and efficacy of human-induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa.

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65rd12/Rpe65rd12 mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65rd12/Rpe65rd12 mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.

Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines

While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic alleles susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G-Wt-G; G-Wt-G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis.

Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.

CAPN5 mutation in hereditary uveitis: the R243L mutation increases calpain catalytic activity and triggers intraocular inflammation in a mouse model

A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.

Clustered Regularly Interspaced Short Palindromic Repeats-Based Genome Surgery for the Treatment of Autosomal Dominant Retinitis Pigmentosa

PURPOSE To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO). DESIGN Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. PARTICIPANTS This study included 2 kinds of human RHO mutation knock-in mouse models: RhoP23H and RhoD190N. In total, 23 RhoP23H/P23H, 43 RhoP23H/+, and 31 RhoD190N/+ mice were used for analysis. METHODS This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. MAIN OUTCOME MEASURES Electroretinographic and histologic analysis. RESULTS The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both RhoP23H and RhoD190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy. CONCLUSIONS As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.

CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa: A Brief Methodology

CRISPR/Cas9 genome engineering is currently the leading genome surgery technology in most genetics laboratories. Combined with other complementary techniques, it serves as a powerful tool for uncovering genotype-phenotype correlations. Here, we describe a simplified protocol that was used in our publication, CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa, providing an overview of each section of the experimental process.

Response to Editas and Intellia: Unexpected mutations after CRISPR-Cas9 editing in vivo

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.Here we provide additional confirmatory data and clarifying discussion, including information about the relatedness of the control and CRISPR-treated mice, as well as sequencing data showing extensive regions with heterozygosity and in some cases more than 2 alleles in CRISPR-treated mice throughout the genome. The control FVB mouse and parents of the CRISPR-treated mice were purchased directly through the JAX genetic stability program and were within one generation of one another. Furthermore, the heterozygous mutations were mostly within 7-10bp adjacent to NGG or NGA nucleotide sequences, the preferred Protospacer Adjacent Motif (PAM) for the SpCas9. The multiple alleles in these cases cannot simply be explained by parental inheritance. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness.

Genome Editing in the Retina: A Case Study in CRISPR for a Patient-Specific Autosomal Dominant Retinitis Pigmentosa Model

The future of precision medicine, genome editing has gained momentum in the field of ophthalmology because of the eye’s amenability to genetic interventions. The eye is an ideal target for gene therapy due to its accessibility, ease of noninvasive monitoring, significant compartmentalization, immunoprivileged status, optical transparency, and the presence of a contralateral control. One of the first gene therapy clinical trials was conducted in the eye for a severe form of early-onset retinal dystrophy called Leber congenital amaurosis, and it has encouraged further exploration of this technique as a viable treatment option for other inherited disorders across medical disciplines. This chapter highlights current ocular gene therapy approaches, clinical and preclinical experiments, and provides a case study of the bench-to-bedside personalized medicine approach taken for a novel and rare retinitis pigmentosa mutation.

Genetic rescue reverses microglial activation in preclinical models of retinitis pigmentosa

Microglia cells (MGCs) play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina. Their activation, however, contributes to the progression of multiple degenerative diseases. Given the potential damage created by MGCs, it is important to better understand their mechanism of activation. Here, we explored the role of MGCs in the context of retinitis pigmentosa (RP) by using four independent preclinical mouse models. For therapeutic modeling, tamoxifen-inducible CreER was introduced to explore changes in MGCs when RP progression halted. The phenotypes of the MGCs were observed using live optical coherence tomography, live autofluorescence, and immunohistochemistry. We found that, regardless of genetic background, MGCs were activated in neurodegenerative conditions and migrated beyond the layers where they are typically found to the inner and outer segments, where degeneration was ongoing. Genetic rescue not only halted degeneration but also deactivated MGCs, regardless of whether the intervention occurred at the early, middle, or late stage of the disease. These findings suggest that halting long-term disease progression may be more successful by downregulating MGC activity while co-administering the therapeutic intervention.