Crispr Genome Surgery in the Retina in Light of Off-Targeting

Cho, Galaxy Y.; Schaefer, Kellie A.; Bassuk, Alexander G.; Tsang, Stephen H.; Mahajan, Vinit B.

doi:10.1097/iae.0000000000002197

Cited by 13 publications

(15 citation statements)

References 118 publications

(167 reference statements)

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“…However, the delivery of a healthy gene allele is not feasible to treat patients with the autosomal dominant disease. Genetic editing with CRISPR/Cas9 is a new approach being studied as a therapeutic for dominant genetic mutations, but there have recently been safety concerns with the potential off‐target cutting of DNA after delivery (Cho et al, ; Kosicki et al, ). Additionally, it is not possible to generate cost‐effective therapeutic vectors for each individual patient mutation.…”

Section: Discussionmentioning

confidence: 99%

“…To cure gain‐of‐function (GOF) disease mutations caused by autosomal dominant genes, the ultimate strategy is to correct the genomic DNA, which is now achievable through the CRISPR/Cas9 system. However, concerns have arisen about the low efficiency and safety of this strategy due to numerous off‐target effects after delivery (Cho, Schaefer, Bassuk, Tsang, & Mahajan, ; Kosicki, Tomberg, & Bradley, ). Additionally, each patient must be sequenced, and their mutation must be known to be able to receive this therapy, and this requires a costly and unique treatment for each individual patient.…”

Section: Introductionmentioning

confidence: 99%

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CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

et al. 2019

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Small molecule pharmacological inhibition of dominant human genetic disease is a feasible treatment that does not rely on the development of individual, patient-specific gene therapy vectors. However, the consequences of protein inhibition as a clinical therapeutic are not well-studied. In advance of human therapeutic trials for CAPN5 vitreoretinopathy, genetic inactivation can be used to infer the effect of protein inhibition in vivo. We created a photoreceptor-specific knockout (KO) mouse for Capn5 and compared the retinal phenotype to both wild-type and an existing Capn5 KO mouse model. In humans, CAPN5 loss-of-function (LOF) gene variants were ascertained in large exome databases from 60,706 unrelated subjects without severe disease phenotypes. Ocular examination of the retina of Capn5 KO mice by histology and electroretinography showed no significant abnormalities. In humans, there were 22 LOF CAPN5 variants located throughout the gene and in all major protein domains. Structural modeling of coding variants showed these LOF variants were nearby known disease-causing variants within the proteolytic core and in regions of high homology between human CAPN5 and 150 homologs, yet the LOF of CAPN5 was tolerated as opposed to gain-of-function disease-causing variants. These results indicate that localized inhibition of CAPN5 is a viable strategy for hyperactivating disease alleles.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

et al. 2019

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“…Here we describe briefly CRISPR mechanism to demonstrate its therapeutic potential for still unapproachable retinal dystrophies. We recommend additional literature for in depth discussion of CRISPR genome surgery [71][72][73], and in the retina [5,23,25,30].…”

Section: Genome Surgerymentioning

confidence: 99%

“…This article reviews recently completed and ongoing clinical trials of gene therapy and briefly describes the future prospects of clustered regularly interspaced short palindromic repeats (CRISPR)based genome surgery. While this review provides a brief summary of gene therapy in the retina, we recommend additional reviews if the reader is interested in additional knowledge on background of gene therapy [27], subretinal injection [28], gene and cell-based therapies for inherited retinal disorders [1,14], AMD gene therapy [29], CRISPR genome surgery in the retina [5,30].…”

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confidence: 99%

Attenuation of Inherited and Acquired Retinal Degeneration Progression with Gene-based Techniques

et al. 2018

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Inherited retinal dystrophies cause progressive vision loss and are major contributors to blindness worldwide. Advances in gene therapy have brought molecular approaches into the realm of clinical trial for these incurable illnesses. Select phase I, II and III trials are complete and provide some promise in terms of functional outcomes and safety; although questions do remain over the durability of their effects and the prevalence of inflammatory reactions. This article reviews gene therapy as it can be applied to inherited retinal dystrophies, provides an update of results from recent clinical trials, and discusses the future prospects of gene therapy and genome surgery. I. Introduction: The retina transmits visual information to the brain, and is thus delicate neural tissue without plasticity [1]. An unfortunate consequence is that cells of the retina affected by inherited dystrophies do not regenerate in humans. For this reason, treatment of retinal degeneration is extremely difficult as photoreceptor death results in irreversible blindness [1, 2]. For approximately 1 in 2000 individuals worldwide, inherited retinal dystrophies (IRDs) cause progressive photoreceptor loss and eventual blindness [3]. The pathophysiology of various §

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“…For the parent Cas9 genome editing system, increasing experimental data suggests that the genome editing is highly specific [ 20 , 44 – 48 ]. Newly developed unbiased profiling techniques further validate the high specificity of this Cas9/sgRNA technology [ 49 – 54 ]. In vivo off-target effects are expected to be low due to epigenetic protection [ 55 , 56 ].…”

Section: Introductionmentioning

confidence: 99%

Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling

Zhang

Arango

et al. 2018

BMC Med Genomics

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BackgroundCRISPR/CAS9 (epi)genome editing revolutionized the field of gene and cell therapy. Our previous study demonstrated that a rapid and robust reactivation of the HIV latent reservoir by a catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM) via HIV long terminal repeat (LTR)-specific MS2-mediated single guide RNAs (msgRNAs) directly induces cellular suicide without additional immunotherapy. However, potential off-target effect remains a concern for any clinical application of Cas9 genome editing and dCas9 epigenome editing. After dCas9 treatment, potential off-target responses have been analyzed through different strategies such as mRNA sequence analysis, and functional screening. In this study, a comprehensive analysis of the host transcriptome including mRNA, lncRNA, and alternative splicing was performed using human cell lines expressing dCas9-SAM and HIV-targeting msgRNAs.ResultsThe control scrambled msgRNA (LTR_Zero), and two LTR-specific msgRNAs (LTR_L and LTR_O) groups show very similar expression profiles of the whole transcriptome. Among 839 identified lncRNAs, none exhibited significantly different expression in LTR_L vs. LTR_Zero group. In LTR_O group, only TERC and scaRNA2 lncRNAs were significantly decreased. Among 142,791 mRNAs, four genes were differentially expressed in LTR_L vs. LTR_Zero group. There were 21 genes significantly downregulated in LTR_O vs. either LTR_Zero or LTR_L group and one third of them are histone related. The distributions of different types of alternative splicing were very similar either within or between groups. There were no apparent changes in all the lncRNA and mRNA transcripts between the LTR_L and LTR_Zero groups.ConclusionThis is an extremely comprehensive study demonstrating the rare off-target effects of the HIV-specific dCas9-SAM system in human cells. This finding is encouraging for the safe application of dCas9-SAM technology to induce target-specific reactivation of latent HIV for an effective “shock-and-kill” strategy.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0394-2) contains supplementary material, which is available to authorized users.

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Crispr Genome Surgery in the Retina in Light of Off-Targeting

Cited by 13 publications

References 118 publications

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

CAPN5 genetic inactivation phenotype supports therapeutic inhibition trials

Attenuation of Inherited and Acquired Retinal Degeneration Progression with Gene-based Techniques

Comprehensive off-target analysis of dCas9-SAM-mediated HIV reactivation via long noncoding RNA and mRNA profiling

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