Crispr/Cas9 HRCRISPR/Cas9 genome surgery for retinal diseases
Introduction
Inherited retinal diseases affect approximately 1 in 3000 people worldwide and include a number of progressive disorders involving photoreceptor degeneration [1], [2]. Many retinal diseases ultimately lead to irreversible blindness. Currently, there are no proven cures for patients suffering from retinal diseases, but developments in medical technologies, pharmacology, and gene and regenerative therapies have yielded promising results [3], [4].
The retina contains several layers of cells that convert light stimuli into electrical signals through phototransduction [5]. Photoreceptors capture photons of light and convert them into electrical signals [6], which are then amplified and transmitted to bipolar cells, inner neuron cells (i.e. amacrine, horizontal), and ganglion cells. The ganglion cells then transmit these electrical impulses to the brain through the optic nerve [7]. The flow of signal from the photoreceptors to the brain is disrupted as photoreceptors degenerate, often leading to irreversible blindness.
One method of treating recessive retinal diseases lies in gene augmentation therapy, which involves supplementing the patient with a copy of the wild type (WT) gene to allow for diseased cells to create functional protein products [8]. Adeno-associated virus (AAV) vectors are often used to deliver the appropriate gene due to their low toxicity and lack of pathogenicity [1]. Gene augmentation therapy in humans showed promising results in the treatment of Leber congenital amaurosis (LCA) caused by mutations in the retinal pigment epithelium-specific 65-kDa protein (RPE65) gene [9], [10], [11]. Researchers demonstrated the long-term survival of the AAV vectors and consistent expression of therapeutic genes in animal disease models [9], [10], [11]. On December 19, 2017, the U.S. Food and Drug Administration (FDA) approved the AAV therapy (named “Luxturna,” or voretigene neparvovec-rzyl) to treat LCA in humans after the phase 3 trial demonstrated safety and efficacy [10], [12].
Despite its successes as a treatment for LCA, AAV gene augmentation therapy currently lacks the ability to treat dominant diseases, as the addition of an exogenous functional gene via gene therapy is insufficient to override the dysfunctional proteins produced by the dominant mutant allele. This is problematic because there are numerous dominant retinal diseases, including but not limited to autosomal dominant retinitis pigmentosa (adRP), pattern macular dystrophies, and Best vitelliform macular dystrophy [13], [14], [15]. For this reason, scientists have turned to alternative methods such as CRISPR/Cas9 genome surgery to overcome this hurdle.
CRISPR/Cas9 can serve as a fundamental tool for translational research on retinal diseases affecting widespread populations, such as retinitis pigmentosa (RP), LCA, and age-related macular degeneration (AMD) [16], [17], [18]. CRISPR/Cas9 technology has been used to create knock-in and knock-out animal disease models geared towards developing treatments for inherited retinal diseases. In this review, we will provide an overview of CRISPR/Cas9, discuss the different types of Cas9 being used in research today, and compare CRISPR/Cas9 to transcription activator-like nucleases (TALENs) and Zinc Finger Nucleases (ZFNs). Next, we will explore how CRISPR/Cas9 has been used to study disease mechanisms of retinal diseases as well as its potential for application in future clinical treatments.
Section snippets
Background of CRISPR/Cas9
The CRISPR/Cas system was first discovered in prokaryotes in the 1990s, and its role in adaptive immunity was further characterized in the 2000s [19], [20], [21], [22]. By incorporating a copy of the pathogenic viral DNA into their own DNA sequences, bacteria retain a genetic memory of former viral invaders and utilize their CRISPR/Cas machinery to cleave and destroy DNA of future viral invaders. Cas9 binds to the DNA target site by recognizing a CRISPR RNA (crRNA) bound to a trans-activating
Using CRISPR/Cas9 for retinal disease modeling
Retinitis pigmentosa (RP) is a form of retinal dystrophy that impairs vision due to the loss of photoreceptor cells, which can lead to irreversible blindness [58]. RP affects 1 in 4000 live births and is genetically heterogeneous, with 3000 mutations and at least 60 genes implicated in causing the disease phenotype [59]. Because of the severe heterogeneity of RP, CRISPR/Cas9 is an efficacious and cost-effective tool that can aid in the production and design of animal models for studying retinal
Conclusion
In this review, we summarized the utility and efficiency of the CRISPR/Cas9 system. Compared to protein-mediated gene editing such as TALEN, which requires the creation of two new TALEN genes, CRISPR/Cas9 is relatively cheaper and simpler to design, as oligos (sgRNAs) are inexpensive and can be easily customized to target genes of interest [54], [56], [57]. In addition, CRISPR/Cas9 is highly specific due to the precise activity of Cas9, which dependably cleaves 3 bp away from the PAM [27]. New
Author contributions
C.L.X. performed the literature searches. C.L.X. and K.S.P. composed the manuscript.
S.H.T. oversaw all aspects of the manuscript preparation and holds final responsibility for contained information.
Conflict of interest
The authors have no conflict of interest to declare.
Acknowledgements and funding
We gratefully thank Y.T. Tsai, W. H. Wu, G. Y. Cho, and J. D. Sengillo for their comments. This paper was supported, in part, by grants from National Eye Institute, NIH; P30EY019007, R01EY018213, R01EY024698, R01EY026682, R21AG050437, National Cancer Institute Core [5P30CA013696]. S.H.T. is a member of the RD-CURE Consortium and is supported by the Tistou and Charlotte Kerstan Foundation, the Schneeweiss Stem Cell Fund, New York State [C029572], the Foundation Fighting Blindness New York
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