Gene editing


Gene editing is a technology that enables scientists to precisely alter human DNA. Much ongoing work is focused on treating genetic diseases by correcting the associated mutations. Some scientists have also used this technology to re-create certain disease models in human stem cells for further research.[1]

Although there are several approaches to edit human DNA, one of the most popular at the moment is the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system.[2] It works like a custom pair of scissors where it can be programmed to cut out an abnormal section of a gene and introduce the correct sequence of letters.

While this technology seems promising, there is still much to learn about the long-term effects it might have on human DNA. It is currently being trialled in a small number of patients affected by a severe blinding childhood condition called Leber congenital amaurosis (LCA) and it is only in the early stages of development for corneal dystrophies.

A short animation on CRISPR/Cas9

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How does CRISPR/Cas9 work?

CRISPR/Cas9 is based on the immune system of a bacteria called Streptococcus pyogenes. The Cas9 protein functions like a pair of molecular scissors and when guided by specially designed markers called guide RNAs, it can cut out the mutated DNA sequence precisely at a pre-determined location. The correct sequence can be provided as a template for the cell’s repair system to introduce into the DNA, enabling it to work normally again, much like using the “cut and paste” function in computers.

In order to deliver the Cas9 protein and its associated guide RNA to the photoreceptor cells in the retina, a similar technique to gene therapy is utilised. It is first packaged into a vehicle (medically known as a vector), usually a virus called adeno-associated virus (AAV) and injected into the retina through surgery (sub-retinal injection). The viruses are rendered harmless initially before introduction to human cells.

In animal models of corneal dystrophies, the Cas9 protein and guide RNA are injected into the stromal layer of the cornea instead. [3]

The normal gene copy is packaged into a harmless virus, which is then injected below the retina through surgery. This allows maximum exposure of the photoreceptors to the injected therapy
Sub-retinal injection technique used for gene therapy

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Is it available to patients now?

The CRISPR/Cas9 system is still being researched as a potential therapy for inherited retinal conditions, with an ongoing phase 1/2 trial for LCA patients (EDIT-101) carrying a specific mutation in the CEP290 gene that is very common in Europe and USA. This mutation introduces an abnormal “stop” signal into the gene and halts protein production prematurely, resulting in a protein which is too short and not functional. As a result, the photoreceptor cells do not work properly from birth and gradually degenerate over time.

Equipped with two guide RNAs that match either end of the abnormal “stop” signal, the Cas9 protein is able to remove this faulty code from the CEP290 gene and restore normal cellular function. EDIT-101 appeared effective and well-tolerated when injected into animal models of the same condition, which has led to the initiation of the current clinical trial.[4]

CRISPR/Cas9 is also being explored as a potential therapeutic option for certain types of corneal dystrophies such as Fuchs endothelial corneal dystrophy (the most common corneal dystrophy worldwide) and Meesmann epithelial corneal dystrophy, but it is still in early stages of development. It may take a number of years before human trials begin.

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Is it safe?

The EDIT-101 trial has only just begun and therefore safety data is unavailable yet. However, the safety profile of the AAV delivery system is well established based on previous retinal gene therapy trials, the most notable one being Luxturna which was approved for the treatment of LCA caused by mutations in the RPE65 gene.[5]

One of the main concerns about CRISPR/Cas9 is the risk of modifying other genetic sequences apart from the intended one (“off-target” editing). With EDIT-101, this risk is minimised by using two guide RNAs and delivering the Cas9 protein with an AAV that specifically targets the photoreceptor cells.[4] Furthermore, the particular CEP290 gene mutation that EDIT-101 aims to correct is only having an effect in the retina. Although pre-clinical experiments using human cells did not reveal any off-target editing, the ongoing clinical trial should provide us with more information in the future.[4]

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1) Off-target editing

The CRISPR/Cas9 system is thought to be more efficient and accurate than other gene editing techniques but a recent study has discovered that extensive genetic rearrangements can be found some distance away from the target genetic sequence.[6] As a result, certain crucial genes may be switched on or off which may have serious consequences on overall health, especially if actively dividing cells (cells that are constantly renewed in the body such as hair, skin and blood) are edited. Scientists are exploring various approaches to mitigate this risk such as making adjustments to the guide RNA and improving the sensitivity and range of tests that are used to assess off-target editing.[7]

2) Unknown long-term effects

Although EDIT-101 was shown to be effective and only caused a mild inflammation in the eye when injected into animal models of LCA, we are still unsure how the human immune system will react to genetically modified cells and whether the effects of gene editing can be sustained over a long period of time.

Related experimental treatments

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  1.  Xue H, Wu J, Li S, Rao MS, Liu Y. Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System. Methods Mol Biol. 2016;1307:173-190
  2.  Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821
  3.  Courtney DG, Moore JE, Atkinson SD, et al. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Ther. Jan 2016;23(1):108-12. doi:10.1038/gt.2015.82
  4.  Maeder ML, Stefanidakis M, Wilson CJ, et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat Med. 2019;25(2):229-233
  5.  Russell S, Bennett J, Wellman JA, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390(10097):849-860
  6.  Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765-771
  7.  Maeder ML, Gersbach CA. Genome-editing Technologies for Gene and Cell Therapy. Mol Ther. 2016;24(3):430-446

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Updated on December 3, 2020
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