A proof-of-principle study has shown that template-free CRISPR/Cas9 gene editing can correct mutations that cause X-linked retinitis pigmentosa in mouse models.
Retinitis pigmentosa is a major cause of inherited blindness globally. It is characterised by the progressive degeneration of retinal photoreceptors, eventually causing loss of vision. There are mutations in over 200 genes that are known to be involved in retinitis pigmentosa.
A particularly severe form of the disease, X-linked retinitis pigmentosa (XLRP), causes retinal disease in the first decades of life. It stems from mutations in genes of the X chromosome. These are most commonly loss-of-function mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Normally, the encoded protein RPGR maintains photoreceptor cell homeostasis and survival in the retina. The C-terminal domain of the ORF15 exon in RPGR is crucial for RPGR function. Frameshift mutations in ORF15 truncate this domain, ultimately causing retinal disease.
ORF15 is a key target for gene replacement therapy since it carries more than 70% of known disease-causing mutations in RPGR. However, previous attempts to engineer and deliver recombinant RPGR constructs have failed due to several obstacles. For instance, its repetitive nature makes RPGR challenging to sequence and prone to spontaneous mutations. Also, gene delivery necessitates caution as RPGR overdosage can be toxic.
Restoring RPGR expression with CRISPR/Cas9
A study, recently published in Gene Therapy, presents a simpler alternative for RPGR gene therapy. A team of researchers at the National Eye Institute USA, developed a simplified CRISPR/Cas9-based genome editing strategy that restores RPGR function.
Most CRISPR/Cas9-based genome editing systems use template-directed, homology-directed repair to replace target mutated sequences with wildtype sequences. However, this approach is highly inefficient. The current design employs non-homologous end joining (NHEJ) instead; the main and more efficient pathway for repairing double-stranded DNA damage. Here, CRISPR/Cas9 is first used to target and induce a double-stranded break in ORF15, which in turn triggers NHEJ. This joins the DNA ends together but introduces random base insertions or deletions in the junctions. In some cells, this results in a shift from the mutated to the normal reading frame, restoring RPGR function.
Using NHEJ in this design is feasible because of two unique features of the ORF15 region targeted by CRISPR/Cas9. First, it is highly tolerant of small indels due to its repetitiveness. Length alterations would not impair overall RPGR function. Further, the region does not contain pyrimidines, meaning that stop codons will not be randomly generated by NHEJ-mediated indels. As this strategy is template-free, it is simpler than template-directed forms of CRISPR/Cas9 gene editing.
A major limitation is that this strategy is only applicable for mutations in genes with similar genomic properties to RPGR.
A potential treatment for retinitis pigmentosa
The researchers tested this template-free design in rd9 mouse models of XLRP. They introduced the CRISPR/Cas9 machinery into mice via subretinal injections of viral vectors or pronuclear injections of ribonucleoprotein complexes. In both somatic and germ cell lines, RPGR-ORF15 expression was corrected in retinal photoreceptors. RPGR mutation-associated early disease phenotypes were also absent in these cells. Promisingly, the researchers did not observe off-target effects.
The design achieved restoration of RPGR-ORF15 expression in only up to 10% of photoreceptors. Nevertheless, this does not preclude the profound impact the therapy would have on patients. Restoring the function of a fraction of photoreceptors is sufficient to support the visual capacity needed for a normal life.
This paper is proof-of-principle for using this template-free CRISPR/Cas9 genome editing approach to correct pathogenic ORF15 mutations in vivo. As ORF15 sequences are poorly conserved, future studies in human models should modify the CRISPR guide RNA sequences. Now, the prospects for safe and effective gene therapy against XLRP are looking brighter.
Image credit: javi_indy – Freepik