A Spanish research team, based at the Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, have published data showing preliminary proof-of-principle for the use of a new gene editing technology aimed at correcting gene mutations that cause Usher’s syndrome. The technology, known as “CRISPR”, was used to correct mutations in the USH2A gene in cells isolated from patients with the disorder. Usher’s is a rare autosomal receive genetic disease caused by mutations in up to 13 distinct genes however, Usher’s type II appears to be the most frequent clinical sub-type. The disorder is characterized by combined visual and hearing impairment and has no current treatment.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has been a significant development within biotechnology in recent years. The gene editing technology derives from observations made in the repetitive sequences isolated from a number of prokaryotic and archaebacteria, first identified in 1987 by Yoshizumi Ishino and Atsuo Nakata, then at Japan’s Research Institute for Microbial Diseases, Osaka University. Following on from the original observations, three papers in 2005 reported that spacer sequences separating individual repetitive sequences appeared to have a phage (bacterial virus) associated origin. Coupled to this were separate observations that viruses were unable to infect cells that carried spacer sequences corresponding to their own genomes. In essence, the system as a whole appeared to represent an un-expected and sophisticated immune system for prokaryotes, essentially a new mechanism that provided an immune memory of previous phage infections, thereby facilitating rapid clearing of subsequent phage invasions that had previously infected the cell. By 2012 a paper published in Science by Jinek and colleagues at the University of California, Berkeley, in collaboration with the University of Vienna and Umeå University in Sweden, harnessed the technology into a system that is “efficient, versatile, and programmable”, with “considerable potential for gene-targeting and genome-editing applications.” Further iterations and development showed how relatively easy the CRISPR system could be used as an RNA-guided platform for specific control of gene expression. When compared to other editing tools, such as zinc finger proteins or TALENs, CRISPR has proved to be remarkably cheaper and less time consuming to work with.
The Spanish research is not the first time the technology has been applied to ophthalmic disease. In 2013, researchers at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, used CRISPR/Cas9 technology to correct a dominant mutation in the mouse crystallin gamma C gene (Crygc), mutations of which may cause significant cataracts. Injection of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant Crygc allele, into animal zygotes, facilitated gene correction via homology-directed repair based on either a supplied oligo or the wild type allele. The researchers reported minimal off-target modifications and confirmed that treated animals transmitted the corrected allele to their progeny.
The results achieved by the Valencia research group showed successful targeting of the c.2299G del mutation in the USH2A gene in which the use of CRISPR, and appropriate corrective DNA sequence, allowed for gene editing through the homology directed repair pathway. Although the rate of repair achieved in patients’ fibroblasts may be too low for in vivo application, several strategies to improve the performance may be considered, including ex-vivo therapy coupled with selection, and virally delivered CRISPR technology. In commenting on the research milestone, the authors of the study concluded that the, “study demonstrates for the first time that correcting the two most prevalent mutations of USH is feasible using the CRISPR/Cas9 system and homologous recombination, thereby offering future promise for repairing these and other mutations in cells from patients.”