Genome engineering

Described as the “biggest biotech discovery of the century”, clustered regularly interspaced short palindromic repeats (CRISPR) has the potential to revolutionise the treatment of genetic disease. The eye is a prime target for the early clinical use of this genome engineering technology, according to reports at the 2016 annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) in Seattle, USA. Genome editing using bacterial CRISPR offers several advantages over previous techniques, including lower cost, higher efficiency, better specificity and greater flexibility, with the potential for multiplex genome editing. Its flexibility and specificity makes CRISPR/Cas genome editing a good fit in today’s era where there is a focus on personalised medicine, noted Alex Hewitt MBBS, PhD, Head, Clinical Genetics, Centre for Eye Research Australia, Melbourne. “Few other medical applications could be considered as personalised as specifically tailoring a gRNA to target an individual patient’s disease-causing variant,” he told EuroTimes. Furthermore, unlike some other gene therapy approaches such as transient siRNA treatment, genome editing with CRISPR/Cas is considered to offer the possibility for a permanent treatment of genetic diseases, said Tara Moore PhD, Director of Biomedical Science Research Institute, Ulster University, Northern Ireland, and Director of R&D at Avellino Labs, Menlo Park, California, USA. Based on characterisation of an individual’s genetic profile, the enzyme system is relatively easy to reprogramme to target a particular disease-causing mutation through development of a personalised gRNA expression vector. Depending on the disease, the goal may be to knock out, repair or replace the mutant allele. Such in vivo genome editing may represent the ultimate dream for application of CRISPR/Cas technology, but it has other potential uses. CRISPR/Cas genome editing could be applied ex vivo for modification of autologous induced pluripotent stem cells (iPSCs) to develop cell replacement therapy for patients with a genetically defined disease, or the patient’s own corneal epithelial stem cells could be genetically corrected ex vivo and transplanted. Additionally, in vitro models are being developed that could facilitate studies of disease pathogenesis or pharmacological screening. A growing number of reports show success is being achieved in all of the above applications. To cite just a few examples, Bassuk et al (Sci Rep. 2016;Jan 27;6:19969) reported using CRISPR/Cas to correct a pathogenic mutation in iPSCs derived from fibroblasts of a patient with retinitis pigmentosa. Work by Dr Hewitt and colleagues provided proof of principle for using CRISPR/Cas to achieve in vivo gene modification of retinal cells in adult transgenic mice, and electroretinography studies showed that the gene editing was achieved without any adverse effects on retinal function. (Invest Ophthalmol Vis Sci. 2016; 57:3470–3476) Others have used the CRISPR/Cas system for in vivo mutation repair in animal models of retinitis pigmentosa (Mol Ther. 2016 Jun 28; Mol Ther. 2016;24(3):556-563). With an interest in heritable corneal diseases, Dr Moore, and colleagues were the first to show in vivo gene editing of a heterozygous disease-causing missense mutation in a humanised mouse model of Meesmann epithelial corneal dystrophy (MECD). (Gene Ther. 2016;23(1):108-112) Now they are working to increase the targeting efficiency of the gene-editing system, optimise its delivery, and characterise disease regression post-intervention. “This approach could work in approximately one-third of mutations known to cause corneal genetic disorders, of which Meesmann is just one, and for a number of the dominant negative retinal disorders,” she said. THE ROAD AHEAD Progress leading to clinical implementation of CRISPR/Cas genome editing faces some technical obstacles, including the need for techniques that can improve targeting specificity and the efficiency of mutation correction. Further investigation of the safety of CRISPR/Cas gene editing is also required before it can move into the clinical arena. “Unwanted gene editing at sites distinct from the intended target remains a concern that needs to be allayed before regulatory bodies are likely to approve any CRISPR/Cas gene therapy trials,” said Dr Moore. There are also ethical issues to address, and in that regard public opinion may influence whether gene therapy becomes a viable option. According to the findings of a global online survey conducted by Dr Hewitt and colleagues, however, there seems to be a favourable majority view about using CRISPR/Cas-based genome editing for curing life-threatening or debilitating diseases in patients of all ages. (Cell Stem Cell. 2016;18(5):569-572). “Nonetheless, it is clear that both somatic and germ line genome editing using CRISPR/Cas must not be rushed into ophthalmic care,” Dr Hewitt warned. Alex Hewitt: hewitt.alex@gmail.com Tara Moore: t.moore@ulster.ac.uk